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AUTHOR Barceló, Xavier and Eichholz, Kian F. and Gonçalves, Inês F. and Garcia, Orquidea and Kelly, Daniel J.
Title Bioprinting of structurally organized meniscal tissue within anisotropic melt electrowritten scaffolds [Abstract]
Year 2023
Journal/Proceedings Acta Biomaterialia
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Abstract
The meniscus is characterised by an anisotropic collagen fibre network which is integral to its biomechanical functionality. The engineering of structurally organized meniscal grafts that mimic the anisotropy of the native tissue remains a significant challenge. In this study, inkjet bioprinting was used to deposit a cell-laden bioink into additively manufactured scaffolds of differing architectures to engineer fibrocartilage grafts with user defined collagen architectures. Polymeric scaffolds consisting of guiding fibre networks with varying aspect ratios (1:1; 1:4; 1:16) were produced using either fused deposition modelling (FDM) or melt electrowriting (MEW), resulting in scaffolds with different internal architectures and fibre diameters. Scaffold architecture was found to influence the spatial organization of the collagen network laid down by the jetted cells, with higher aspect ratios (1:4 and 1:16) supporting the formation of structurally anisotropic tissues. The MEW scaffolds supported the development of a fibrocartilaginous tissue with compressive mechanical properties similar to that of native meniscus, while the anisotropic tensile properties of these constructs could be tuned by altering the fibre network aspect ratio. This MEW framework was then used to generate scaffolds with spatially distinct fibre patterns, which in turn supported the development of heterogenous tissues consisting of isotropic and anisotropic collagen networks. Such bioprinted tissues could potentially form the basis of new treatment options for damaged and diseased meniscal tissue. Statement of significance This study describes a multiple tool biofabrication strategy which enables the engineering of spatially organized fibrocartilage tissues. The architecture of MEW scaffolds can be tailored to not only modulate the directionality of the collagen fibres laid down by cells, but also to tune the anisotropic tensile mechanical properties of the resulting constructs, thereby enabling the engineering of biomimetic meniscal-like tissues. Furthermore, the inherent flexibility of MEW enables the development of zonally defined and potentially patient-specific implants.
AUTHOR Ainsworth, Madison Jade and Chirico, Nino and de Ruijter, Mylène and Hrynevich, Andrei and Dokter, Inge and Sluijter, Joost P. G. and Malda, Jos and van Mil, Alain and Castilho, Miguel
Title Convergence of melt electrowriting and extrusion-based bioprinting for vascular patterning of a myocardial construct [Abstract]
Year 2023
Journal/Proceedings Biofabrication
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To progress cardiac tissue engineering strategies closer to the clinic, thicker constructs are required to meet the functional need following a cardiac event. Consequently, pre-vascularization of these constructs needs to be investigated to ensure survival and optimal performance of implantable engineered heart tissue. The aim of this research is to investigate the potential of combining extrusion-based bioprinting (EBB) and melt electrowriting for the fabrication of a myocardial construct with a precisely patterned pre-vascular pathway. Gelatin methacryloyl (GelMA) was investigated as a base hydrogel for the respective myocardial and vascular bioinks with collagen, Matrigel and fibrinogen as interpenetrating polymers to support myocardial functionality. Subsequently, extrusion-based printability and viability were investigated to determine the optimal processing parameters for printing into melt electrowritten meshes. Finally, an anatomically inspired vascular pathway was implemented in a dual EBB set-up into melt electrowritten meshes, creating a patterned pre-vascularized myocardial construct. It was determined that a blend of 5% GelMA and 0.8 mg·ml−1 collagen with a low crosslinked density was optimal for myocardial cellular arrangement and alignment within the constructs. For the vascular fraction, the optimized formulation consisted of 5% GelMA, 0.8 mg·ml−1 collagen and 1 mg·ml−1 fibrinogen with a higher crosslinked density, which led to enhanced vascular cell connectivity. Printability assessment confirmed that the optimized bioinks could effectively fill the microfiber mesh while supporting cell viability (∼70%). Finally, the two bioinks were applied using a dual EBB system for the fabrication of a pre-vascular pathway with the shape of a left anterior descending artery within a myocardial construct, whereby the distinct cell populations could be visualized in their respective patterns up to D14. This research investigated the first step towards developing a thick engineered cardiac tissue construct in which a pre-vascularization pathway is fabricated within a myocardial construct.
AUTHOR Daghrery, Arwa and Ferreira, Jessica A. and Xu, Jinping and Golafshan, Nasim and Kaigler, Darnell and Bhaduri, Sarit B. and Malda, Jos and Castilho, Miguel and Bottino, Marco C.
Title Tissue-specific melt electrowritten polymeric scaffolds for coordinated regeneration of soft and hard periodontal tissues [Abstract]
Year 2023
Journal/Proceedings Bioactive Materials
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Abstract
Periodontitis is a chronic inflammatory condition that often causes serious damage to tooth-supporting tissues. The limited successful outcomes of clinically available approaches underscore the need for therapeutics that cannot only provide structural guidance to cells but can also modulate the local immune response. Here, three-dimensional melt electrowritten (i.e., poly(ε-caprolactone)) scaffolds with tissue-specific attributes were engineered to guide differentiation of human-derived periodontal ligament stem cells (hPDLSCs) and mediate macrophage polarization. The investigated tissue-specific scaffold attributes comprised fiber morphology (aligned vs. random) and highly-ordered architectures with distinct strand spacings (small 250 μm and large 500 μm). Macrophages exhibited an elongated morphology in aligned and highly-ordered scaffolds, while maintaining their round-shape on randomly-oriented fibrous scaffolds. Expressions of periostin and IL-10 were more pronounced on the aligned and highly-ordered scaffolds. While hPDLSCs on the scaffolds with 500 μm strand spacing show higher expression of osteogenic marker (Runx2) over 21 days, cells on randomly-oriented fibrous scaffolds showed upregulation of M1 markers. In an orthotopic mandibular fenestration defect model, findings revealed that the tissue-specific scaffolds (i.e., aligned fibers for periodontal ligament and highly-ordered 500 μm strand spacing fluorinated calcium phosphate [F/CaP]-coated fibers for bone) could enhance the mimicking of regeneration of natural periodontal tissues.
AUTHOR Daghrery, Arwa and Ferreira, Jessica A. and Xu, Jinping and Golafshan, Nasim and Kaigler, Darnell and Bhaduri, Sarit B. and Malda, Jos and Castilho, Miguel and Bottino, Marco C.
Title Tissue-specific melt electrowritten polymeric scaffolds for coordinated regeneration of soft and hard periodontal tissues [Abstract]
Year 2023
Journal/Proceedings Bioactive Materials
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DOI/URL URL DOI
Abstract
Periodontitis is a chronic inflammatory condition that often causes serious damage to tooth-supporting tissues. The limited successful outcomes of clinically available approaches underscore the need for therapeutics that cannot only provide structural guidance to cells but can also modulate the local immune response. Here, three-dimensional melt electrowritten (i.e., poly(ε-caprolactone)) scaffolds with tissue-specific attributes were engineered to guide differentiation of human-derived periodontal ligament stem cells (hPDLSCs) and mediate macrophage polarization. The investigated tissue-specific scaffold attributes comprised fiber morphology (aligned vs. random) and highly-ordered architectures with distinct strand spacings (small 250 μm and large 500 μm). Macrophages exhibited an elongated morphology in aligned and highly-ordered scaffolds, while maintaining their round-shape on randomly-oriented fibrous scaffolds. Expressions of periostin and IL-10 were more pronounced on the aligned and highly-ordered scaffolds. While hPDLSCs on the scaffolds with 500 μm strand spacing show higher expression of osteogenic marker (Runx2) over 21 days, cells on randomly-oriented fibrous scaffolds showed upregulation of M1 markers. In an orthotopic mandibular fenestration defect model, findings revealed that the tissue-specific scaffolds (i.e., aligned fibers for periodontal ligament and highly-ordered 500 μm strand spacing fluorinated calcium phosphate [F/CaP]-coated fibers for bone) could enhance the mimicking of regeneration of natural periodontal tissues.
AUTHOR Daghrery, Arwa and Ferreira, Jessica A. and Xu, Jinping and Golafshan, Nasim and Kaigler, Darnell and Bhaduri, Sarit B. and Malda, Jos and Castilho, Miguel and Bottino, Marco C.
Title Tissue-specific melt electrowritten polymeric scaffolds for coordinated regeneration of soft and hard periodontal tissues [Abstract]
Year 2023
Journal/Proceedings Bioactive Materials
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DOI/URL URL DOI
Abstract
Periodontitis is a chronic inflammatory condition that often causes serious damage to tooth-supporting tissues. The limited successful outcomes of clinically available approaches underscore the need for therapeutics that cannot only provide structural guidance to cells but can also modulate the local immune response. Here, three-dimensional melt electrowritten (i.e., poly(ε-caprolactone)) scaffolds with tissue-specific attributes were engineered to guide differentiation of human-derived periodontal ligament stem cells (hPDLSCs) and mediate macrophage polarization. The investigated tissue-specific scaffold attributes comprised fiber morphology (aligned vs. random) and highly-ordered architectures with distinct strand spacings (small 250 μm and large 500 μm). Macrophages exhibited an elongated morphology in aligned and highly-ordered scaffolds, while maintaining their round-shape on randomly-oriented fibrous scaffolds. Expressions of periostin and IL-10 were more pronounced on the aligned and highly-ordered scaffolds. While hPDLSCs on the scaffolds with 500 μm strand spacing show higher expression of osteogenic marker (Runx2) over 21 days, cells on randomly-oriented fibrous scaffolds showed upregulation of M1 markers. In an orthotopic mandibular fenestration defect model, findings revealed that the tissue-specific scaffolds (i.e., aligned fibers for periodontal ligament and highly-ordered 500 μm strand spacing fluorinated calcium phosphate [F/CaP]-coated fibers for bone) could enhance the mimicking of regeneration of natural periodontal tissues.
AUTHOR Daghrery, Arwa and Ferreira, Jessica A. and Xu, Jinping and Golafshan, Nasim and Kaigler, Darnell and Bhaduri, Sarit B. and Malda, Jos and Castilho, Miguel and Bottino, Marco C.
Title Tissue-specific melt electrowritten polymeric scaffolds for coordinated regeneration of soft and hard periodontal tissues [Abstract]
Year 2023
Journal/Proceedings Bioactive Materials
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DOI/URL URL DOI
Abstract
Periodontitis is a chronic inflammatory condition that often causes serious damage to tooth-supporting tissues. The limited successful outcomes of clinically available approaches underscore the need for therapeutics that cannot only provide structural guidance to cells but can also modulate the local immune response. Here, three-dimensional melt electrowritten (i.e., poly(ε-caprolactone)) scaffolds with tissue-specific attributes were engineered to guide differentiation of human-derived periodontal ligament stem cells (hPDLSCs) and mediate macrophage polarization. The investigated tissue-specific scaffold attributes comprised fiber morphology (aligned vs. random) and highly-ordered architectures with distinct strand spacings (small 250 μm and large 500 μm). Macrophages exhibited an elongated morphology in aligned and highly-ordered scaffolds, while maintaining their round-shape on randomly-oriented fibrous scaffolds. Expressions of periostin and IL-10 were more pronounced on the aligned and highly-ordered scaffolds. While hPDLSCs on the scaffolds with 500 μm strand spacing show higher expression of osteogenic marker (Runx2) over 21 days, cells on randomly-oriented fibrous scaffolds showed upregulation of M1 markers. In an orthotopic mandibular fenestration defect model, findings revealed that the tissue-specific scaffolds (i.e., aligned fibers for periodontal ligament and highly-ordered 500 μm strand spacing fluorinated calcium phosphate [F/CaP]-coated fibers for bone) could enhance the mimicking of regeneration of natural periodontal tissues.
AUTHOR Terpstra, Margo L. and Li, Jinyu and Mensinga, Anneloes and de Ruijter, Myl{`{e}}ne and van Rijen, Mattie H. P. and Androulidakis, Charalampos and Galiotis, Costas and Papantoniou, Ioannis and Matsusaki, Michiya and Malda, Jos and Levato, Riccardo
Title Bioink with cartilage-derived extracellular matrix microfibers enables spatial control of vascular capillary formation in bioprinted constructs [Abstract]
Year 2022
Journal/Proceedings Biofabrication
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Microvasculature is essential for the exchange of gas and nutrient for most tissues in our body. Some tissue structures such as the meniscus presents spatially confined blood vessels adjacent to non-vascularized regions. In biofabrication, mimicking the spatial distribution of such vascular components is paramount, as capillary ingrowth into non-vascularized tissues can lead to tissue matrix alterations and subsequent pathology. Multi-material three-dimensional (3D) bioprinting strategies have the potential to resolve anisotropic tissue features, although building complex constructs comprising stable vascularized and non-vascularized regions remains a major challenge to date. In this study, we developed endothelial cell-laden pro- and anti-angiogenic bioinks, supplemented with bioactive matrix-derived microfibers (MFs) that were created from type I collagen sponges (col-1) and cartilage decellularized extracellular matrix (CdECM), respectively. Human umbilical vein endothelial cell (HUVEC)-driven capillary networks started to form 2 d after bioprinting. Supplementing cartilage-derived MFs to endothelial-cell laden bioinks reduced the total length of neo-microvessels by 29%, and the number of microvessel junctions by 37% after 14 d, compared to bioinks with pro-angiogenic col-1 MFs. As a proof of concept, the bioinks were bioprinted into an anatomical meniscus shape with a biomimetic vascularized outer and non-vascularized inner region, using a gellan gum microgel suspension bath. These 3D meniscus-like constructs were cultured up to 14 d, with in the outer zone the HUVEC-, mural cell-, and col-1 MF-laden pro-angiogenic bioink, and in the inner zone a meniscus progenitor cell (MPC)- and CdECM MF-laden anti-angiogenic bioink, revealing successful spatial confinement of the nascent vascular network only in the outer zone. Further, to co-facilitate both microvessel formation and MPC-derived matrix formation, we formulated cell culture medium conditions with a temporal switch. Overall, this study provides a new strategy that could be applied to develop zonal biomimetic meniscal constructs. Moreover, the use of ECM-derived MFs to promote or inhibit capillary networks opens new possibilities for the biofabrication of tissues with anisotropic microvascular distribution. These have potential for many applications including in vitro models of vascular-to-avascular tissue interfaces, cancer progression, and for testing anti-angiogenic therapies.
AUTHOR Dufour, A. and Gallostra, X. Barceló and O'Keeffe, C. and Eichholz, K. and Von Euw, S. and Garcia, O. and Kelly, D. J.
Title Integrating melt electrowriting and inkjet bioprinting for engineering structurally organized articular cartilage [Abstract]
Year 2022
Journal/Proceedings Biomaterials
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Successful cartilage engineering requires the generation of biological grafts mimicking the structure, composition and mechanical behaviour of the native tissue. Here melt electrowriting (MEW) was used to produce arrays of polymeric structures whose function was to orient the growth of cellular aggregates spontaneously generated within these structures, and to provide tensile reinforcement to the resulting tissues. Inkjet printing was used to deposit defined numbers of cells into MEW structures, which self-assembled into an organized array of spheroids within hours, ultimately generating a hybrid tissue that was hyaline-like in composition. Structurally, the engineered cartilage mimicked the histotypical organization observed in skeletally immature synovial joints. This biofabrication framework was then used to generate scaled-up (50 mm × 50 mm) cartilage implants containing over 3,500 cellular aggregates in under 15 min. After 8 weeks in culture, a 50-fold increase in the compressive stiffness of these MEW reinforced tissues were observed, while the tensile properties were still dominated by the polymer network, resulting in a composite construct demonstrating tension-compression nonlinearity mimetic of the native tissue. Helium ion microscopy further demonstrated the development of an arcading collagen network within the engineered tissue. This hybrid bioprinting strategy provides a versatile and scalable approach to engineer cartilage biomimetic grafts for biological joint resurfacing.
AUTHOR Daghrery, Arwa and Ferreira, Jessica A. and de Souza Araújo, Isaac J. and Clarkson, Brian H. and Eckert, George J. and Bhaduri, Sarit B. and Malda, Jos and Bottino, Marco C.
Title A Highly Ordered, Nanostructured Fluorinated CaP-Coated Melt Electrowritten Scaffold for Periodontal Tissue Regeneration [Abstract]
Year 2021
Journal/Proceedings Advanced Healthcare Materials
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Abstract Periodontitis is a chronic inflammatory, bacteria-triggered disorder affecting nearly half of American adults. Although some level of tissue regeneration is realized, its low success in complex cases demands superior strategies to amplify regenerative capacity. Herein, highly ordered scaffolds are engineered via Melt ElectroWriting (MEW), and the effects of strand spacing, as well as the presence of a nanostructured fluorinated calcium phosphate (F/CaP) coating on the adhesion/proliferation, and osteogenic differentiation of human-derived periodontal ligament stem cells, are investigated. Upon initial cell-scaffold interaction screening aimed at defining the most suitable design, MEW poly(ε-caprolactone) scaffolds with 500 µm strand spacing are chosen. Following an alkali treatment, scaffolds are immersed in a pre-established solution to allow for coating formation. The presence of a nanostructured F/CaP coating leads to a marked upregulation of osteogenic genes and attenuated bacterial growth. In vivo findings confirm that the F/CaP-coated scaffolds are biocompatible and lead to periodontal regeneration when implanted in a rat mandibular periodontal fenestration defect model. In aggregate, it is considered that this work can contribute to the development of personalized scaffolds capable of enabling tissue-specific differentiation of progenitor cells, and thus guide simultaneous and coordinated regeneration of soft and hard periodontal tissues, while providing antimicrobial protection.
AUTHOR He, Shaolong and Radeke, Carmen and Jacobsen, Jette and Lind, Johan Ulrik and Mu, Huiling
Title Multi-material 3D printing of programmable and stretchable oromucosal patches for delivery of saquinavir [Abstract]
Year 2021
Journal/Proceedings International Journal of Pharmaceutics
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Oromucosal patches for drug delivery allow fast onset of action and ability to circumvent hepatic first pass metabolism of drugs. While conventional fabrication methods such as solvent casting or hot melt extrusion are ideal for scalable production of low-cost delivery patches, these methods chiefly allow for simple, homogenous patch designs. As alternative, a multi-material direct-ink-write 3D printing for rapid fabrication of complex oromucosal patches with unique design features was demonstrated in the present study. Specifically, three print-materials: an acidic saquinavir-loaded hydroxypropyl methylcellulose ink, an alkaline effervescent sodium carbonate-loaded ink, and a methyl cellulose backing material were combined in various designs. The CO2 content and pH of the microenvironment were controlled by adjusting the number of alkaline layers in the patch. Additionally, the rigid and brittle patches were converted to compliant and stretchable patches by implementing mesh-like designs. Our results illustrate how 3D printing can be used for rapid design and fabrication of multifunctional or customized oromucosal patches with tailored dosages and changed drug permeation.
AUTHOR Asulin, Masha and Michael, Idan and Shapira, Assaf and Dvir, Tal
Title One-Step 3D Printing of Heart Patches with Built-In Electronics for Performance Regulation [Abstract]
Year 2021
Journal/Proceedings Advanced Science
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Abstract Three dimensional (3D) printing of heart patches usually provides the ability to precisely control cell location in 3D space. Here, one-step 3D printing of cardiac patches with built-in soft and stretchable electronics is reported. The tissue is simultaneously printed using three distinct bioinks for the cells, for the conducting parts of the electronics and for the dielectric components. It is shown that the hybrid system can withstand continuous physical deformations as those taking place in the contracting myocardium. The electronic patch is flexible, stretchable, and soft, and the electrodes within the printed patch are able to monitor the function of the engineered tissue by providing extracellular potentials. Furthermore, the system allowed controlling tissue function by providing electrical stimulation for pacing. It is envisioned that such transplantable patches may regain heart contractility and allow the physician to monitor the implant function as well as to efficiently intervene from afar when needed.
AUTHOR Madiedo-Podvrsan, Sabrina and Belaïdi, Jean-Philippe and Desbouis, Stephanie and Simonetti, Lucie and Ben-Khalifa, Youcef and Soeur, Jérémie and Rielland, Maïté
Title Utilization of patterned bioprinting for heterogeneous and physiologically representative reconstructed epidermal skin models [Abstract]
Year 2021
Journal/Proceedings Scientific Reports
Reftype Madiedo-Podvrsan2021
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Organotypic skin tissue models have decades of use for basic research applications, the treatment of burns, and for efficacy/safety evaluation studies. The complex and heterogeneous nature of native human skin however creates difficulties for the construction of physiologically comparable organotypic models. Within the present study, we utilized bioprinting technology for the controlled deposition of separate keratinocyte subpopulations to create a reconstructed epidermis with two distinct halves in a single insert, each comprised of a different keratinocyte sub-population, in order to better model heterogonous skin and reduce inter-sample variability. As an initial proof-of-concept, we created a patterned epidermal skin model using GPF positive and negative keratinocyte subpopulations, both printed into 2 halves of a reconstructed skin insert, demonstrating the feasibility of this approach. We then demonstrated the physiological relevance of this bioprinting technique by generating a heterogeneous model comprised of dual keratinocyte population with either normal or low filaggrin expression. The resultant model exhibited a well-organized epidermal structure with each half possessing the phenotypic characteristics of its constituent cells, indicative of a successful and stable tissue reconstruction. This patterned skin model aims to mimic the edge of lesions as seen in atopic dermatitis or ichthyosis vulgaris, while the use of two populations within a single insert allows for paired statistics in evaluation studies, likely increasing study statistical power and reducing the number of models required per study. This is the first report of human patterned epidermal model using a predefined bioprinted designs, and demonstrates the relevance of bioprinting to faithfully reproduce human skin microanatomy.
AUTHOR Browning, James R. and Derr, Paige and Derr, Kristy and Doudican, Nicole and Michael, Sam and Lish, Samantha R. and Taylor, Nicholas A. and Krueger, James G. and Ferrer, Marc and Carucci, John A. and Gareau, Daniel S.
Title A 3D biofabricated cutaneous squamous cell carcinoma tissue model with multi-channel confocal microscopy imaging biomarkers to quantify antitumor effects of chemotherapeutics in tissue [Abstract]
Year 2020
Journal/Proceedings Oncotarget; Vol 11, No 27
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// James R. Browning 1 , Paige Derr 2 , Kristy Derr 2 , Nicole Doudican 3 , Sam Michael 2 , Samantha R. Lish 1 , Nicholas A. Taylor 3 , James G. Krueger 1 , Marc Ferrer 2 , John A. Carucci 3 and Daniel S. Gareau 1 1 Laboratory for Investigative Dermatology, The Rockefeller University, New York, New York, USA 2 National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA 3 The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA Correspondence to: Daniel S. Gareau, email: dgareau@rockefeller.edu Keywords: squamous cell carcinoma; screening; 3D printing; in vitro model; confocal microscopy Received: January 05, 2020     Accepted: April 03, 2020     Published: July 07, 2020 ABSTRACT Cutaneous squamous cell carcinoma (cSCC) causes approximately 10,000 deaths annually in the U. S. Current therapies are largely ineffective against metastatic and locally advanced cSCC. There is a need to identify novel, effective, and less toxic small molecule cSCC therapeutics. We developed a 3-dimensional bioprinted skin (3DBPS) model of cSCC tumors together with a microscopy assay to test chemotherapeutic effects in tissue. The full thickness SCC tissue model was validated using hematoxylin and eosin (H&E) and immunohistochemical histological staining, confocal microscopy, and cDNA microarray analysis. A nondestructive, 3D fluorescence confocal imaging assay with tdTomato-labeled A431 SCC and ZsGreen-labeled keratinocytes was developed to test efficacy and general toxicity of chemotherapeutics. Fluorescence-derived imaging biomarkers indicated that 50% of cancer cells were killed in the tissue after 1?M 5-Fluorouracil 48-hour treatment, compared to a baseline of 12% for untreated controls. The imaging biomarkers also showed that normal keratinocytes were less affected by treatment (11% killed) than the untreated tissue, which had no significant killing effect. Data showed that 5-Fluorouracil selectively killed cSCC cells more than keratinocytes. Our 3DBPS assay platform provides cellular-level measurement of cell viability and can be adapted to achieve nondestructive high-throughput screening (HTS) in bio-fabricated tissues.
AUTHOR Benmeridja, Lara and De Moor, Lise and De Maere, Elisabeth and Vanlauwe, Florian and Ryx, Michelle and Tytgat, Liesbeth and Vercruysse, Chris and Dubruel, Peter and Van Vlierberghe, Sandra and Blondeel, Phillip and Declercq, Heidi
Title High-throughput fabrication of vascularized adipose microtissues for 3D bioprinting [Abstract]
Year 2020
Journal/Proceedings Journal of Tissue Engineering and Regenerative Medicine
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Abstract For patients with soft tissue defects, repair with autologous in vitro engineered adipose tissue could be a promising alternative to current surgical therapies. A volume-persistent engineered adipose tissue construct under in vivo conditions can only be achieved by early vascularization after transplantation. The combination of 3D bioprinting technology with self-assembling microvascularized units as building blocks can potentially answer the need for a microvascular network. In the present study, co-culture spheroids combining adipose-derived stem cells (ASC) and human umbilical vein endothelial cells (HUVEC) were created with an ideal geometry for bioprinting. When applying the favourable seeding technique and condition, compact viable spheroids were obtained, demonstrating high adipogenic differentiation and capillary-like network formation after 7 and 14 days of culture, as shown by live/dead analysis, immunohistochemistry and RT-qPCR. Moreover, we were able to successfully 3D bioprint the encapsulated spheroids, resulting in compact viable spheroids presenting capillary-like structures, lipid droplets and spheroid outgrowth after 14 days of culture. This is the first study that generates viable high-throughput (pre-)vascularized adipose microtissues as building blocks for bioprinting applications using a novel ASC/HUVEC co-culture spheroid model, which enables both adipogenic differentiation while simultaneously supporting the formation of prevascular-like structures within engineered tissues in vitro.
AUTHOR Dubey, Nileshkumar and Ferreira, Jessica A. and Daghrery, Arwa and Aytac, Zeynep and Malda, Jos and Bhaduri, Sarit B. and Bottino, Marco C.
Title Highly Tunable Bioactive Fiber-Reinforced Hydrogel for Guided Bone Regeneration [Abstract]
Year 2020
Journal/Proceedings Acta Biomaterialia
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One of the most damaging pathologies that affects the health of both soft and hard tissues around the tooth is periodontitis. Clinically, periodontal tissue destruction has been managed by an integrated approach involving elimination of injured tissues followed by regenerative strategies with bone substitutes and/or barrier membranes. Regrettably, a barrier membrane with predictable mechanical integrity and multifunctional therapeutic features has yet to be established. Herein, we report a fiber-reinforced hydrogel with unprecedented tunability in terms of mechanical competence and therapeutic features by integration of highly porous poly(ε-caprolactone) fibrous mesh(es) with well-controlled 3D architecture into bioactive amorphous magnesium phosphate-laden gelatin methacryloyl hydrogels. The presence of amorphous magnesium phosphate and PCL mesh in the hydrogel can control the mechanical properties and improve the osteogenic ability, opening a tremendous opportunity in guided bone regeneration (GBR). Results demonstrate that the presence of PCL meshes fabricated via melt electrowriting can delay hydrogel degradation preventing soft tissue invasion and providing the mechanical barrier to allow time for slower migrating progenitor cells to participate in bone regeneration due to their ability to differentiate into bone-forming cells. Altogether, our approach offers a platform technology for the development of the next-generation of GBR membranes with tunable mechanical and therapeutic properties to amplify bone regeneration in compromised sites.
AUTHOR Peiffer, Quentin C. and de Ruijter, Mylène and van Duijn, Joost and Crottet, Denis and Dominic, Ernst and Malda, Jos and Castilho, Miguel
Title Melt electrowriting onto anatomically relevant biodegradable substrates: Resurfacing a diarthrodial joint [Abstract]
Year 2020
Journal/Proceedings Materials & Design
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Three-dimensional printed hydrogel constructs with well-organized melt electrowritten (MEW) fibre-reinforcing scaffolds have been demonstrated as a promising regenerative approach to treat small cartilage defects. Here, we investige how to translate the fabrication of small fibre-reinforced structures on flat surfaces to anatomically relevant structures. In particular, the accurate deposition of MEW-fibres onto curved surfaces of conductive and non-conductive regenerative biomaterials is studied. This study reveals that clinically relevant materials with low conductivities are compatible with resurfacing with organized MEW fibres. Importantly, accurate patterning on non-flat surfaces was successfully shown, provided that a constant electrical field strength and an electrical force normal to the substrate material is maintained. Furthermore, the application of resurfacing the geometry of the medial human femoral condyle is confirmed by the fabrication of a personalised osteochondral implant. The implant composed of an articular cartilage-resident chondroprogenitor cells (ACPCs)-laden hydrogel reinforced with a well-organized MEW scaffold retained its personalised shape, improved its compressive properties and supported neocartilage formation after 28 days in vitro culture. Overall, this study establishes the groundwork for translating MEW from planar and non-resorbable material substrates to anatomically relevant geometries and regenerative materials that the regenerative medicine field aims to create.
AUTHOR Afanasenkau, Dzmitry and Kalinina, Daria and Lyakhovetskii, Vsevolod and Tondera, Christoph and Gorsky, Oleg and Moosavi, Seyyed and Pavlova, Natalia and Merkulyeva, Natalia and Kalueff, Allan V. and Minev, Ivan R. and Musienko, Pavel
Title Rapid prototyping of soft bioelectronic implants for use as neuromuscular interfaces [Abstract]
Year 2020
Journal/Proceedings Nature Biomedical Engineering
Reftype Afanasenkau2020
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Abstract
Neuromuscular interfaces are required to translate bioelectronic technologies for application in clinical medicine. Here, by leveraging the robotically controlled ink-jet deposition of low-viscosity conductive inks, extrusion of insulating silicone pastes and in situ activation of electrode surfaces via cold-air plasma, we show that soft biocompatible materials can be rapidly printed for the on-demand prototyping of customized electrode arrays well adjusted to specific anatomical environments, functions and experimental models. We also show, with the monitoring and activation of neuronal pathways in the brain, spinal cord and neuromuscular system of cats, rats and zebrafish, that the printed bioelectronic interfaces allow for long-term integration and functional stability. This technology might enable personalized bioelectronics for neuroprosthetic applications.
AUTHOR Noor, Nadav and Shapira, Assaf and Edri, Reuven and Gal, Idan and Wertheim, Lior and Dvir, Tal
Title 3D Printing of Personalized Thick and Perfusable Cardiac Patches and Hearts [Abstract]
Year 2019
Journal/Proceedings Advanced Science
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Abstract Generation of thick vascularized tissues that fully match the patient still remains an unmet challenge in cardiac tissue engineering. Here, a simple approach to 3D-print thick, vascularized, and perfusable cardiac patches that completely match the immunological, cellular, biochemical, and anatomical properties of the patient is reported. To this end, a biopsy of an omental tissue is taken from patients. While the cells are reprogrammed to become pluripotent stem cells, and differentiated to cardiomyocytes and endothelial cells, the extracellular matrix is processed into a personalized hydrogel. Following, the two cell types are separately combined with hydrogels to form bioinks for the parenchymal cardiac tissue and blood vessels. The ability to print functional vascularized patches according to the patient's anatomy is demonstrated. Blood vessel architecture is further improved by mathematical modeling of oxygen transfer. The structure and function of the patches are studied in vitro, and cardiac cell morphology is assessed after transplantation, revealing elongated cardiomyocytes with massive actinin striation. Finally, as a proof of concept, cellularized human hearts with a natural architecture are printed. These results demonstrate the potential of the approach for engineering personalized tissues and organs, or for drug screening in an appropriate anatomical structure and patient-specific biochemical microenvironment.
AUTHOR Daly, Andrew C. and Kelly, Daniel J.
Title Biofabrication of spatially organised tissues by directing the growth of cellular spheroids within 3D printed polymeric microchambers [Abstract]
Year 2019
Journal/Proceedings Biomaterials
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Abstract
Successful tissue engineering requires the generation of human scale implants that mimic the structure, composition and mechanical properties of native tissues. Here, we report a novel biofabrication strategy that enables the engineering of structurally organised tissues by guiding the growth of cellular spheroids within arrays of 3D printed polymeric microchambers. With the goal of engineering stratified articular cartilage, inkjet bioprinting was used to deposit defined numbers of mesenchymal stromal cells (MSCs) and chondrocytes into pre-printed microchambers. These jetted cell suspensions rapidly underwent condensation within the hydrophobic microchambers, leading to the formation of organised arrays of cellular spheroids. The microchambers were also designed to provide boundary conditions to these spheroids, guiding their growth and eventual fusion, leading to the development of stratified cartilage tissue with a depth-dependant collagen fiber architecture that mimicked the structure of native articular cartilage. Furthermore, the composition and biomechanical properties of the bioprinted cartilage was also comparable to the native tissue. Using multi-tool biofabrication, we were also able to engineer anatomically accurate, human scale, osteochondral templates by printing this microchamber system on top of a hypertrophic cartilage region designed to support endochondral bone formation and then maintaining the entire construct in long-term bioreactor culture to enhance tissue development. This bioprinting strategy provides a versatile and scalable approach to engineer structurally organised cartilage tissues for joint resurfacing applications.
AUTHOR Derr, Kristy and Zou, Jinyun and Luo, Keren and Song, Min Jae and Sittampalam, G. Sitta and Zhou, Chao and Michael, Samuel and Ferrer, Marc and Derr, Paige
Title Fully 3D Bioprinted Skin Equivalent Constructs with Validated Morphology and Barrier Function [Abstract]
Year 2019
Journal/Proceedings Tissue Engineering Part C: Methods
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Abstract
Development of high throughput, reproducible, three-dimensional bioprinted skin equivalents that are morphologically and functionally comparable to native skin tissue is advancing research in skin diseases, and providing a physiologically relevant platform for the development of therapeutics, transplants for regenerative medicine, and testing of skin products like cosmetics. Current protocols for the production of engineered skin rafts are limited in their ability to control three dimensional geometry of the structure and contraction leading to variability of skin function between constructs. Here we describe a method for the biofabrication of skin equivalents that are fully bioprinted using an open market bioprinter, made with commercially available primary cells and natural hydrogels. The unique hydrogel formulation allows for the production of a human-like skin equivalent with minimal lateral tissue contraction in a multiwell plate format, thus making them suitable for high throughput bioprinting in a single print with fast print and relatively short incubation times. The morphology and barrier function of the fully three-dimensional bioprinted skin equivalents are validated by immunohistochemistry staining, optical coherence tomography, and permeation assays.
AUTHOR Gonzalez-Fernandez, T. and Rathan, S. and Hobbs, C. and Pitacco, P. and Freeman, F. E. and Cunniffe, G. M. and Dunne, N. J. and McCarthy, H. O. and Nicolosi, V. and O'Brien, F. J. and Kelly, D. J.
Title Pore-forming bioinks to enable Spatio-temporally defined gene delivery in bioprinted tissues [Abstract]
Year 2019
Journal/Proceedings Journal of Controlled Release
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Abstract
The regeneration of complex tissues and organs remains a major clinical challenge. With a view towards bioprinting such tissues, we developed a new class of pore-forming bioink to spatially and temporally control the presentation of therapeutic genes within bioprinted tissues. By blending sacrificial and stable hydrogels, we were able to produce bioinks whose porosity increased with time following printing. When combined with amphipathic peptide-based plasmid DNA delivery, these bioinks supported enhanced non-viral gene transfer to stem cells in vitro. By modulating the porosity of these bioinks, it was possible to direct either rapid and transient (pore-forming bioinks), or slower and more sustained (solid bioinks) transfection of host or transplanted cells in vivo. To demonstrate the utility of these bioinks for the bioprinting of spatially complex tissues, they were next used to zonally position stem cells and plasmids encoding for either osteogenic (BMP2) or chondrogenic (combination of TGF-β3, BMP2 and SOX9) genes within networks of 3D printed thermoplastic fibers to produce mechanically reinforced, gene activated constructs. In vivo, these bioprinted tissues supported the development of a vascularised, bony tissue overlaid by a layer of stable cartilage. When combined with multiple-tool biofabrication strategies, these gene activated bioinks can enable the bioprinting of a wide range of spatially complex tissues.
AUTHOR de Ruijter, Mylène and Ribeiro, Alexandre and Dokter, Inge and Castilho, Miguel and Malda, Jos
Title Simultaneous Micropatterning of Fibrous Meshes and Bioinks for the Fabrication of Living Tissue Constructs [Abstract]
Year 2018
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract Fabrication of biomimetic tissues holds much promise for the regeneration of cells or organs that are lost or damaged due to injury or disease. To enable the generation of complex, multicellular tissues on demand, the ability to design and incorporate different materials and cell types needs to be improved. Two techniques are combined: extrusion-based bioprinting, which enables printing of cell-encapsulated hydrogels; and melt electrowriting (MEW), which enables fabrication of aligned (sub)-micrometer fibers into a single-step biofabrication process. Composite structures generated by infusion of MEW fiber structures with hydrogels have resulted in mechanically and biologically competent constructs; however, their preparation involves a two-step fabrication procedure that limits freedom of design of microfiber architectures and the use of multiple materials and cell types. How convergence of MEW and extrusion-based bioprinting allows fabrication of mechanically stable constructs with the spatial distributions of different cell types without compromising cell viability and chondrogenic differentiation of mesenchymal stromal cells is demonstrated for the first time. Moreover, this converged printing approach improves freedom of design of the MEW fibers, enabling 3D fiber deposition. This is an important step toward biofabrication of voluminous and complex hierarchical structures that can better resemble the characteristics of functional biological tissues.
AUTHOR Cunniffe, Gráinne and Gonzalez-Fernandez, Tomas and Daly, Andrew and Nelson Sathy, Binulal and Jeon, Oju and Alsberg, Eben and J. Kelly, Daniel
Title Three-Dimensional Bioprinting of Polycaprolactone Reinforced Gene Activated Bioinks for Bone Tissue Engineering [Abstract]
Year 2017
Journal/Proceedings Tissue Engineering Part A
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Abstract
Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-g-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bonemarrow-derived mesenchymal stemcells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization andmineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs.
AUTHOR Yao, Y. and Raymond, J. E. and Kauffmann, F. and Maekawa, S. and Sugai, J. V. and Lahann, J. and Giannobile, W. V.
Title Multicompartmental Scaffolds for Coordinated Periodontal Tissue Engineering [Abstract]
Year 2023
Journal/Proceedings Journal of Dental Research
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Abstract
Successful periodontal repair and regeneration requires the coordinated responses from soft and hard tissues as well as the soft tissue–to–bone interfaces. Inspired by the hierarchical structure of native periodontal tissues, tissue engineering technology provides unique opportunities to coordinate multiple cell types into scaffolds that mimic the natural periodontal structure in vitro. In this study, we designed and fabricated highly ordered multicompartmental scaffolds by melt electrowriting, an advanced 3-dimensional (3D) printing technique. This strategy attempted to mimic the characteristic periodontal microenvironment through multicompartmental constructs comprising 3 tissue-specific regions: 1) a bone compartment with dense mesh structure, 2) a ligament compartment mimicking the highly aligned periodontal ligaments (PDLs), and 3) a transition region that bridges the bone and ligament, a critical feature that differentiates this system from mono- or bicompartmental alternatives. The multicompartmental constructs successfully achieved coordinated proliferation and differentiation of multiple cell types in vitro within short time, including both ligamentous- and bone-derived cells. Long-term 3D coculture of primary human osteoblasts and PDL fibroblasts led to a mineral gradient from calcified to uncalcified regions with PDL-like insertions within the transition region, an effect that is challenging to achieve with mono- or bicompartmental platforms. This process effectively recapitulates the key feature of interfacial tissues in periodontium. Collectively, this tissue-engineered approach offers a fundament for engineering periodontal tissue constructs with characteristic 3D microenvironments similar to native tissues. This multicompartmental 3D printing approach is also highly compatible with the design of next-generation scaffolds, with both highly adjustable compartmentalization properties and patient-specific shapes, for multitissue engineering in complex periodontal defects.
AUTHOR Silberman, Eric and Oved, Hadas and Namestnikov, Michael and Shapira, Assaf and Dvir, Tal
Title Post-Maturation Reinforcement of 3d-Printed Vascularized Cardiac Tissues [Abstract]
Year 2023
Journal/Proceedings Advanced Materials
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Abstract
Abstract Despite advances in biomaterials engineering, a large gap remains between the weak mechanical properties that can be achieved with natural materials and the strength of synthetic materials. Here, we present a method for reinforcing an engineered cardiac tissue fabricated from differentiated iPSCs and an ECM-based hydrogel in a manner that is fully biocompatible. The reinforcement occurs as a post-fabrication step, which allows for the use of 3D printing technology to generate thick, fully cellularized, and vascularized cardiac tissues. After tissue assembly and during the maturation process in a soft hydrogel, a small, tissue-penetrating reinforcer is deployed, leading to a significant increase in the tissue's mechanical properties. The tissue's robustness is demonstrated by injecting the tissue in a simulated minimally invasive procedure and showing that the tissue is functional and undamaged at the nano-, micro-, and macro-scales. This article is protected by copyright. All rights reserved
AUTHOR Ainsworth, Madison J. and Lotz, Oliver and Gilmour, Aaron and Zhang, Anyu and Chen, Michael J. and McKenzie, David R. and Bilek, Marcela M. M. and Malda, Jos and Akhavan, Behnam and Castilho, Miguel
Title Covalent Protein Immobilization on 3D-Printed Microfiber Meshes for Guided Cartilage Regeneration [Abstract]
Year 2022
Journal/Proceedings Advanced Functional Materials
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Abstract
Abstract Current biomaterial-based strategies explored to treat articular cartilage defects have failed to provide adequate physico-chemical cues in order to guide functional tissue regeneration. Here, it is hypothesized that atmospheric-pressure plasma (APPJ) treatment and melt electrowriting (MEW) will produce microfiber support structures with covalently-immobilized transforming growth factor beta-1 (TGFβ1) that can stimulate the generation of functional cartilage tissue. The effect of APPJ operational speeds to activate MEW polycaprolactone meshes for immobilization of TGFβ1 is first investigated and chondrogenic differentiation and neo-cartilage production are assessed in vitro. All APPJ speeds test enhanced hydrophilicity of the meshes, with the slow treatment speed having significantly less CC/CH and more COOH than the untreated meshes. APPJ treatment increases TGFβ1 loading efficiency. Additionally, in vitro experiments highlight that APPJ-based TGFβ1 attachment to the scaffolds is more advantageous than direct supplementation within the medium. After 28 days of culture, the group with immobilized TGFβ1 has significantly increased compressive modulus (more than threefold) and higher glycosaminoglycan production (more than fivefold) than when TGFβ1 is supplied through the medium. These results demonstrate that APPJ activation allows reagent-free, covalent immobilization of TGFβ1 on microfiber meshes and, importantly, that the biofunctionalized meshes can stimulate neo-cartilage matrix formation. This opens new perspectives for guided tissue regeneration.
AUTHOR Park, Hae Sang and Lee, Ji Seung and Kim, Chang-Beom and Lee, Kwang-Ho and Hong, In-Sun and Jung, Harry and Lee, Hanna and Lee, Young Jin and Ajiteru, Olatunji and Sultan, Md Tipu and Lee, Ok Joo and Kim, Soon Hee and Park, Chan Hum
Title Fluidic integrated 3D bioprinting system to sustain cell viability towards larynx fabrication [Abstract]
Year 2022
Journal/Proceedings Bioengineering & Translational Medicine
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Abstract
Abstract Herein, we report the first study to create a three-dimensional (3D) bioprinted artificial larynx for whole-laryngeal replacement. Our 3D bio-printed larynx was generated using extrusion-based 3D bioprinter with rabbit's chondrocyte-laden gelatin methacryloyl (GelMA)/glycidyl-methacrylated hyaluronic acid (GMHA) hybrid bioink. We used a polycaprolactone (PCL) outer framework incorporated with pores to achieve the structural strength of printed constructs, as well as to provide a suitable microenvironment to support printed cells. Notably, we established a novel fluidics supply (FS) system that simultaneously supplies basal medium together with a 3D bioprinting process, thereby improving cell survival during the printing process. Our results showed that the FS system enhanced post-printing cell viability, which enabled the generation of a large-scale cell-laden artificial laryngeal framework. Additionally, the incorporation of the PCL outer framework with pores and inner hydrogel provides structural stability and sufficient nutrient/oxygen transport. An animal study confirmed that the transplanted 3D bio-larynx successfully maintained the airway. With further development, our new strategy holds great potential for fabricating human-scale larynxes with in vivo-like biological functions for laryngectomy patients.
AUTHOR Freeman, Fiona E. and Pitacco, Pierluca and van Dommelen, Lieke H. A. and Nulty, Jessica and Browe, David C. and Shin, Jung-Youn and Alsberg, Eben and Kelly, Daniel J.
Title 3D bioprinting spatiotemporally defined patterns of growth factors to tightly control tissue regeneration [Abstract]
Year 2020
Journal/Proceedings Science Advances
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Abstract
Therapeutic growth factor delivery typically requires supraphysiological dosages, which can cause undesirable off-target effects. The aim of this study was to 3D bioprint implants containing spatiotemporally defined patterns of growth factors optimized for coupled angiogenesis and osteogenesis. Using nanoparticle functionalized bioinks, it was possible to print implants with distinct growth factor patterns and release profiles spanning from days to weeks. The extent of angiogenesis in vivo depended on the spatial presentation of vascular endothelial growth factor (VEGF). Higher levels of vessel invasion were observed in implants containing a spatial gradient of VEGF compared to those homogenously loaded with the same total amount of protein. Printed implants containing a gradient of VEGF, coupled with spatially defined BMP-2 localization and release kinetics, accelerated large bone defect healing with little heterotopic bone formation. This demonstrates the potential of growth factor printing, a putative point of care therapy, for tightly controlled tissue regeneration.
AUTHOR Lee, Ji Seung and Park, Hae Sang and Jung, Harry and Lee, Hanna and Hong, Heesun and Lee, Young Jin and Suh, Ye Ji and Lee, Ok Joo and Kim, Soon Hee and Park, Chan Hum
Title 3D-printable photocurable bioink for cartilage regeneration of tonsil-derived mesenchymal stem cells [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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Abstract
Cartilage regeneration is challenging because of the poor intrinsic self-repair capacity of avascular tissue. Three-dimensional (3D) bioprinting has gained significant attention in the field of tissue engineering and is a promising technology to overcome current difficulties in cartilage regeneration. Although bioink is an essential component of bioprinting technology, several challenges remain in satisfying different requirements for ideal bioink, including biocompatibility and printability based on specific biological requirements. Gelatin and hyaluronic acid (HA) have been shown to be ideal biomimetic hydrogel sources for cartilage regeneration. However, controlling their structure, mechanical properties, biocompatibility, and degradation rate for cartilage repair remains a challenge. Here, we show a photocurable bioink created by hybridization of gelatin methacryloyl (GelMA) and glycidyl-methacrylated HA (GMHA) for material extrusion 3D bioprinting in cartilage regeneration. GelMA and GMHA were mixed in various ratios, and the mixture of 7% GelMA and 5% GMHA bioink (G7H5) demonstrated the most reliable mechanical properties, rheological properties, and printability. This G7H5 bioink allowed us to build a highly complex larynx structure, including the hyoid bone, thyroid cartilage, cricoid cartilage, arytenoid cartilage, and cervical trachea. This bioink also provided an excellent microenvironment for chondrogenesis of tonsil-derived mesenchymal stem cells (TMSCs) in vitro and in vivo. In summary, this study presents the ideal formulation of GelMA/GMHA hybrid bioink to generate a well-suited photocurable bioink for cartilage regeneration of TMSCs using a material extrusion bioprinter, and could be applied to cartilage tissue engineering.
AUTHOR Liu, Xue and Michael, Samuel and Bharti, Kapil and Ferrer, Marc and Song, Min Jae
Title A biofabricated vascularized skin model of atopic dermatitis for preclinical studies [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Abstract
Three-dimensional (3D) biofabrication techniques enable the production of multicellular tissue models as assay platforms for drug screening. The increased cellular and physiological complexity in these 3D tissue models should recapitulate the relevant biological environment found in the body. Here we describe the use of 3D bioprinting techniques to fabricate skin equivalent tissues of varying physiological complexity, including human epidermis, non-vascularized and vascularized full-thickness skin tissue equivalents, in a multi-well platform to enable drug screening. Human keratinocytes, fibroblasts, and pericytes, and induced pluripotent stem cell (iPSC)-derived endothelial cells were used in the biofabrication process to produce the varying complexity. The skin equivalents exhibit the correct structural markers of dermis and epidermis stratification, with physiological functions of the skin barrier. The robustness, versatility and reproducibility of the biofabrication techniques are further highlighted by the generation of atopic dermatitis (AD)-disease like tissues. These AD models demonstrate several clinical hallmarks of the disease, including: (i) spongiosis and hyperplasia; (ii) early and terminal expression of differentiation proteins; and (iii) increases in levels of pro-inflammatory cytokines. We show the pre-clinical relevance of the biofabricated AD tissue models to correct disease phenotype by testing the effects of dexamethasone, an anti-inflammatory corticosteroid, and three Janus Kinase inhibitors from clinical trials for AD. This study demonstrates the development of a versatile and reproducible bioprinting approach to create human skin equivalents with a range of cellular complexity for disease modelling. In addition, we establish several assay readouts that are quantifiable, robust, AD relevant, and can be scaled up for compound screening. The results show that the cellular complexity of the tissues develops a more physiologically relevant AD disease model. Thus, the skin models in this study offer an in vitro approach for the rapid understanding of pathological mechanisms, and testing for efficacy of action and toxic effects of drugs.
AUTHOR Colle, Julien and Blondeel, Phillip and De Bruyne, Axelle and Bochar, Silke and Tytgat, Liesbeth and Vercruysse, Chris and Van Vlierberghe, Sandra and Dubruel, Peter and Declercq, Heidi
Title Bioprinting predifferentiated adipose-derived mesenchymal stem cell spheroids with methacrylated gelatin ink for adipose tissue engineering [Abstract]
Year 2020
Journal/Proceedings Journal of Materials Science: Materials in Medicine
Reftype Colle2020
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Abstract
The increasing number of mastectomies results in a greater demand for breast reconstruction characterized by simplicity and a low complication profile. Reconstructive surgeons are investigating tissue engineering (TE) strategies to overcome the current surgical drawbacks. 3D bioprinting is the rising technique for the fabrication of large tissue constructs which provides a potential solution for unmet clinical needs in breast reconstruction building on decades of experience in autologous fat grafting, adipose-derived mesenchymal stem cell (ASC) biology and TE. A scaffold was bioprinted using encapsulated ASC spheroids in methacrylated gelatin ink (GelMA). Uniform ASC spheroids with an ideal geometry and diameter for bioprinting were formed, using a high-throughput non-adhesive agarose microwell system. ASC spheroids in adipogenic differentiation medium (ADM) were evaluated through live/dead staining, histology (HE, Oil Red O), TEM and RT-qPCR. Viable spheroids were obtained for up to 14 days post-printing and showed multilocular microvacuoles and successful differentiation toward mature adipocytes shown by gene expression analysis. Moreover, spheroids were able to assemble at random in GelMA, creating a macrotissue. Combining the advantage of microtissues to self-assemble and the controlled organization by bioprinting technologies, these ASC spheroids can be useful as building blocks for the engineering of soft tissue implants.
AUTHOR Daly, Andrew C. and Pitacco, Pierluca and Nulty, Jessica and Cunniffe, Gráinne M. and Kelly, Daniel J.
Title 3D printed microchannel networks to direct vascularisation during endochondral bone repair [Abstract]
Year 2018
Journal/Proceedings Biomaterials
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Abstract
Bone tissue engineering strategies that recapitulate the developmental process of endochondral ossification offer a promising route to bone repair. Clinical translation of such endochondral tissue engineering strategies will require overcoming a number of challenges, including the engineering of large and often anatomically complex cartilage grafts, as well as the persistence of core regions of avascular cartilage following their implantation into large bone defects. Here 3D printing technology is utilized to develop a versatile and scalable approach to guide vascularisation during endochondral bone repair. First, a sacrificial pluronic ink was used to 3D print interconnected microchannel networks in a mesenchymal stem cell (MSC) laden gelatin-methacryloyl (GelMA) hydrogel. These constructs (with and without microchannels) were next chondrogenically primed in vitro and then implanted into critically sized femoral bone defects in rats. The solid and microchanneled cartilage templates enhanced bone repair compared to untreated controls, with the solid cartilage templates (without microchannels) supporting the highest levels of total bone formation. However, the inclusion of 3D printed microchannels was found to promote osteoclast/immune cell invasion, hydrogel degradation, and vascularisation following implantation. In addition, the endochondral bone tissue engineering strategy was found to support comparable levels of bone healing to BMP-2 delivery, whilst promoting lower levels of heterotopic bone formation, with the microchanneled templates supporting the lowest levels of heterotopic bone formation. Taken together, these results demonstrate that 3D printed hypertrophic cartilage grafts represent a promising approach for the repair of complex bone fractures, particularly for larger defects where vascularisation will be a key challenge.
AUTHOR Ng, Wei Long and Qi, Jovina Tan Zhi and Yeong, Wai Yee and Naing, May Win
Title Proof-of-concept: 3D bioprinting of pigmented human skin constructs [Abstract]
Year 2018
Journal/Proceedings Biofabrication
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Abstract
Three-dimensional (3D) pigmented human skin constructs have been fabricated using a 3D bioprinting approach. The 3D pigmented human skin constructs are obtained from using three different types of skin cells (keratinocytes, melanocytes and fibroblasts from three different skin donors) and they exhibit similar constitutive pigmentation (pale pigmentation) as the skin donors. A two-step drop-on-demand bioprinting strategy facilitates the deposition of cell droplets to emulate the epidermal melanin units (pre-defined patterning of keratinocytes and melanocytes at the desired positions) and manipulation of the microenvironment to fabricate 3D biomimetic hierarchical porous structures found in native skin tissue. The 3D bioprinted pigmented skin constructs are compared to the pigmented skin constructs fabricated by conventional a manual-casting approach; in-depth characterization of both the 3D pigmented skin constructs has indicated that the 3D bioprinted skin constructs have a higher degree of resemblance to native skin tissue in term of the presence of well-developed stratified epidermal layers and the presence of a continuous layer of basement membrane proteins as compared to the manually-cast samples. The 3D bioprinting approach facilitates the development of 3D in vitro pigmented human skin constructs for potential toxicology testing and fundamental cell biology research.
AUTHOR Huang, Boyang and Wang, Yaxin and Vyas, Cian and Bartolo, Paulo
Title Crystal Growth of 3D Poly(ε-caprolactone) Based Bone Scaffolds and Its Effects on the Physical Properties and Cellular Interactions [Abstract]
Year 2022
Journal/Proceedings Advanced Science
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Abstract
Abstract Extrusion additive manufacturing is widely used to fabricate polymer-based 3D bone scaffolds. However, the insight views of crystal growths, scaffold features and eventually cell-scaffold interactions are still unknown. In this work, melt and solvent extrusion additive manufacturing techniques are used to produce scaffolds considering highly analogous printing conditions. Results show that the scaffolds produced by these two techniques present distinct physiochemical properties, with melt-printed scaffolds showing stronger mechanical properties and solvent-printed scaffolds showing rougher surface, higher degradation rate, and faster stress relaxation. These differences are attributed to the two different crystal growth kinetics, temperature-induced crystallization (TIC) and strain-induced crystallization (SIC), forming large/integrated spherulite-like and a small/fragmented lamella-like crystal regions respectively. The stiffer substrate of melt-printed scaffolds contributes to higher ratio of nuclear Yes-associated protein (YAP) allocation, favoring cell proliferation and differentiation. Faster relaxation and degradation of solvent-printed scaffolds result in dynamic surface, contributing to an early-stage faster osteogenesis differentiation.
AUTHOR Kessel, Benjamin and Lee, Mihyun and Bonato, Angela and Tinguely, Yann and Tosoratti, Enrico and Zenobi-Wong, Marcy
Title 3D Bioprinting of Macroporous Materials Based on Entangled Hydrogel Microstrands [Abstract]
Year 2020
Journal/Proceedings Advanced Science
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Abstract
Abstract Hydrogels are excellent mimetics of mammalian extracellular matrices and have found widespread use in tissue engineering. Nanoporosity of monolithic bulk hydrogels, however, limits mass transport of key biomolecules. Microgels used in 3D bioprinting achieve both custom shape and vastly improved permissivity to an array of cell functions, however spherical-microbead-based bioinks are challenging to upscale, are inherently isotropic, and require secondary crosslinking. Here, bioinks based on high-aspect-ratio hydrogel microstrands are introduced to overcome these limitations. Pre-crosslinked, bulk hydrogels are deconstructed into microstrands by sizing through a grid with apertures of 40–100 µm. The microstrands are moldable and form a porous, entangled structure, stable in aqueous medium without further crosslinking. Entangled microstrands have rheological properties characteristic of excellent bioinks for extrusion bioprinting. Furthermore, individual microstrands align during extrusion and facilitate the alignment of myotubes. Cells can be placed either inside or outside the hydrogel phase with >90% viability. Chondrocytes co-printed with the microstrands deposit abundant extracellular matrix, resulting in a modulus increase from 2.7 to 780.2 kPa after 6 weeks of culture. This powerful approach to deconstruct bulk hydrogels into advanced bioinks is both scalable and versatile, representing an important toolbox for 3D bioprinting of architected hydrogels.
AUTHOR Kokkinis, Dimitri and Bouville, Florian and Studart, André R.
Title 3D Printing of Materials with Tunable Failure via Bioinspired Mechanical Gradients [Abstract]
Year 2018
Journal/Proceedings Advanced Materials
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Abstract
Abstract Mechanical gradients are useful to reduce strain mismatches in heterogeneous materials and thus prevent premature failure of devices in a wide range of applications. While complex graded designs are a hallmark of biological materials, gradients in manmade materials are often limited to 1D profiles due to the lack of adequate fabrication tools. Here, a multimaterial 3D‐printing platform is developed to fabricate elastomer gradients spanning three orders of magnitude in elastic modulus and used to investigate the role of various bioinspired gradient designs on the local and global mechanical behavior of synthetic materials. The digital image correlation data and finite element modeling indicate that gradients can be effectively used to manipulate the stress state and thus circumvent the weakening effect of defect‐rich interfaces or program the failure behavior of heterogeneous materials. Implementing this concept in materials with bioinspired designs can potentially lead to defect‐tolerant structures and to materials whose tunable failure facilitates repair of biomedical implants, stretchable electronics, or soft robotics.
AUTHOR Farazin, Ashkan and Zhang, Chunwei and Gheisizadeh, Amirhossein and Shahbazi, Aminadel
Title 3D bio-printing for use as bone replacement tissues: A review of biomedical application [Abstract]
Year 2023
Journal/Proceedings Biomedical Engineering Advances
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Abstract
Since we are able to use 3D printers, producing porous metal scaffolds become very easy. Contrary to usual methods, 3D printing of porous scaffolds is determined by a controllable and precise manufacturing process. That property allows us to form customized prefabricated implants for individual patients and make a regular pore distribution at the micro-scale as same as the structure of a bone, design of a structure like bone is very complicated because the pores of that structure must have enough space for cell attachment and proliferation. The reaction of cells and bone ingrowth can influence the effect of 3D printed porous metal scaffolds on bone ingrowth. This review introduces 3D printing techniques brief and focuses on the factors that potentially influence bone ingrowth into 3D printed porous metal scaffolds like materials, pore size, porosity, pore structure, surface modification, and mechanical properties. In each section, we described the mechanisms underlying cell-scaffold interactions in detail also there is a short introduction of clinical application of 3D printing. After all, there is a list that shows the most appropriate parameters for a flawless porous metal scaffold, and it is lead to finding a combination of these parameters that foretaste good bone ingrowth.
AUTHOR Liu, Chun and Dai, Ting and Wu, Xiaoyu and Ma, Jiayi and Liu, Jun and Wu, Siyu and Yang, Lei and Zhao, Hongbin
Title 3D bioprinting of cell-laden nano-attapulgite/gelatin methacrylate composite hydrogel scaffolds for bone tissue repair [Abstract]
Year 2023
Journal/Proceedings Journal of Materials Science & Technology
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Abstract
Bone tissue engineering (BTE) has proven to be a promising strategy for bone defect repair. Due to its excellent biological properties, gelatin methacrylate (GelMA) hydrogels have been used as bioinks for 3D bioprinting in some BTE studies to produce scaffolds for bone regeneration. However, applications for load-bearing defects are limited by poor mechanical properties and a lack of bioactivity. In this study, 3D printing technology was used to create nano-attapulgite (nano-ATP)/GelMA composite hydrogels loaded into mouse bone mesenchymal stem cells (BMSCs) and mouse umbilical vein endothelial cells (MUVECs). The bioprintability, physicochemical properties, and mechanical properties were all thoroughly evaluated. Our findings showed that nano-ATP groups outperform the control group in terms of printability, indicating that nano-ATP is beneficial for printability. Additionally, after incorporation with nano-ATP, the mechanical strength of the composite hydrogels was significantly improved, resulting in adequate mechanical properties for bone regeneration. The presence of nano-ATP in the scaffolds has also been studied for cell-material interactions. The findings show that cells within the scaffold not only have high viability but also a clear proclivity to promote osteogenic differentiation of BMSCs. Besides, the MUVECs-loaded composite hydrogels demonstrated increased angiogenic activity. A cranial defect model was also developed to evaluate the bone repair capability of scaffolds loaded with rat BMSCs. According to histological analysis, cell-laden nano-ATP composite hydrogels can effectively improve bone regeneration and promote angiogenesis. This study demonstrated the potential of nano-ATP for bone tissue engineering, which should also increase the clinical practicality of nano-ATP.
AUTHOR Cernencu, Alexandra I. and Vlasceanu, George M. and Serafim, Andrada and Pircalabioru, Gratiela and Ionita, Mariana
Title 3D double-reinforced graphene oxide – nanocellulose biomaterial inks for tissue engineered constructs [Abstract]
Year 2023
Journal/Proceedings RSC Adv.
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Abstract
The advent of improved fabrication technologies{,} particularly 3D printing{,} has enabled the engineering of bone tissue for patient-specific healing and the fabrication of in vitro tissue models for ex vivo testing. However{,} inks made from natural polymers often fall short in terms of mechanical strength{,} stability{,} and the induction of osteogenesis. Our research focused on developing novel printable formulations using a gelatin/pectin polymeric matrix that integrate synergistic reinforcement components i.e. graphene oxide (GO) and oxidized nanocellulose fibers (CNF). Using 3D printing technology and the aforementioned biomaterial composite inks{,} bone-like scaffolds were created. To simulate critical-sized flaws and demonstrate scaffold fidelity{,} 3D scaffolds were successfully printed using formulations with varied GO concentrations (0.25{,} 0.5{,} and 1% wt with respect to polymer content). The addition of GO to hydrogel inks enhanced not only the compressive modulus but also the printability and scaffold fidelity compared to the pure colloid-gelatin/pectin system. Due to its strong potential for 3D bioprinting{,} the sample containing 0.5% GO is shown to have the greatest perspectives for bone tissue models and tissue engineering applications.
AUTHOR Li, Jianfeng and Reimers, Armin and Dang, Ka My and Brunk, Michael G. K. and Drewes, Jonas and Hirsch, Ulrike M. and Willems, Christian and Schmelzer, Christian E. H. and Groth, Thomas and Nia, Ali Shaygan and Feng, Xinliang and Adelung, Rainer and Sacher, Wesley D. and Schütt, Fabian and Poon, Joyce K. S.
Title 3D printed neural tissues with in situ optical dopamine sensors [Abstract]
Year 2023
Journal/Proceedings Biosensors and Bioelectronics
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Engineered neural tissues serve as models for studying neurological conditions and drug screening. Besides observing the cellular physiological properties, in situ monitoring of neurochemical concentrations with cellular spatial resolution in such neural tissues can provide additional valuable insights in models of disease and drug efficacy. In this work, we demonstrate the first three-dimensional (3D) tissue cultures with embedded optical dopamine (DA) sensors. We developed an alginate/Pluronic F127 based bio-ink for human dopaminergic brain tissue printing with tetrapodal-shaped-ZnO microparticles (t-ZnO) additive as the DA sensor. DA quenches the autofluorescence of t-ZnO in physiological environments, and the reduction of the fluorescence intensity serves as an indicator of the DA concentration. The neurons that were 3D printed with the t-ZnO showed good viability, and extensive 3D neural networks were formed within one week after printing. The t-ZnO could sense DA in the 3D printed neural network with a detection limit of 0.137 μM. The results are a first step toward integrating tissue engineering with intensiometric biosensing for advanced artificial tissue/organ monitoring.
AUTHOR Wang, Bin and Barceló, Xavier and Von Euw, Stanislas and Kelly, Daniel J.
Title 3D printing of mechanically functional meniscal tissue equivalents using high concentration extracellular matrix inks [Abstract]
Year 2023
Journal/Proceedings Materials Today Bio
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Abstract
Decellularized extracellular matrix (dECM) has emerged as a promising biomaterial in the fields of tissue engineering and regenerative medicine due to its ability to provide specific biochemical and biophysical cues supportive of the regeneration of diverse tissue types. Such biomaterials have also been used to produce tissue-specific inks and bioinks for 3D printing applications. However, a major limitation associated with the use of such dECM materials is their poor mechanical properties, which limits their use in load-bearing applications such as meniscus regeneration. In this study, native porcine menisci were solubilized and decellularized using different methods to produce highly concentrated dECM inks of differing biochemical content and printability. All dECM inks displayed shear thinning and thixotropic properties, with increased viscosity and improved printability observed at higher pH levels, enabling the 3D printing of anatomically defined meniscal implants. With additional crosslinking of the dECM inks following thermal gelation at pH 11, it was possible to fabricate highly elastic meniscal tissue equivalents with compressive mechanical properties similar to the native tissue. These improved mechanical properties at higher pH correlated with the development of a denser network of smaller diameter collagen fibers. These constructs also displayed repeatable loading and unloading curves when subjected to long-term cyclic compression tests. Moreover, the printing of dECM inks at the appropriate pH promoted a preferential alignment of the collagen fibers. Altogether, these findings demonstrate the potential of 3D printing of highly concentrated meniscus dECM inks to produce mechanically functional and biocompatible implants for meniscal tissue regeneration. This approach could be applied to a wide variety of different biological tissues, enabling the 3D printing of tissue mimics with diverse applications from tissue engineering to surgical planning.
AUTHOR Dairaghi, Jacob and Benito Alston, Claudia and Cadle, Rachel and Rogozea, Dan and Solorio, Luis and Barco, Clark T. and Moldovan, Nicanor I.
Title A dual osteoconductive-osteoprotective implantable device for vertical alveolar ridge augmentation [Abstract]
Year 2023
Journal/Proceedings Frontiers in Dental Medicine
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Repair of large oral bone defects such as vertical alveolar ridge augmentation could benefit from the rapidly developing additive manufacturing technology used to create personalized osteoconductive devices made from porous tricalcium phosphate/hydroxyapatite (TCP/HA)-based bioceramics. These devices can be also used as hydrogel carriers to improve their osteogenic potential. However, the TCP/HA constructs are prone to brittle fracture, therefore their use in clinical situations is difficult. As a solution, we propose the protection of this osteoconductive multi-material (herein called “core”) with a shape-matched “cover” made from biocompatible poly-ɛ-caprolactone (PCL), which is a ductile, and thus more resistant polymeric material. In this report, we present a workflow starting from patient-specific medical scan in Digital Imaging and Communications in Medicine (DICOM) format files, up to the design and 3D printing of a hydrogel-loaded porous TCP/HA core and of its corresponding PCL cover. This cover could also facilitate the anchoring of the device to the patient's defect site via fixing screws. The large, linearly aligned pores in the TCP/HA bioceramic core, their sizes, and their filling with an alginate hydrogel were analyzed by micro-CT. Moreover, we created a finite element analysis (FEA) model of this dual-function device, which permits the simulation of its mechanical behavior in various anticipated clinical situations, as well as optimization before surgery. In conclusion, we designed and 3D-printed a novel, structurally complex multi-material osteoconductive-osteoprotective device with anticipated mechanical properties suitable for large-defect oral bone regeneration.
AUTHOR Rikkers, Margot and Nguyen, H. Chien and Golafshan, Nasim and de Ruijter, Mylène and Levato, Riccardo and Vonk, Lucienne A. and van Egmond, Nienke and Castilho, Miguel and Custers, Roel J. H. and Malda, Jos
Title A Gap-Filling, Regenerative Implant for Open-Wedge Osteotomy [Abstract]
Year 2023
Journal/Proceedings Journal of Cartilage & Joint Preservation
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Purpose In patients suffering from unilateral osteoarthritis in the knee, an osteotomy can provide symptomatic relief and postpone the need for replacement of the joint. Nevertheless, open-wedge osteotomies (OWO) around the knee joint face several challenges like postoperative pain and bone non-union. In this study, the aim was to design, fabricate, and evaluate a gap-filling implant for OWO using an osteoinductive and degradable biomaterial. Methods Design of porous wedge-shaped implants was based on computed tomography (CT) scans of cadaveric legs. Implants were 3D printed using a magnesium strontium phosphate-polycaprolactone (MgPSr-PCL) biomaterial ink. Standardized scaffolds with different inter-fibre spacing (IFS) were mechanically characterized and osteoinductive properties of the biomaterial were assessed in vitro. Finally, human-sized implants with different heights (5 mm, 10 mm, 15 mm) were designed and fabricated for ex vivo implantation during three OWO procedures in human cadaveric legs. Results Implants printed with an interior of IFS-1.0 resulted in scaffolds that maintained top and bottom porosity, while the interior of the implant exhibited significant mechanical stability. Bone marrow concentrate and culture expanded mesenchymal stromal cells attached to the MgPSr-PCL material and proliferated over 21 days in culture. The production of osteogenic markers alkaline phosphatase activity, calcium, and osteocalcin was promoted in all culture conditions, independent of osteogenic induction medium. Finally, three OWO procedures were planned and fabricated wedges were implanted ex vivo during the procedures. A small fraction of one side of the wedges was resected to assure fit into the proximal biplanar osteotomy gap. Pre-planned wedge heights were maintained after implantation as measured by micro-CT. Conclusion To conclude, personalized implants for implantation in open-wedge osteotomies were successfully designed and manufactured. The implant material supported osteogenesis of MSCs and BMC in vitro and full-size implants were successfully implemented into the surgical procedure, without compromising pre-planned wedge height.
AUTHOR Daskalakis, Evangelos and Hassan, Mohamed H. and Omar, Abdalla M. and Acar, Anil A. and Fallah, Ali and Cooper, Glen and Weightman, Andrew and Blunn, Gordon and Koc, Bahattin and Bartolo, Paulo
Title Accelerated Degradation of Poly-ε-caprolactone Composite Scaffolds for Large Bone Defects [Abstract]
Year 2023
Journal/Proceedings Polymers
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This research investigates the accelerated hydrolytic degradation process of both anatomically designed bone scaffolds with a pore size gradient and a rectangular shape (biomimetically designed scaffolds or bone bricks). The effect of material composition is investigated considering poly-ε-caprolactone (PCL) as the main scaffold material, reinforced with ceramics such as hydroxyapatite (HA), β-tricalcium phosphate (TCP) and bioglass at a concentration of 20 wt%. In the case of rectangular scaffolds, the effect of pore size (200 μm, 300 μm and 500 μm) is also investigated. The degradation process (accelerated degradation) was investigated during a period of 5 days in a sodium hydroxide (NaOH) medium. Degraded bone bricks and rectangular scaffolds were measured each day to evaluate the weight loss of the samples, which were also morphologically, thermally, chemically and mechanically assessed. The results show that the PCL/bioglass bone brick scaffolds exhibited faster degradation kinetics in comparison with the PCL, PCL/HA and PCL/TCP bone bricks. Furthermore, the degradation kinetics of rectangular scaffolds increased by increasing the pore size from 500 μm to 200 μm. The results also indicate that, for the same material composition, bone bricks degrade slower compared with rectangular scaffolds. The scanning electron microscopy (SEM) images show that the degradation process was faster on the external regions of the bone brick scaffolds (600 μm pore size) compared with the internal regions (200 μm pore size). The thermal gravimetric analysis (TGA) results show that the ceramic concentration remained constant throughout the degradation process, while differential scanning calorimetry (DSC) results show that all scaffolds exhibited a reduction in crystallinity (Xc), enthalpy (Δm) and melting temperature (Tm) throughout the degradation process, while the glass transition temperature (Tg) slightly increased. Finally, the compression results show that the mechanical properties decreased during the degradation process, with PCL/bioglass bone bricks and rectangular scaffolds presenting higher mechanical properties with the same design in comparison with the other materials.
AUTHOR Anupama Sekar, J. and Velayudhan, Shiny and Anil Kumar, P. R.
Title Biocompatibility evaluation of antioxidant cocktail loaded gelatin methacrylamide as bioink for extrusion-based 3D bioprinting [Abstract]
Year 2023
Journal/Proceedings Biomedical Materials
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Three-dimensional (3D) liver bioprinting is a promising technique for creating 3D liver models that can be used for in vitro drug testing, hepatotoxicity studies, and transplantation. The functional performance of 3D bioprinted liver constructs are limited by the lack of cell–cell interactions, which calls for the creation of bioprinted tissue constructs with high cell densities. This study reports the fabrication of 3D bioprinted liver constructs using a novel photocrosslinkable gelatin methacrylamide (GelMA)-based bioink formulation. However, the formation of excess free radicals during photoinitiation poses a challenge, particularly during photocrosslinking of large constructs with high cell densities. Hence, we designed a bioink formulation comprising the base polymer GelMA loaded with an antioxidant cocktail containing vitamin C (L-ascorbic acid (AA)) and vitamin E (α-tocopherol (α-Toc)). We confirmed that the combination of antioxidants loaded in GelMA enhanced the ability to scavenge intracellular reactive oxygen species formed during photocrosslinking. The GelMA formulation was evaluated for biocompatibility in vitro and in vivo. These results demonstrated that the bioink had adequate rheological characteristics and was biocompatible. Furthermore, when compared to bioprinted constructs with lower cell density, high-density primary rat hepatocyte constructs demonstrated improved cell-cell interactions and liver-specific functions like albumin and urea secretion, which increased 5-fold and 2.5-fold, respectively.
AUTHOR Ostrovidov, Serge and Ramalingam, Murugan and Bae, Hojae and Orive, Gorka and Fujie, Toshinori and Shi, Xuetao and Kaji, Hirokazu
Title Bioprinting and biomaterials for dental alveolar tissue regeneration. [Abstract]
Year 2023
Journal/Proceedings Frontiers in bioengineering and biotechnology
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Three dimensional (3D) bioprinting is a powerful tool, that was recently applied to tissue engineering. This technique allows the precise deposition of cells encapsulated in supportive bioinks to fabricate complex scaffolds, which are used to repair targeted tissues. Here, we review the recent developments in the application of 3D bioprinting to dental tissue engineering. These tissues, including teeth, periodontal ligament, alveolar bones, and dental pulp, present cell types and mechanical properties with great heterogeneity, which is challenging to reproduce in vitro. After highlighting the different bioprinting methods used in regenerative dentistry, we reviewed the great variety of bioink formulations and their effects on cells, which have been established to support the development of these tissues. We discussed the different advances achieved in the fabrication of each dental tissue to provide an overview of the current state of the methods. We conclude with the remaining challenges and future needs.
AUTHOR Barceló, Xavier and Garcia, Orquidea and Kelly, Daniel J.
Title Chondroitinase ABC Treatment Improves the Organization and Mechanics of 3D Bioprinted Meniscal Tissue [Abstract]
Year 2023
Journal/Proceedings ACS Biomater. Sci. Eng.
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The meniscus is a fibrocartilage tissue that is integral to the correct functioning of the knee joint. The tissue possesses a unique collagen fiber architecture that is integral to its biomechanical functionality. In particular, a network of circumferentially aligned collagen fibers function to bear the high tensile forces generated in the tissue during normal daily activities. The limited regenerative capacity of the meniscus has motivated increased interest in meniscus tissue engineering; however, the in vitro generation of structurally organized meniscal grafts with a collagen architecture mimetic of the native meniscus remains a significant challenge. Here we used melt electrowriting (MEW) to produce scaffolds with defined pore architectures to impose physical boundaries upon cell growth and extracellular matrix production. This enabled the bioprinting of anisotropic tissues with collagen fibers preferentially oriented parallel to the long axis of the scaffold pores. Furthermore, temporally removing glycosaminoglycans (sGAGs) during the early stages of in vitro tissue development using chondroitinase ABC (cABC) was found to positively impact collagen network maturation. Specially we found that temporal depletion of sGAGs is associated with an increase in collagen fiber diameter without any detrimental effect on the development of a meniscal tissue phenotype or subsequent extracellular matrix production. Moreover, temporal cABC treatment supported the development of engineered tissues with superior tensile mechanical properties compared to empty MEW scaffolds. These findings demonstrate the benefit of temporal enzymatic treatments when engineering structurally anisotropic tissues using emerging biofabrication technologies such as MEW and inkjet bioprinting.
AUTHOR Golafshan, Nasim and Castilho, Miguel and Daghrery, Arwa and Alehosseini, Morteza and van de Kemp, Tom and Krikonis, Konstantinos and de Ruijter, Mylene and Dal-Fabbro, Renan and Dolatshahi-Pirouz, Alireza and Bhaduri, Sarit B. and Bottino, Marco C. and Malda, Jos
Title Composite Graded Melt Electrowritten Scaffolds for Regeneration of the Periodontal Ligament-to-Bone Interface
Year 2023
Journal/Proceedings ACS Appl. Mater. Interfaces
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AUTHOR Fuchs, Andreas and Bartolf-Kopp, Michael and Böhm, Hartmut and Straub, Anton and Kübler, Alexander C. and Linz, Christian and Gbureck, Uwe
Title Composite grafts made of polycaprolactone fiber mats and oil-based calcium phosphate cement pastes for the reconstruction of cranial and maxillofacial defects [Abstract]
Year 2023
Journal/Proceedings Clinical Oral Investigations
Reftype Fuchs2023
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Synthetic bone substitutes which can be adapted preoperatively and patient specific may be helpful in various bony defects in the field of oral- and maxillofacial surgery. For this purpose, composite grafts made of self-setting and oil-based calcium phosphate cement (CPC) pastes, which were reinforced with 3D-printed polycaprolactone (PCL) fiber mats were manufactured.
AUTHOR Kumar, Virender and Kaur, Harmanpreet and Kumari, Anuradha and Hooda, Goldy and Garg, Vandana and Dureja, Harish
Title Drug delivery and testing via 3D printing [Abstract]
Year 2023
Journal/Proceedings Bioprinting
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3D printing first came into existence in the year 1984. Since then, it has found significant use in various fields, including pharmaceutical industries.3D printing is a process of manufacturing products by depositing materials layer by layer. Thus, also called additive manufacturing. Additive manufacturing provides patient-specific formulation, an advantage over conventional drug design methods. 3D printing helps in the designing of complex structures. Since the approval of the first 3D-printed tablet, this field has gained popularity. In this review, various techniques used in 3D printing have been discussed. This article further gives insight into the recent research done on AM technology. There is also some discussion about the formulations made for pediatric patients using AM technology. Different types of drug delivery systems mentioned in this work are oral, vaginal, rectal, oro-mucosal, transdermal, and implant. Further drug testing devices, including 3D-printed organoids and organ-on-chip models, have been discussed. Finally, it gives information about the future direction of this technology.
AUTHOR Nalesso, Paulo Roberto Lopes and Vedovatto, Matheus and Gregório, Julia Eduarda Schneider and Huang, Boyang and Vyas, Cian and Santamaria-Jr, Milton and Bártolo, Paulo and Caetano, Guilherme Ferreira
Title Early In Vivo Osteogenic and Inflammatory Response of 3D Printed Polycaprolactone/Carbon Nanotube/Hydroxyapatite/Tricalcium Phosphate Composite Scaffolds [Abstract]
Year 2023
Journal/Proceedings Polymers
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The development of advanced biomaterials and manufacturing processes to fabricate biologically and mechanically appropriate scaffolds for bone tissue is a significant challenge. Polycaprolactone (PCL) is a biocompatible and degradable polymer used in bone tissue engineering, but it lacks biofunctionalization. Bioceramics, such as hydroxyapatite (HA) and β tricalcium phosphate (β-TCP), which are similar chemically to native bone, can facilitate both osteointegration and osteoinduction whilst improving the biomechanics of a scaffold. Carbon nanotubes (CNTs) display exceptional electrical conductivity and mechanical properties. A major limitation is the understanding of how PCL-based scaffolds containing HA, TCP, and CNTs behave in vivo in a bone regeneration model. The objective of this study was to evaluate the use of three-dimensional (3D) printed PCL-based composite scaffolds containing CNTs, HA, and β-TCP during the initial osteogenic and inflammatory response phase in a critical bone defect rat model. Gene expression related to early osteogenesis, the inflammatory phase, and tissue formation was evaluated using quantitative real-time PCR (RT-qPCR). Tissue formation and mineralization were assessed by histomorphometry. The CNT+HA/TCP group presented higher expression of osteogenic genes after seven days. The CNT+HA and CNT+TCP groups stimulated higher gene expression for tissue formation and mineralization, and pro- and anti-inflammatory genes after 14 and 30 days. Moreover, the CNT+TCP and CNT+HA/TCP groups showed higher gene expressions related to M1 macrophages. The association of CNTs with ceramics at 10wt% (CNT+HA/TCP) showed lower expressions of inflammatory genes and higher osteogenic, presenting a positive impact and balanced cell signaling for early bone formation. The association of CNTs with both ceramics promoted a minor inflammatory response and faster bone tissue formation.
AUTHOR Helaehil, Júlia Venturini and Helaehil, Luiza Venturini and Alves, Laryssa Fernanda and Huang, Boyang and Santamaria-Jr, Milton and Bartolo, Paulo and Caetano, Guilherme Ferreira
Title Electrical Stimulation Therapy and HA/TCP Composite Scaffolds Modulate the Wnt Pathways in Bone Regeneration of Critical-Sized Defects [Abstract]
Year 2023
Journal/Proceedings Bioengineering
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Critical bone defects are the most difficult challenges in the area of tissue repair. Polycaprolactone (PCL) scaffolds, associated with hydroxyapatite (HA) and tricalcium phosphate (TCP), are reported to have an enhanced bioactivity. Moreover, the use of electrical stimulation (ES) has overcome the lack of bioelectricity at the bone defect site and compensated the endogenous electrical signals. Such treatments could modulate cells and tissue signaling pathways. However, there is no study investigating the effects of ES and bioceramic composite scaffolds on bone tissue formation, particularly in the view of cell signaling pathway. This study aims to investigate the application of HA/TCP composite scaffolds and ES and their effects on the Wingless-related integration site (Wnt) pathway in critical bone repair. Critical bone defects (25 mm2) were performed in rats, which were divided into four groups: PCL, PCL + ES, HA/TCP and HA/TCP + ES. The scaffolds were grafted at the defect site and applied with the ES application twice a week using 10 µA of current for 5 min. Bone samples were collected for histomorphometry, immunohistochemistry and molecular analysis. At the Wnt canonical pathway, HA/TCP and HA/TCP + ES groups showed higher Wnt1 and β-catenin gene expression levels, especially HA/TCP. Moreover, HA/TCP + ES presented higher Runx2, Osterix and Bmp-2 levels. At the Wnt non-canonical pathway, HA/TCP group showed higher voltage-gated calcium channel (Vgcc), calmodulin-dependent protein kinase II, and Wnt5a genes expression, while HA/TCP + ES presented higher protein expression of VGCC and calmodulin (CaM) at the same period. The decrease in sclerostin and osteopontin genes expressions and the lower bone sialoprotein II in the HA/TCP + ES group may be related to the early bone remodeling. This study shows that the use of ES modulated the Wnt pathways and accelerated the osteogenesis with improved tissue maturation.
AUTHOR Mungenast, Lena and Nieminen, Ronya and Gaiser, Carine and Faia-Torres, Ana Bela and Rühe, Jürgen and Suter-Dick, Laura
Title Electrospun decellularized extracellular matrix scaffolds promote the regeneration of injured neurons [Abstract]
Year 2023
Journal/Proceedings Biomaterials and Biosystems
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Traumatic injury to the spinal cord (SCI) causes the transection of neurons, formation of a lesion cavity, and remodeling of the microenvironment by excessive extracellular matrix (ECM) deposition and scar formation leading to a regeneration-prohibiting environment. Electrospun fiber scaffolds have been shown to simulate the ECM and increase neural alignment and neurite outgrowth contributing to a growth-permissive matrix. In this work, electrospun ECM-like fibers providing biochemical and topological cues are implemented into a scaffold to represent an oriented biomaterial suitable for the alignment and migration of neural cells in order to improve spinal cord regeneration. The successfully decellularized spinal cord ECM (dECM), with no visible cell nuclei and dsDNA content < 50 ng/mg tissue, showed preserved ECM components, such as glycosaminoglycans and collagens. Serving as the biomaterial for 3D printer-assisted electrospinning, highly aligned and randomly distributed dECM fiber scaffolds (< 1 µm fiber diameter) were fabricated. The scaffolds were cytocompatible and supported the viability of a human neural cell line (SH-SY5Y) for 14 days. Cells were selectively differentiated into neurons, as confirmed by immunolabeling of specific cell markers (ChAT, Tubulin ß), and followed the orientation given by the dECM scaffolds. After generating a lesion site on the cell-scaffold model, cell migration was observed and compared to reference poly-ε-caprolactone fiber scaffolds. The aligned dECM fiber scaffold promoted the fastest and most efficient lesion closure, indicating superior cell guiding capabilities of dECM-based scaffolds. The strategy of combining decellularized tissues with controlled deposition of fibers to optimize biochemical and topographical cues opens the way for clinically relevant central nervous system scaffolding solutions.
AUTHOR Tan, Yadong and Fan, Shijie and Wu, Xiaoyu and Liu, Menggege and Dai, Ting and Liu, Chun and Ni, Su and Wang, Jiafeng and Yuan, Xiuchen and Zhao, Hongbin and Weng, Yiping
Title Fabrication of a three-dimensional printed gelatin/sodium alginate/nano-attapulgite composite polymer scaffold loaded with leonurine hydrochloride and its effects on osteogenesis and vascularization [Abstract]
Year 2023
Journal/Proceedings International Journal of Biological Macromolecules
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Bone tissue engineering scaffolds have made significant progress in treating bone defects in recent decades. However, the lack of a vascular network within the scaffold limits bone formation after implantation in vivo. Recent research suggests that leonurine hydrochloride (LH) can promote healing in full-thickness cutaneous wounds by increasing vessel formation and collagen deposition. Gelatin and Sodium Alginate are both polymers. ATP is a magnesium silicate chain mineral. In this study, a Gelatin/Sodium Alginate/Nano-Attapulgite composite hydrogel was used as the base material first, and the Gelatin/Sodium Alginate/Nano-Attapulgite composite polymer scaffold loaded with LH was then created using 3D printing technology. Finally, LH was grafted onto the base material by an amide reaction to construct a scaffold loaded with LH to achieve long-term LH release. When compared to pure polymer scaffolds, in vitro results showed that LH-loaded scaffolds promoted the differentiation of BMSCs into osteoblasts, as evidenced by increased expression of osteogenic key genes. The results of in vivo tissue staining revealed that the drug-loaded scaffold promoted both angiogenesis and bone formation. Collectively, these findings suggest that LH-loaded Gelatin/Sodium Alginate/Nano-Attapulgite composite hydrogel scaffolds are a potential therapeutic strategy and can assist bone regeneration.
AUTHOR Pereira, Inês and Lopez-Martinez, Maria J. and Villasante, Aranzazu and Introna, Clelia and Tornero, Daniel and Canals, Josep M. and Samitier, Josep
Title Hyaluronic acid-based bioink improves the differentiation and network formation of neural progenitor cells [Abstract]
Year 2023
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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Introduction: Three-dimensional (3D) bioprinting is a promising technique for the development of neuronal in vitro models because it controls the deposition of materials and cells. Finding a biomaterial that supports neural differentiation in vitro while ensuring compatibility with the technique of 3D bioprinting of a self-standing construct is a challenge.Methods: In this study, gelatin methacryloyl (GelMA), methacrylated alginate (AlgMA), and hyaluronic acid (HA) were examined by exploiting their biocompatibility and tunable mechanical properties to resemble the extracellular matrix (ECM) and to create a suitable material for printing neural progenitor cells (NPCs), supporting their long-term differentiation. NPCs were printed and differentiated for up to 15 days, and cell viability and neuronal differentiation markers were assessed throughout the culture.Results and Discussion: This composite biomaterial presented the desired physical properties to mimic the ECM of the brain with high water intake, low stiffness, and slow degradation while allowing the printing of defined structures. The viability rates were maintained at approximately 80% at all time points. However, the levels of β-III tubulin marker increased over time, demonstrating the compatibility of this biomaterial with neuronal cell culture and differentiation. Furthermore, these cells showed increased maturation with corresponding functional properties, which was also demonstrated by the formation of a neuronal network that was observed by recording spontaneous activity via Ca2+ imaging.
AUTHOR Kaneda, Giselle and Chan, Julie L. and Castaneda, Chloe M. and Papalamprou, Angela and Sheyn, Julia and Shelest, Oksana and Huang, Dave and Kluser, Nadine and Yu, Victoria and Ignacio, Gian C. and Gertych, Arkadiusz and Yoshida, Ryu and Metzger, Melodie and Tawackoli, Wafa and Vernengo, Andrea and Sheyn, Dmitriy
Title iPSC-derived tenocytes seeded on microgrooved 3D printed scaffolds for Achilles Tendon Regeneration [Abstract]
Year 2023
Journal/Proceedings Journal of Orthopaedic Research
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AbstractTendons and ligaments have a poor innate healing capacity, yet account for 50% of musculoskeletal injuries in the US. Full structure and function restoration post-injury remains an unmet clinical need. This study aimed to assess the application of novel 3D printed scaffolds and induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) overexpressing the transcription factor Scleraxis (SCX, iMSCSCX+) as a new strategy for tendon defect repair. The polycaprolactone (PCL) scaffolds were fabricated by extrusion through a patterned nozzle or conventional round nozzle. Scaffolds were seeded with iMSCSCX+ and outcomes were assessed in vitro via gene expression analysis and immunofluorescence. In vivo, rat Achilles tendon defects were repaired with iMSCSCX+-seeded microgrooved scaffolds, microgrooved scaffolds only, or suture only and assessed via gait, gene expression, biomechanical testing, histology, and immunofluorescence.iMSCSCX+-seeded on microgrooved scaffolds showed upregulation of tendon markers and increased organization and linearity of cells compared to non-patterned scaffolds in vitro. In vivo gait analysis showed improvement in the Scaffold+iMSCSCX+-treated group compared to the controls. Tensile testing of the tendons demonstrated improved biomechanical properties of the Scaffold+iMSCSCX+ group compared to the controls. Histology and immunofluorescence demonstrated more regular tissue formation in the Scaffold+iMSCSCX+ group.This article is protected by copyright. All rights reserved.
AUTHOR Ege, Duygu and Hasirci, Vasif
Title Is 3D Printing Promising for Osteochondral Tissue Regeneration? [Abstract]
Year 2023
Journal/Proceedings ACS Appl. Bio Mater.
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Osteochondral tissue regeneration is quite difficult to achieve due to the complexity of its organization. In the design of these complex multilayer structures, a fabrication method, 3D printing, started to be employed, especially by using extrusion, stereolithography and inkjet printing approaches. In this paper, the designs are discussed including biphasic, triphasic, and gradient structures which aim to mimic the cartilage and the calcified cartilage and the whole osteochondral tissue closely. In the first section of the review paper, 3D printing of hydrogels including gelatin methacryloyl (GelMa), alginate, and polyethylene glycol diacrylate (PEGDA) are discussed. However, their physical and biological properties need to be augmented, and this generally is achieved by blending the hydrogel with other, more durable, less hydrophilic, polymers. These scaffolds are very suitable to carry growth factors, such as TGF-β1, to further stimulate chondrogenesis. The bone layer is mimicked by printing calcium phosphates (CaPs) or bioactive glasses together with the hydrogels or as a component of another polymer layer. The current research findings indicate that polyester (i.e. polycaprolactone (PCL), polylactic acid (PLA) and poly(lactide-co-glycolide) (PLGA)) reinforced hydrogels may more successfully mimic the complex structure of osteochondral tissue. Moreover, more recent printing methods such as melt electrowriting (MEW), are being used to integrate polyester fibers to enhance the mechanical properties of hydrogels. Additionally, polyester scaffolds that are 3D printed without hydrogels are discussed after the hydrogel-based scaffolds. In this review paper, the relevant studies are analyzed and discussed, and future work is recommended with support of tables of designed scaffolds. The outcome of the survey of the field is that 3D printing has significant potential to contribute to osteochondral tissue repair.
AUTHOR Mamidi, Narsimha and Ijadi, Fatemeh and Norahan, Mohammad Hadi
Title Leveraging the Recent Advancements in GelMA Scaffolds for Bone Tissue Engineering: An Assessment of Challenges and Opportunities [Abstract]
Year 2023
Journal/Proceedings Biomacromolecules
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Abstract
The field of bone tissue engineering has seen significant advancements in recent years. Each year, over two million bone transplants are performed globally, and conventional treatments, such as bone grafts and metallic implants, have their limitations. Tissue engineering offers a new level of treatment, allowing for the creation of living tissue within a biomaterial framework. Recent advances in biomaterials have provided innovative approaches to rebuilding bone tissue function after damage. Among them, gelatin methacryloyl (GelMA) hydrogel is emerging as a promising biomaterial for supporting cell proliferation and tissue regeneration, and GelMA has exhibited exceptional physicochemical and biological properties, making it a viable option for clinical translation. Various methods and classes of additives have been used in the application of GelMA for bone regeneration, with the incorporation of nanofillers or other polymers enhancing its resilience and functional performance. Despite promising results, the fabrication of complex structures that mimic the bone architecture and the provision of balanced physical properties for both cell and vasculature growth and proper stiffness for load bearing remain as challenges. In terms of utilizing osteogenic additives, the priority should be on versatile components that promote angiogenesis and osteogenesis while reinforcing the structure for bone tissue engineering applications. This review focuses on recent efforts and advantages of GelMA-based composite biomaterials for bone tissue engineering, covering the literature from the last five years.
AUTHOR Ianchis, Raluca and Marin, Maria Minodora and Alexa, Rebeca Leu and Gifu, Ioana Catalina and Alexandrescu, Elvira and Pircalabioru, Gratiela Gradisteanu and Vlasceanu, George Mihail and Teodorescu, George Mihail and Serafim, Andrada and Preda, Silviu and Nistor, Cristina Lavinia and Petcu, Cristian
Title Nanoclay-reinforced alginate/salecan composite inks for 3D printing applications
Year 2023
Journal/Proceedings IJB
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AUTHOR Sabzevari, Alireza and Rayat Pisheh, Hossein and Ansari, Mojtaba and Salati, Amir
Title Progress in bioprinting technology for tissue regeneration [Abstract]
Year 2023
Journal/Proceedings Journal of Artificial Organs
Reftype Sabzevari2023
DOI/URL DOI
Abstract
In recent years, due to the increase in diseases that require organ/tissue transplantation and the limited donor, on the other hand, patients have lost hope of recovery and organ transplantation. Regenerative medicine is one of the new sciences that promises a bright future for these patients by providing solutions to repair, improve function, and replace tissue. One of the technologies used in regenerative medicine is three-dimensional (3D) bioprinters. Bioprinting is a new strategy that is the basis for starting a global revolution in the field of medical sciences and has attracted much attention. 3D bioprinters use a combination of advanced biology and cell science, computer science, and materials science to create complex bio-hybrid structures for various applications. The capacity to use this technology can be demonstrated in regenerative medicine to make various connective tissues, such as skin, cartilage, and bone. One of the essential parts of a 3D bioprinter is the bio-ink. Bio-ink is a combination of biologically active molecules, cells, and biomaterials that make the printed product. In this review, we examine the main bioprinting strategies, such as inkjet printing, laser, and extrusion-based bioprinting, as well as some of their applications.
AUTHOR Pan, Yiwen and Chen, Shaoqing and Meng, Yanyan and He, Mu and Liu, Chun and Wang, Cheli and Ni, Xinye
Title Study on 3D-Printed Emodin/Nano-Hydroxyapatite Scaffolds Promoting Bone Regeneration by Supporting Osteoblast Proliferation and Macrophage M2 Polarization [Abstract]
Year 2023
Journal/Proceedings ACS Appl. Polym. Mater.
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Abstract
The treatment of bone defects caused by diseases, trauma, or tumor has always been a great clinical challenge. Implantation of bone biomaterials into bone defect areas is an effective method for bone injury repair. In this study, we used three-dimensional (3D) printing technology to prepare nano-hydroxyapatite (nHA)/sodium alginate (SA)/gelatin (Gel) hydrogel scaffolds loaded with different ratios (0, 0.13, 0.26, 0.39, 0.53, and 0.79‰) of emodin (EM) (EM/nHA/SA/Gel). Scanning electron microscopy showed that the scaffolds had a smooth surface without fracture and nHA was evenly distributed on the surface. The cell proliferation and migration results showed that the 0.39‰ EM group, in particular, could significantly promote the proliferation and migration of mouse embryonic osteoblast precursor (MC3T3-E1) cells and significantly increase the mRNA expression of osteogenic differentiation-related genes (bone morphogenetic protein/BMP-2, BMP-9, osteocalcin). In addition, the 0.39‰ EM group exhibited the best effect on osteogenic differentiation-related proteins (alkaline phosphatase, Runx 2, OSX). The expression of M2 polarization-related genes (arginase-1, CD206) also significantly increased after the treatment with the 0.39‰ EM group. Micro-CT showed that in the rat skull defect model, the EM/nHA/SA/Gel scaffold group significantly promoted bone regeneration after being implanted into the skull for 30 days. Our results indicate that the EM/nHA/SA/Gel hydrogel scaffolds can not only directly promote the proliferation and differentiation of osteoblasts but also indirectly promote osteogenic differentiation by supporting M2 polarization of macrophages. EM/nHA/SA/Gel hydrogel scaffolds are potential bone tissue engineering materials for bone regeneration.
AUTHOR Cai, Runxuan and Gimenez-Camino, Naroa and Xiao, Ming and Bi, Shuguang and DiVito, Kyle A.
Title Technological advances in three-dimensional skin tissue engineering
Year 2023
Journal/Proceedings REVIEWS ON ADVANCED MATERIALS SCIENCE
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AUTHOR Zhang, Yubei and Raza, Ali and Xue, Ya-Qi and Yang, Ganggang and Hayat, Uzma and Yu, Jingwen and Liu, Chang and Wang, Hua-Jie and Wang, Jin-Ye
Title Water-responsive 4D printing based on self-assembly of hydrophobic protein “Zein” for the control of degradation rate and drug release [Abstract]
Year 2023
Journal/Proceedings Bioactive Materials
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Abstract
Four-dimensional (4D) printing is a promising technology that provides solutions for compelling needs in various fields. Most of the reported 4D printed systems are based on the temporal shape transformation of printed subjects. Induction of temporal heterogenicity in functions in addition to shape may extend the scope of 4D printing. Herein, we report a 4D printing approach using plant protein (zein) gel inspired by the amyloid fibrils formation mechanism. The printing of zein gel in a specialized layered-Carbopol supporting bath with different water concentrations in an ethanol-water mixture modulates hydrophobic and hydrogen bonding that causes temporal changes in functions. The part of the construct printed in a supporting bath with higher water content exhibits higher drug loading, faster drug release and degradation than those printed in the supporting bath with lower water content. Tri-segment conduit and butterfly-shaped construct with two asymmetrical wings are printed using this system to evaluate biomedical function as nerve conduit and drug delivery system. 4D printed conduits are also effective as a drug-eluting urethral stent in the porcine model. Overall, this study extends the concept of 4D printing beyond shape transformation and presents an approach of fabricating specialized baths for 4D printing that can also be extended to other materials to obtain 4D printed medical devices with translational potential.
AUTHOR Xue, Ya-Qi and Zhang, Yu-Cheng and Zhang, Yu-Bei and Wang, Jin-Ye
Title Zein-based 3D tubular constructs with tunable porosity for 3D cell culture and drug delivery [Abstract]
Year 2023
Journal/Proceedings Biomedical Engineering Advances
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Abstract
Manufacturing tubular constructs with tunable porosity can mimic the vascular structure, not only for supplying nutrients and removing metabolites to support long-term 3D cell culture but also for delivering bioactive components and drugs to tissues. There are few reports on the second purpose through 3D printing. In this study, bio-inspired tubular constructs with permeability were achieved using zein-based ink, forming structures with tunable porosity via the 3D printing technique. The parameters, e.g., zein content, with/without the addition of porogen, and drying conditions, were optimized to control the porous structure and porosity of the printed tubes. The inner wall of the resultant tube supported the adhesion of endothelial cells. A perfusion system was designed, and the penetrability of zein-based tubular constructs was demonstrated by the dialysis test. Moreover, perfusion of cell culture media and the anti-cancer drug in cell-laden hydrogels with tubular structure resulted in 3-day of 3D cell culture with a higher survival rate, and the drug was delivered to local cells around the tubular constructs, respectively. This is a new report on the preparation of 3D-printed tubular constructs using zein as the biomaterial inks with tunable porosity and porous structure, providing a general system for 3D cell culture, 3D drugs screening/pharmacokinetics in vitro, and tissue engineering.
AUTHOR Solis, Daphene Marques and Czekanski, Aleksander
Title 3D and 4D additive manufacturing techniques for vascular-like structures – A review [Abstract]
Year 2022
Journal/Proceedings Bioprinting
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Abstract
The critical shortage in organ donors is a problem facing patients and health care systems worldwide. The most promising solution to this crisis is the artificial production of organs and tissues, known as tissue engineering. Significant advances in this field have led to the commercial production of artificial skin and cartilage, but limitations remain in the production of thicker tissues (i.e., >200 μm thickness). The challenge of producing thicker tissues relates to the inability to establish and maintain a vascular system, which is the basic requirement for artificially producing any vital organ. Given the importance of these structures, a major scientific effort is underway to better understand how to reproduce a vascular system. This review presents the most recent advances in the manufacturing of vascular-like structures, especially techniques involving additive manufacturing, or 3D and 4D printing. This review starts with a primer on the classification, composition, and mechanical proprieties of blood vessels, offering the reader a better understanding of the challenges involved in the artificial production of vessels. The review then discusses the methodologies, technologies, and materials used and available for the manufacturing of the vascular system.
AUTHOR Pitacco, Pierluca and Sadowska, Joanna M. and O'Brien, Fergal J. and Kelly, Daniel J.
Title 3D bioprinting of cartilaginous templates for large bone defect healing [Abstract]
Year 2022
Journal/Proceedings Acta Biomaterialia
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Abstract
Damaged or diseased bone can be treated using autografts or a range of different bone grafting biomaterials, however limitations with such approaches has motivated increased interest in developmentally inspired bone tissue engineering (BTE) strategies that seek to recapitulate the process of endochondral ossification (EO) as a means of regenerating critically sized defects. The clinical translation of such strategies will require the engineering of scaled-up, geometrically defined hypertrophic cartilage grafts that can be rapidly vascularised and remodelled into bone in mechanically challenging defect environments. The goal of this study was to 3D bioprint mechanically reinforced cartilaginous templates and to assess their capacity to regenerate critically sized femoral bone defects. Human mesenchymal stem/stromal cells (hMSCs) were incorporated into fibrin based bioinks and bioprinted into polycaprolactone (PCL) frameworks to produce mechanically reinforced constructs. Chondrogenic priming of such hMSC laden constructs was required to support robust vascularisation and graft mineralisation in vivo following their subcutaneous implantation into nude mice. With a view towards maximising their potential to support endochondral bone regeneration, we next explored different in vitro culture regimes to produce chondrogenic and early hypertrophic engineered grafts. Following their implantation into femoral bone defects within transiently immunosuppressed rats, such bioprinted constructs were rapidly remodelled into bone in vivo, with early hypertrophic constructs supporting higher levels of vascularisation and bone formation compared to the chondrogenic constructs. Such early hypertrophic bioprinted constructs also supported higher levels of vascularisation and spatially distinct patterns of new formation compared to BMP-2 loaded collagen scaffolds (here used as a positive control). In conclusion, this study demonstrates that fibrin based bioinks support chondrogenesis of hMSCs in vitro, which enables the bioprinting of mechanically reinforced hypertrophic cartilaginous templates capable of supporting large bone defect regeneration. These results support the use of 3D bioprinting as a strategy to scale-up the engineering of developmentally inspired templates for BTE. Statement of significance Despite the promise of developmentally inspired tissue engineering strategies for bone regeneration, there are still challenges that need to be addressed to enable clinical translation. This work reports the development and assessment (in vitro and in vivo) of a 3D bioprinting strategy to engineer mechanically-reinforced cartilaginous templates for large bone defect regeneration using human MSCs. Using distinct in vitro priming protocols, it was possible to generate cartilage grafts with altered phenotypes. More hypertrophic grafts, engineered in vitro using TGF-β3 and BMP-2, supported higher levels of blood vessel infiltration and accelerated bone regeneration in vivo. This study also identifies some of the advantages and disadvantages of such endochondral bone TE strategies over the direct delivery of BMP-2 from collagen-based scaffolds.
AUTHOR Govindharaj, Mano and Hashimi, Noura Al and Soman, Soja Saghar and Kanwar, Susheem and Vijayavenkataraman, Sanjairaj
Title 3D Bioprinting of human Mesenchymal Stem Cells in a novel tunic decellularized ECM bioink for Cartilage Tissue Engineering [Abstract]
Year 2022
Journal/Proceedings Materialia
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Abstract
Tunicates are marine organisms renowned for their thick, leathery exoskeleton called tunic. This tunic is composed of an extracellular matrix packed with protein-cellulose complexes and sulfated polysaccharides, making it a charming biomaterial choice for cartilage tissue engineering. In this study, P.nigra tunicate was collected and processed to obtain its rich decellularized extracellular matrix (dECM). The dECM was either seeded with human mesenchymal stem cells (hMSCs) as is or underwent further processing to form a hydrogel for 3D bioprinting. The characterization of tunic dECM was achieved by FTIR, XRD, TGA, Raman spectroscopy, SEM and tensile mechanical analysis. Biological compatibility and staining were done by live/dead, alamar blue, alcian blue, safranin O and PCR gene expression. After decellularization, the tunic dECM scaffold preserved the natural honeycomb-shaped microstructure, as well as its functional cellulose and protein groups. Both the tunic dECM scaffolds and bioprinted scaffolds showed enhanced metabolic activity, cell proliferation and chondrogenic differentiation. Combining both the mechanical robustness and biocompatibility, the bioink is able to fill the elusive gap in cartilage regeneration. This study offers a new potential source of dECM scaffolds and bioinks which are both biologically compatible and mechanically stable, making it a one stop shop for cartilage tissue engineering.
AUTHOR Leu Alexa, Rebeca and Cucuruz, Andreia and Ghițulică, Cristina-Daniela and Voicu, Georgeta and Stamat (Balahura), Liliana-Roxana and Dinescu, Sorina and Vlasceanu, George Mihail and Stavarache, Cristina and Ianchis, Raluca and Iovu, Horia and Costache, Marieta
Title 3D Printable Composite Biomaterials Based on GelMA and Hydroxyapatite Powders Doped with Cerium Ions for Bone Tissue Regeneration [Abstract]
Year 2022
Journal/Proceedings International Journal of Molecular Sciences
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Abstract
The main objective was to produce 3D printable hydrogels based on GelMA and hydroxyapatite doped with cerium ions with potential application in bone regeneration. The first part of the study regards the substitution of Ca2+ ions from hydroxyapatite structure with cerium ions (Ca10-xCex(PO4)6(OH)2, xCe = 0.1, 0.3, 0.5). The second part followed the selection of the optimal concentration of HAp doped, which will ensure GelMA-based scaffolds with good biocompatibility, viability and cell proliferation. The third part aimed to select the optimal concentrations of GelMA for the 3D printing process (20%, 30% and 35%). In vitro biological assessment presented the highest level of cell viability and proliferation potency of GelMA-HC5 composites, along with a low cytotoxic potential, highlighting the beneficial effects of cerium on cell growth, also supported by Live/Dead results. According to the 3D printing experiments, the 30% GelMA enriched with HC5 was able to generate 3D scaffolds with high structural integrity and homogeneity, showing the highest suitability for the 3D printing process. The osteogenic differentiation experiments confirmed the ability of 30% GelMA-3% HC5 scaffold to support and efficiently maintain the osteogenesis process. Based on the results, 30% GelMA-3% HC5 3D printed scaffolds could be considered as biomaterials with suitable characteristics for application in bone tissue engineering.
AUTHOR Qin, Wen and Li, Chenkai and Liu, Chun and Wu, Siyu and Liu, Jun and Ma, Jiayi and Chen, Wenyang and Zhao, Hongbin and Zhao, Xiubo
Title 3D printed biocompatible graphene oxide, attapulgite, and collagen composite scaffolds for bone regeneration [Abstract]
Year 2022
Journal/Proceedings Journal of Biomaterials Applications
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Abstract
Tissue-engineered bone material is one of the effective methods to repair bone defects, but the application is restricted in clinical because of the lack of excellent scaffolds that can induce bone regeneration as well as the difficulty in making scaffolds with personalized structures. 3D printing is an emerging technology that can fabricate bespoke 3D scaffolds with precise structure. However, it is challenging to develop the scaffold materials with excellent printability, osteogenesis ability, and mechanical strength. In this study, graphene oxide (GO), attapulgite (ATP), type I collagen (Col I) and polyvinyl alcohol were used as raw materials to prepare composite scaffolds via 3D bioprinting. The composite materials showed excellent printability. The microcosmic architecture and properties was characterized by scanning electron microscopy, Fourier transform infrared and thermal gravimetric analyzer, respectively. To verify the biocompatibility of the scaffolds, the viability, proliferation and osteogenic differentiation of Bone Marrow Stromal Cells (BMSCs) on the scaffolds were assessed by CCK-8, Live/Dead staining and Real-time PCR in vitro. The composited scaffolds were then implanted into the skull defects on rat for bone regeneration. Hematoxylin-eosin staining, Masson staining and immunohistochemistry staining were carried out in vivo to evaluate the regeneration of bone tissue.The results showed that GO/ATP/COL scaffolds have been demonstrated to possess controlled porosity, water absorption, biodegradability and good apatite-mineralization ability. The scaffold consisting of 0.5% GO/ATP/COL have excellent biocompatibility and was able to promote the growth, proliferation and osteogenic differentiation of mouse BMSCs in vitro. Furthermore, the 0.5% GO/ATP/COL scaffolds were also able to promote bone regeneration of in rat skull defects. Our results illustrated that the 3D printed GO/ATP/COL composite scaffolds have good mechanical properties, excellent cytocompatibility for enhanced mouse BMSCs adhesion, proliferation, and osteogenic differentiation. All these advantages made it potential as a promising biomaterial for osteogenic reconstruction.
AUTHOR Leu Alexa, Rebeca and Cucuruz, Andreia and Ghițulică, Cristina-Daniela and Voicu, Georgeta and Stamat (Balahura), Liliana-Roxana and Dinescu, Sorina and Vlasceanu, George Mihail and Iovu, Horia and Serafim, Andrada and Ianchis, Raluca and Ciocan, Lucian-Toma and Costache, Marieta
Title 3D Printed Composite Scaffolds of GelMA and Hydroxyapatite Nanopowders Doped with Mg/Zn Ions to Evaluate the Expression of Genes and Proteins of Osteogenic Markers [Abstract]
Year 2022
Journal/Proceedings Nanomaterials
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Abstract
As bone diseases and defects are constantly increasing, the improvement of bone regeneration techniques is constantly evolving. The main purpose of this scientific study was to obtain and investigate biomaterials that can be used in tissue engineering. In this respect, nanocomposite inks of GelMA modified with hydroxyapatite (HA) substituted with Mg and Zn were developed. Using a 3D bioprinting technique, scaffolds with varying shapes and dimensions were obtained. The following analyses were used in order to study the nanocomposite materials and scaffolds obtained by the 3D printing technique: Fourier transform infrared spectrometry and X-ray diffraction (XRD), scanning electron microscopy (SEM), and micro-computed tomography (Micro-CT). The swelling and dissolvability of each scaffold were also studied. Biological studies, osteopontin (OPN), and osterix (OSX) gene expression evaluations were confirmed at the protein levels, using immunofluorescence coupled with confocal microscopy. These findings suggest the positive effect of magnesium and zinc on the osteogenic differentiation process. OSX fluorescent staining also confirmed the capacity of GelMA-HM5 and GelMA-HZ5 to support osteogenesis, especially of the magnesium enriched scaffold.
AUTHOR Yang, Xue and Li, Shuai and Ren, Ya and Qiang, Lei and Liu, Yihao and Wang, Jinwu and Dai, Kerong
Title 3D printed hydrogel for articular cartilage regeneration [Abstract]
Year 2022
Journal/Proceedings Composites Part B: Engineering
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Abstract
Tissue engineering is a promising strategy for damaged cartilage tissue repair. Three-dimensional (3D) printed hydrogel exhibits great potential in cartilage tissue engineering for fabricating 3D cell culture scaffolds, owing to its similarity to the extracellular matrix (ECM). Numerous hydrogels have been tested for 3D printing in vitro articular cartilage tissues, including natural and synthetic hydrogels that mimic their in vivo counterparts. The advancement of materials science and 3D printing techniques enables a wide range of fabrication strategies that produce cartilage tissues with delicate structures and on multiple scales. Stimuli-responsive hydrogels, which rely on the external environment to transform to a desired structure or dimension, have likewise been widely studied in tissue engineering. This review summarizes the characteristics, functions, and research conducted on 3D printed hydrogels by categorizing cutting-edge hydrogel materials commonly used in cartilage tissue engineering and their complexes. The challenges and application prospects of hydrogels in cartilage tissue engineering are described. Novel composite hydrogels must be investigated to meet the requirements of native articular cartilage in the aspects of structure, scale, mechanical properties, among others. Combining stimuli-responsive hydrogels with biological scaffolds also shows great potential in various applications, including but not limited to articular cartilage, vascularization, and osteochondral repair.
AUTHOR Hashimi, Noura Sayed Al and Soman, Soja Saghar and Govindharaj, Mano and Vijayavenkataraman, Sanjairaj
Title 3D printing of complex architected metamaterial structures by simple material extrusion for bone tissue engineering [Abstract]
Year 2022
Journal/Proceedings Materials Today Communications
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Abstract
Triply periodic minimal surfaces (TPMS) are gaining popularity as scaffolds for bioapplications due to their unique structure, offering strong mechanical properties and biomorphic surfaces which enhance cell attachment and proliferation. In this work, polymer TPMS sheet lattices were printed using a well-known yet unprecedented technique of manufacturing such structures; which is material extrusion (specifically, pneumatic melt extrusion). This method offers a one step, straightforward yet reliable way to print complex porous structures while retaining design accuracy and significantly simplifying the process. Multiple primitive, gyroid and cubic structures were designed using MSLattice and Solidworks with 70% porosity and 2×2×3 unit cells. The scaffolds were printed by melt extrusion of polycaprolactone (PCL) at different parameters to establish the optimal settings. Morphological features (pore size and strut thickness) were determined using scanning electron microscopy (SEM) and the accuracy of print was determined by comparing to the design, showing high print accuracy and minimal percentage errors of less than 15% in all prints. Uniaxial compression testing was used to demonstrate the different deformation processes of the scaffolds and evaluate their mechanical properties, with primitive having the highest modulus and gyroid the highest yield strength. Finally, cell viability was quantified by alamar blue cell viability assay and visualized by SEM, displaying significant increase in cell proliferation and attachment, specifically in the primitive structure. Herein we will explain the challenges faced with design and print optimization and how we overcame them, making this work the first of its kind in material extrusion (pneumatic melt extrusion) printing of TPMS scaffolds.
AUTHOR Dairaghi, Jacob and Rogozea, Dan and Cadle, Rachel and Bustamante, Joseph and Moldovan, Leni and Petrache, Horia I. and Moldovan, Nicanor I.
Title 3D Printing of Human Ossicle Models for the Biofabrication of Personalized Middle Ear Prostheses [Abstract]
Year 2022
Journal/Proceedings Applied Sciences
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Abstract
The middle ear bones (‘ossicles’) may become severely damaged due to accidents or to diseases. In these situations, the most common current treatments include replacing them with cadaver-derived ossicles, using a metal (usually titanium) prosthesis, or introducing bridges made of biocompatible ceramics. Neither of these solutions is ideal, due to the difficulty in finding or producing shape-matching replacements. However, the advent of additive manufacturing applications to biomedical problems has created the possibility of 3D-printing anatomically correct, shape- and size-personalized ossicle prostheses. To demonstrate this concept, we generated and printed several models of ossicles, as solid, porous, or soft material structures. These models were first printed with a plottable calcium phosphate/hydroxyapatite paste by extrusion on a solid support or embedded in a Carbopol hydrogel bath, followed by temperature-induced hardening. We then also printed an ossicle model with this ceramic in a porous format, followed by loading and crosslinking an alginate hydrogel within the pores, which was validated by microCT imaging. Finally, ossicle models were printed using alginate as well as a cell-containing nanocellulose-based bioink, within the supporting hydrogel bath. In selected cases, the devised workflow and the printouts were tested for repeatability. In conclusion, we demonstrate that moving beyond simplistic geometric bridges to anatomically realistic constructs is possible by 3D printing with various biocompatible materials and hydrogels, thus opening the way towards the in vitro generation of personalized middle ear prostheses for implantation.
AUTHOR Dairaghi, Jacob and Rogozea, Dan and Cadle, Rachel and Bustamante, Joseph and Moldovan, Leni and Petrache, Horia I. and Moldovan, Nicanor I.
Title 3D Printing of Human Ossicle Models for the Biofabrication of Personalized Middle Ear Prostheses [Abstract]
Year 2022
Journal/Proceedings Applied Sciences
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DOI/URL URL DOI
Abstract
The middle ear bones (‘ossicles’) may become severely damaged due to accidents or to diseases. In these situations, the most common current treatments include replacing them with cadaver-derived ossicles, using a metal (usually titanium) prosthesis, or introducing bridges made of biocompatible ceramics. Neither of these solutions is ideal, due to the difficulty in finding or producing shape-matching replacements. However, the advent of additive manufacturing applications to biomedical problems has created the possibility of 3D-printing anatomically correct, shape- and size-personalized ossicle prostheses. To demonstrate this concept, we generated and printed several models of ossicles, as solid, porous, or soft material structures. These models were first printed with a plottable calcium phosphate/hydroxyapatite paste by extrusion on a solid support or embedded in a Carbopol hydrogel bath, followed by temperature-induced hardening. We then also printed an ossicle model with this ceramic in a porous format, followed by loading and crosslinking an alginate hydrogel within the pores, which was validated by microCT imaging. Finally, ossicle models were printed using alginate as well as a cell-containing nanocellulose-based bioink, within the supporting hydrogel bath. In selected cases, the devised workflow and the printouts were tested for repeatability. In conclusion, we demonstrate that moving beyond simplistic geometric bridges to anatomically realistic constructs is possible by 3D printing with various biocompatible materials and hydrogels, thus opening the way towards the in vitro generation of personalized middle ear prostheses for implantation.
AUTHOR Dairaghi, Jacob and Rogozea, Dan and Cadle, Rachel and Bustamante, Joseph and Moldovan, Leni and Petrache, Horia I. and Moldovan, Nicanor I.
Title 3D Printing of Human Ossicle Models for the Biofabrication of Personalized Middle Ear Prostheses [Abstract]
Year 2022
Journal/Proceedings Applied Sciences
Reftype
DOI/URL URL DOI
Abstract
The middle ear bones (‘ossicles’) may become severely damaged due to accidents or to diseases. In these situations, the most common current treatments include replacing them with cadaver-derived ossicles, using a metal (usually titanium) prosthesis, or introducing bridges made of biocompatible ceramics. Neither of these solutions is ideal, due to the difficulty in finding or producing shape-matching replacements. However, the advent of additive manufacturing applications to biomedical problems has created the possibility of 3D-printing anatomically correct, shape- and size-personalized ossicle prostheses. To demonstrate this concept, we generated and printed several models of ossicles, as solid, porous, or soft material structures. These models were first printed with a plottable calcium phosphate/hydroxyapatite paste by extrusion on a solid support or embedded in a Carbopol hydrogel bath, followed by temperature-induced hardening. We then also printed an ossicle model with this ceramic in a porous format, followed by loading and crosslinking an alginate hydrogel within the pores, which was validated by microCT imaging. Finally, ossicle models were printed using alginate as well as a cell-containing nanocellulose-based bioink, within the supporting hydrogel bath. In selected cases, the devised workflow and the printouts were tested for repeatability. In conclusion, we demonstrate that moving beyond simplistic geometric bridges to anatomically realistic constructs is possible by 3D printing with various biocompatible materials and hydrogels, thus opening the way towards the in vitro generation of personalized middle ear prostheses for implantation.
AUTHOR Hilgeroth, Philipp S. and Thümmler, Justus F. and Binder, Wolfgang H.
Title 3D Printing of Triamcinolone Acetonide in Triblock Copolymers of Styrene–Isobutylene–Styrene as a Slow-Release System [Abstract]
Year 2022
Journal/Proceedings Polymers
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Abstract
Additive manufacturing has a wide range of applications and has opened up new methods of drug formulation, in turn achieving attention in medicine. We prepared styrene–isobutylene–styrene triblock copolymers (SIBS; Mn = 10 kDa–25 kDa, PDI 1,3–1,6) as a drug carrier for triamcinolone acetonide (TA), further processed by fused deposition modeling to create a solid drug release system displaying improved bioavailability and applicability. Living carbocationic polymerization was used to exert control over block length and polymeric architecture. Thermorheological properties of the SIBS polymer (22.3 kDa, 38 wt % S) were adjusted to the printability of SIBS/TA mixtures (1–5% of TA), generating an effective release system effective for more than 60 days. Continuous drug release and morphological investigations were conducted to probe the influence of the 3D printing process on the drug release, enabling 3D printing as a formulation method for a slow-release system of Triamcinolone.
AUTHOR Mao, Qiuyi and Zhu, Bowen and Zhuang, Hai and Bu, Shoushan
Title 3D-Printing Assisted SF-SA Based MgP Hybrid Hydrogel Scaffold for Bone Tissue Engineering [Abstract]
Year 2022
Journal/Proceedings Frontiers in Materials
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Abstract
A new prototype of hybrid silk fibroin and sodium alginate (SF-SA) based osteogenic hydrogel scaffold with a concentration of 2.5% magnesium phosphate (MgP) based gel was prepared with the assistance of an extrusion-based three-dimensional (3D) printing machine in this study. To determine the optimum ratio of MgP-based gel in the hydrogel, a series of physical and biochemical experiments were performed to determine the proper concentration of MgP in two-dimensional hydrogel films, as well as the cell compatibility with these materials in sequence. The SF-SA hydrogel with 2.5wt% magnesium phosphate (SF-SA/MgP) stood out and then was used to fabricate 3D hydrogel scaffolds according to the consequences of the experiments, with SF-SA hydrogel as a control. Then the morphology and osteogenic activity of the scaffolds were further characterized by field emission scanning electron microscope (SEM), calcium mineralization staining, and reverse transcription-polymerase chain reaction (rt-PCR). The SF-SA/MgP hydrogel scaffold promoted the adhesion of rat mesenchymal stem cells with higher degrees of efficiency under dynamic culture conditions. After co-culturing in an osteogenic differentiation medium, cells seeded on SF-SA/MgP hydrogel scaffold were shown to have better performance on osteogenesis in the early stage than the control group. This work illustrates that the 3D structures of hybrid SF-SA/MgP hydrogel are promising headstones for osteogenic tissue engineering.
AUTHOR Kitana, Waseem and Apsite, Indra and Hazur, Jonas and Boccaccini, Aldo R. and Ionov, Leonid
Title 4D Biofabrication of T-Shaped Vascular Bifurcation [Abstract]
Year 2022
Journal/Proceedings Advanced Materials Technologies
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Abstract
Abstract 4D Biofabrication – a pioneering biofabrication technique – involves the automated fabrication of 3D constructs that are dynamic and show shape-transformation capability. Although current 4D biofabrication methods are highly promising for the fabrication of vascular elements such as tubes, the fabrication of tubular junctions is still highly challenging. Here, for the first time, a 4D biofabrication-based concept for the fabrication of a T-shaped vascular bifurcation using 3D printed shape-changing layers based on a mathematical model is reported. The formation of tubular structures with various diameters is achieved by precisely controlling the parameters (e.g. crosslinking time). Consequently, the 3D printed films show self-transformation into a T-junction upon immersion in water with a diameter of a few millimeters. Perfusion of the tubular T-junction with an aqueous medium simulating blood flow through vessels shows minimal leakages with a maximum flow velocity of 0.11 m s–1. Furthermore, human umbilical vein endothelial cells seeded on the inner surface of the plain T-junction show outstanding growth properties and excellent cell viability. The achieved diameters are comparable to the native blood vessels, which is still a challenge in 3D biofabrication. This approach paves the way for the fabrication of fully automatic self-actuated vascular bifurcations as vascular grafts.
AUTHOR Anand, Resmi and Amoli, Mehdi Salar and Huysecom, An-Sofie and Amorim, Paulo Alexandre and Agten, Hannah and Geris, Liesbet and Bloemen, Veerle
Title A tunable gelatin-hyaluronan dialdehyde/methacryloyl gelatin interpenetrating polymer network hydrogel for additive tissue manufacturing [Abstract]
Year 2022
Journal/Proceedings Biomedical Materials
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DOI/URL DOI
Abstract
Methacryloyl gelatin (GelMA) is a versatile material for bioprinting because of its tunable physical properties and inherent bioactivity. Bioprinting of GelMA is often met with challenges such as lower viscosity of GelMA inks due to higher methacryloyl substitution and longer physical gelation time at room temperature. In this study, a tunable interpenetrating polymer network (IPN) hydrogel was prepared from gelatin-hyaluronan dialdehyde (Gel-HDA) Schiff’s polymer, and 100% methacrylamide substituted GelMA for biofabrication through extrusion based bioprinting. Temperature sweep rheology measurements show a higher sol-gel transition temperature for IPN (30 °C) compared to gold standard GelMA (27 °C). Furthermore, to determine the tunability of the IPN hydrogel, several IPN samples were prepared by combining different ratios of Gel-HDA and GelMA achieving a compressive modulus ranging from 20.6 ± 2.48 KPa to 116.7 ± 14.80 KPa. Our results showed that the mechanical properties and printability at room temperature could be tuned by adjusting the ratios of GelMA and Gel-HDA. To evaluate cell response to the material, MC3T3-E1 mouse pre-osteoblast cells were embedded in hydrogels and 3D-printed, demonstrating excellent cell viability and proliferation after 10 d of 3D in vitro culture, making the IPN an interesting bioink for the fabrication of 3D constructs for tissue engineering applications.
AUTHOR Özcan, Mutlu and Magini, Eduarda Blasi and Volpato, Guilherme Maziero and Cruz, Ariadne and Volpato, Claudia Angela Maziero
Title Additive Manufacturing Technologies for Fabrication of Biomaterials for Surgical Procedures in Dentistry: A Narrative Review [Abstract]
Year 2022
Journal/Proceedings Journal of Prosthodontics
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DOI/URL DOI
Abstract
Abstract Purpose To screen and critically appraise available literature regarding additive manufacturing technologies for bone graft material fabrication in dentistry. Material and Methods PubMed and Scopus were searched up to May 2021. Studies reporting the additive manufacturing techniques to manufacture scaffolds for intraoral bone defect reconstruction were considered eligible. A narrative review was synthesized to discuss the techniques for bone graft material fabrication in dentistry and the biomaterials used. Results The databases search resulted in 933 articles. After removing duplicate articles (128 articles), the titles and abstracts of the remaining articles (805 articles) were evaluated. A total of 89 articles were included in this review. Reading these articles, 5 categories of additive manufacturing techniques were identified: material jetting, powder bed fusion, vat photopolymerization, binder jetting, and material extrusion. Conclusions Additive manufacturing technologies for bone graft material fabrication in dentistry, especially 3D bioprinting approaches, have been successfully used to fabricate bone graft material with distinct compositions.
AUTHOR Murab, Sumit and Gupta, Aastha and Włodarczyk-Biegun, Małgorzata Katarzyna and Kumar, Anuj and van Rijn, Patrick and Whitlock, Patrick and Han, Sung Soo and Agrawal, Garima
Title Alginate based hydrogel inks for 3D bioprinting of engineered orthopedic tissues [Abstract]
Year 2022
Journal/Proceedings Carbohydrate Polymers
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DOI/URL URL DOI
Abstract
3D printed hydrogels have emerged as a novel tissue engineering and regeneration platform due to their ability to provide a suitable environment for cell growth. To obtain a well-defined scaffold with good post-printing shape fidelity, a proper hydrogel ink formulation plays a crucial role. In this regard, alginate has received booming interest owing to its biocompatibility, biodegradability, easy functionalization, and fast gelling behavior. Hence, this review highlights the significance of alginate-based hydrogel inks for fabricating 3D printed scaffolds for bone and cartilage regeneration. Herein, we discuss the fundamentals of direct extrusion 3D bioprinting method and provide a comprehensive overview of various alginate-based hydrogel ink formulations that have been used so far. We also summarize the requirements of hydrogel inks and 3D printed scaffolds to achieve similarity to the native tissue environment. Finally, we discuss the challenges, and research directions relevant for future clinical translation.
AUTHOR Song, Min Jae and Quinn, Russ and Nguyen, Eric and Hampton, Christopher and Sharma, Ruchi and Park, Tea Soon and Koster, Céline and Voss, Ty and Tristan, Carlos and Weber, Claire and Singh, Anju and Dejene, Roba and Bose, Devika and Chen, Yu-Chi and Derr, Paige and Derr, Kristy and Michael, Sam and Barone, Francesca and Chen, Guibin and Boehm, Manfred and Maminishkis, Arvydas and Singec, Ilyas and Ferrer, Marc and Bharti, Kapil
Title Bioprinted 3D outer retina barrier uncovers RPE-dependent choroidal phenotype in advanced macular degeneration [Abstract]
Year 2022
Journal/Proceedings Nature Methods
Reftype Song2022
DOI/URL DOI
Abstract
Age-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retina-barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch’s membrane, and choriocapillaris. The mechanisms of AMD initiation and progression remain poorly understood owing to the lack of physiologically relevant human oBRB models. To this end, we engineered a native-like three-dimensional (3D) oBRB tissue (3D-oBRB) by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB model, a fully-polarized RPE monolayer provides barrier resistance, induces choriocapillaris fenestration, and supports the formation of Bruch’s-membrane-like structure by inducing changes in gene expression in cells of the choroid. Complement activation in the 3D-oBRB triggers dry AMD phenotypes (including subRPE lipid-rich deposits called drusen and choriocapillaris degeneration), and HIF-α stabilization or STAT3 overactivation induce choriocapillaris neovascularization and type-I wet AMD phenotype. The 3D-oBRB provides a physiologically relevant model to studying RPE-choriocapillaris interactions under healthy and diseased conditions.
AUTHOR Pontiggia, Luca and Hengel, Ingmar A.J. Van and Klar, Agnes and Rütsche, Dominic and Nanni, Monica and Scheidegger, Andreas and Figi, Sandro and Reichmann, Ernst and Moehrlen, Ueli and Biedermann, Thomas
Title Bioprinting and plastic compression of large pigmented and vascularized human dermo-epidermal skin substitutes by means of a new robotic platform [Abstract]
Year 2022
Journal/Proceedings Journal of Tissue Engineering
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DOI/URL URL DOI
Abstract
Extensive availability of engineered autologous dermo-epidermal skin substitutes (DESS) with functional and structural properties of normal human skin represents a goal for the treatment of large skin defects such as severe burns. Recently, a clinical phase I trial with this type of DESS was successfully completed, which included patients own keratinocytes and fibroblasts. Yet, two important features of natural skin were missing: pigmentation and vascularization. The first has important physiological and psychological implications for the patient, the second impacts survival and quality of the graft. Additionally, accurate reproduction of large amounts of patient’s skin in an automated way is essential for upscaling DESS production. Therefore, in the present study, we implemented a new robotic unit (called SkinFactory) for 3D bioprinting of pigmented and pre-vascularized DESS using normal human skin derived fibroblasts, blood- and lymphatic endothelial cells, keratinocytes, and melanocytes. We show the feasibility of our approach by demonstrating the viability of all the cells after printing in vitro, the integrity of the reconstituted capillary network in vivo after transplantation to immunodeficient rats and the anastomosis to the vascular plexus of the host. Our work has to be considered as a proof of concept in view of the implementation of an extended platform, which fully automatize the process of skin substitution: this would be a considerable improvement of the treatment of burn victims and patients with severe skin lesions based on patients own skin derived cells.
AUTHOR Saghar Soman, Soja and Govindraj, Mano and Al Hashimi, Noura and Zhou, Jiarui and Vijayavenkataraman, Sanjairaj
Title Bioprinting of Human Neural Tissues Using a Sustainable Marine Tunicate-Derived Bioink for Translational Medicine Applications [Abstract]
Year 2022
Journal/Proceedings International Journal of Bioprinting; Vol 8, No 4 (2022)DO - 10.18063/ijb.v8i4.604
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DOI/URL URL
Abstract
Bioprinting of nervous tissue is a major challenge in the bioprinting field due to its soft consistency and complex architecture. The first step in efficient neural bioprinting is the design and optimization of printable bioinks which favor the growth and differentiation of neural tissues by providing the mechanophysiological properties of the native tissue microenvironment. However, till date, limited studies have been conducted to make tissue specific bioinks. Here, we report a novel bioink formulation specifically designed for bioprinting and differentiation of neural stem cells (NSCs) to peripheral neurons, using a marine tunicate-derived hydrogel and Matrigel. The formulation resulted in seamless bioprinting of NSCs with minimal processing time from bioink preparation to in vitro culture. The tissues exhibited excellent post-printing viability and cell proliferation along with a precise peripheral nerve morphology on in vitro differentiation. The cultured tissues showed significant cell recovery after subjecting to a freeze-thaw cycle of −80 to 37°C, indicating the suitability of the method for developing tissues compatible for long-term storage and transportation for clinical use. The study provides a robust method to use a sustainable bioink for three-dimensional bioprinting of neural tissues for translational medicine applications.
AUTHOR Saghar Soman, Soja and Govindraj, Mano and Al Hashimi, Noura and Zhou, Jiarui and Vijayavenkataraman, Sanjairaj
Title Bioprinting of Human Neural Tissues Using a Sustainable Marine Tunicate-Derived Bioink for Translational Medicine Applications [Abstract]
Year 2022
Journal/Proceedings International Journal of Bioprinting; Vol 8, No 4 (2022)DO - 10.18063/ijb.v8i4.604
Reftype
DOI/URL URL
Abstract
Bioprinting of nervous tissue is a major challenge in the bioprinting field due to its soft consistency and complex architecture. The first step in efficient neural bioprinting is the design and optimization of printable bioinks which favor the growth and differentiation of neural tissues by providing the mechanophysiological properties of the native tissue microenvironment. However, till date, limited studies have been conducted to make tissue specific bioinks. Here, we report a novel bioink formulation specifically designed for bioprinting and differentiation of neural stem cells (NSCs) to peripheral neurons, using a marine tunicate-derived hydrogel and Matrigel. The formulation resulted in seamless bioprinting of NSCs with minimal processing time from bioink preparation to in vitro culture. The tissues exhibited excellent post-printing viability and cell proliferation along with a precise peripheral nerve morphology on in vitro differentiation. The cultured tissues showed significant cell recovery after subjecting to a freeze-thaw cycle of −80 to 37°C, indicating the suitability of the method for developing tissues compatible for long-term storage and transportation for clinical use. The study provides a robust method to use a sustainable bioink for three-dimensional bioprinting of neural tissues for translational medicine applications.
AUTHOR Saghar Soman, Soja and Govindraj, Mano and Al Hashimi, Noura and Zhou, Jiarui and Vijayavenkataraman, Sanjairaj
Title Bioprinting of Human Neural Tissues Using a Sustainable Marine Tunicate-Derived Bioink for Translational Medicine Applications [Abstract]
Year 2022
Journal/Proceedings International Journal of Bioprinting; Vol 8, No 4 (2022)DO - 10.18063/ijb.v8i4.604
Reftype
DOI/URL URL
Abstract
Bioprinting of nervous tissue is a major challenge in the bioprinting field due to its soft consistency and complex architecture. The first step in efficient neural bioprinting is the design and optimization of printable bioinks which favor the growth and differentiation of neural tissues by providing the mechanophysiological properties of the native tissue microenvironment. However, till date, limited studies have been conducted to make tissue specific bioinks. Here, we report a novel bioink formulation specifically designed for bioprinting and differentiation of neural stem cells (NSCs) to peripheral neurons, using a marine tunicate-derived hydrogel and Matrigel. The formulation resulted in seamless bioprinting of NSCs with minimal processing time from bioink preparation to in vitro culture. The tissues exhibited excellent post-printing viability and cell proliferation along with a precise peripheral nerve morphology on in vitro differentiation. The cultured tissues showed significant cell recovery after subjecting to a freeze-thaw cycle of −80 to 37°C, indicating the suitability of the method for developing tissues compatible for long-term storage and transportation for clinical use. The study provides a robust method to use a sustainable bioink for three-dimensional bioprinting of neural tissues for translational medicine applications.
AUTHOR Saghar Soman, Soja and Govindraj, Mano and Al Hashimi, Noura and Zhou, Jiarui and Vijayavenkataraman, Sanjairaj
Title Bioprinting of Human Neural Tissues Using a Sustainable Marine Tunicate-Derived Bioink for Translational Medicine Applications [Abstract]
Year 2022
Journal/Proceedings International Journal of Bioprinting; Vol 8, No 4 (2022)DO - 10.18063/ijb.v8i4.604
Reftype
DOI/URL URL
Abstract
Bioprinting of nervous tissue is a major challenge in the bioprinting field due to its soft consistency and complex architecture. The first step in efficient neural bioprinting is the design and optimization of printable bioinks which favor the growth and differentiation of neural tissues by providing the mechanophysiological properties of the native tissue microenvironment. However, till date, limited studies have been conducted to make tissue specific bioinks. Here, we report a novel bioink formulation specifically designed for bioprinting and differentiation of neural stem cells (NSCs) to peripheral neurons, using a marine tunicate-derived hydrogel and Matrigel. The formulation resulted in seamless bioprinting of NSCs with minimal processing time from bioink preparation to in vitro culture. The tissues exhibited excellent post-printing viability and cell proliferation along with a precise peripheral nerve morphology on in vitro differentiation. The cultured tissues showed significant cell recovery after subjecting to a freeze-thaw cycle of −80 to 37°C, indicating the suitability of the method for developing tissues compatible for long-term storage and transportation for clinical use. The study provides a robust method to use a sustainable bioink for three-dimensional bioprinting of neural tissues for translational medicine applications.
AUTHOR Daskalakis, Evangelos and Huang, Boyang and Vyas, Cian and Acar, Anil A. and Liu, Fengyuan and Fallah, Ali and Cooper, Glen and Weightman, Andrew and Blunn, Gordon and Koç, Bahattin and Bartolo, Paulo
Title Bone Bricks: The Effect of Architecture and Material Composition on the Mechanical and Biological Performance of Bone Scaffolds [Abstract]
Year 2022
Journal/Proceedings ACS Omega
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DOI/URL DOI
Abstract
Large bone loss injuries require high-performance scaffolds with an architecture and material composition resembling native bone. However, most bone scaffold studies focus on three-dimensional (3D) structures with simple rectangular or circular geometries and uniform pores, not able to recapitulate the geometric characteristics of the native tissue. This paper addresses this limitation by proposing novel anatomically designed scaffolds (bone bricks) with nonuniform pore dimensions (pore size gradients) designed based on new lay-dawn pattern strategies. The gradient design allows one to tailor the properties of the bricks and together with the incorporation of ceramic materials allows one to obtain structures with high mechanical properties (higher than reported in the literature for the same material composition) and improved biological characteristics.
AUTHOR Fl{'{e}}geau, Killian and Puiggali-Jou, Anna and Zenobi-Wong, Marcy
Title Cartilage tissue engineering by extrusion bioprinting utilizing porous hyaluronic acid microgel bioinks [Abstract]
Year 2022
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
3D bioprinting offers an excellent opportunity to provide tissue-engineered cartilage to microtia patients. However, hydrogel-based bioinks are hindered by their dense and cell-restrictive environment, impairing tissue development and ultimately leading to mechanical failure of large scaffolds in vivo. Granular hydrogels, made of annealed microgels, offer a superior alternative to conventional bioinks, with their improved porosity and modularity. We have evaluated the ability of enzymatically crosslinked hyaluronic acid (HA) microgel bioinks to form mature cartilage in vivo. Microgel bioinks were formed by mechanically sizing bulk HA-tyramine hydrogels through meshes with aperture diameters of 40, 100 or 500 µm. Annealing of the microgels was achieved by crosslinking residual tyramines. Secondary crosslinked scaffolds were stable in solution and showed tunable porosity from 9% to 21%. Bioinks showed excellent rheological properties and were used to print different objects. Printing precision was found to be directly correlated to microgel size. As a proof of concept, freeform reversible embedding of suspended hydrogels printing with gelation triggered directly in the bath was performed to demonstrate the versatility of the method. The granular hydrogels support the homogeneous development of mature cartilage-like tissues in vitro with mechanical stiffening up to 200 kPa after 63 d. After 6 weeks of in vivo implantation, small-diameter microgels formed stable constructs with low immunogenicity and continuous tissue maturation. Conversely, increasing the microgel size resulted in increased inflammatory response, with limited stability in vivo. This study reports the development of new microgel bioinks for cartilage tissue biofabrication and offers insights into the foreign body reaction towards porous scaffolds implantation.
AUTHOR Cao, Chuanliang and Huang, Pengren and Prasopthum, Aruna and Parsons, Andrew J. and Ai, Fanrong and Yang, Jing
Title Characterisation of bone regeneration in 3D printed ductile PCL/PEG/hydroxyapatite scaffolds with high ceramic microparticle concentrations [Abstract]
Year 2022
Journal/Proceedings Biomater. Sci.
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DOI/URL DOI
Abstract
3D printed bioactive glass or bioceramic particle reinforced composite scaffolds for bone tissue engineering currently suffer from low particle concentration (100% breaking strain) by adding poly(ethylene glycol) which is biocompatible and FDA approved. The scaffolds require no post-printing washing to remove hazardous components. More exposure of HA microparticles on strut surfaces is enabled by incorporating higher HA concentrations. Compared to scaffolds with 72 wt% HA{,} scaffolds with higher HA content (90 wt%) enhance matrix formation but not new bone volume after 12 weeks implantation in rat calvarial defects. Histological analyses demonstrate that bone regeneration within the 3D printed scaffolds is via intramembranous ossification and starts in the central region of pores. Fibrous tissue that resembles non-union tissue within bone fractures is formed within pores that do not have new bone. The amount of blood vessels is similar between scaffolds with mainly fibrous tissue and those with more bone tissue{,} suggesting vascularization is not a deciding factor for determining the type of tissues regenerated within the pores of 3D printed scaffolds. Multinucleated immune cells are commonly present in all scaffolds surrounding the struts{,} suggesting a role of managing inflammation in bone regeneration within 3D printed scaffolds.
AUTHOR Monaco, Graziana and Qawasmi, Feras and El Haj, Alicia J. and Forsyth, Nicolas R. and Stoddart, Martin J.
Title Chondrogenic differentiation of human bone marrow MSCs in osteochondral implants under kinematic mechanical load is dependent on the underlying osteo component [Abstract]
Year 2022
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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DOI/URL DOI
Abstract
Chondrogenic models utilizing human mesenchymal stromal cells (hMSCs) are often simplistic, with a single cell type and the absence of mechanical stimulation. Considering the articulating joint as an organ it would be beneficial to include more complex stimulation. Within this study we applied clinically relevant kinematic load to biphasic constructs. In each case, the upper layer consisted of fibrin embedded hMSCs retained within an elastomeric polyurethane (PU) scaffold. These were randomly assigned to five base scaffolds, a cell-free fibrin PU base, viable bone, decellularized bone, 3D printed calcium phosphate or clinically used cement. This allowed the study of cross talk between viable bone and chondrogenically differentiating MSCs, while controlling for the change in stiffness of the base material. Data obtained showed that the bulk stiffness of the construct was not the defining factor in the response obtained, with viable and decellularized bone producing similar results to the softer PU base. However, the stiff synthetic materials led to reduced chondrogenesis and increased calcification in the upper MSC seeded layer. This demonstrates that the underlying base material must be considered when driving chondrogenesis of human cells using a clinically relevant loading protocol. It also indicates that the material used for bony reconstruction of osteochondral defects may influence subsequent chondrogenic potential.
AUTHOR Clua-Ferré, Laura and de Chiara, Francesco and Rodríguez-Comas, Júlia and Comelles, Jordi and Martinez, Elena and Godeau, Amelie Luise and García-Alamán, Ainhoa and Gasa, Rosa and Ramón-Azcón, Javier
Title Collagen-Tannic Acid Spheroids for β-Cell Encapsulation Fabricated Using a 3D Bioprinter [Abstract]
Year 2022
Journal/Proceedings Advanced Materials Technologies
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DOI/URL DOI
Abstract
Abstract Type 1 Diabetes results from autoimmune response elicited against β-cell antigens. Nowadays, insulin injections remain the leading therapeutic option. However, injection treatment fails to emulate the highly dynamic insulin release that β-cells provide. 3D cell-laden microspheres have been proposed during the last years as a major platform for bioengineering insulin-secreting constructs for tissue graft implantation and a model for in vitro drug screening platforms. Current microsphere fabrication technologies have several drawbacks: the need for an oil phase containing surfactants, diameter inconsistency of the microspheres, and high time-consuming processes. These technologies have widely used alginate for its rapid gelation, high processability, and low cost. However, its low biocompatible properties do not provide effective cell attachment. This study proposes a high-throughput methodology using a 3D bioprinter that employs an ECM-like microenvironment for effective cell-laden microsphere production to overcome these limitations. Crosslinking the resulting microspheres with tannic acid prevents collagenase degradation and enhances spherical structural consistency while allowing the diffusion of nutrients and oxygen. The approach allows customization of microsphere diameter with extremely low variability. In conclusion, a novel bio-printing procedure is developed to fabricate large amounts of reproducible microspheres capable of secreting insulin in response to extracellular glucose stimuli.
AUTHOR Man, Kenny and Barroso, Inês A. and Brunet, Mathieu Y. and Peacock, Ben and Federici, Angelica S. and Hoey, David A. and Cox, Sophie C.
Title Controlled Release of Epigenetically-Enhanced Extracellular Vesicles from a GelMA/Nanoclay Composite Hydrogel to Promote Bone Repair [Abstract]
Year 2022
Journal/Proceedings International Journal of Molecular Sciences
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DOI/URL URL DOI
Abstract
Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Although EVs’ potential for bone regeneration has been shown, issues associated with their therapeutic potency and short half-life in vivo hinders their clinical utility. Epigenetic reprogramming with the histone deacetylase inhibitor Trichostatin A (TSA) has been reported to promote the osteoinductive potency of osteoblast-derived EVs. Gelatin methacryloyl (GelMA) hydrogels functionalised with the synthetic nanoclay laponite (LAP) have been shown to effectively bind, stabilise, and improve the retention of bioactive factors. This study investigated the potential of utilising a GelMA-LAP hydrogel to improve local retention and control delivery of epigenetically enhanced osteoblast-derived EVs as a novel bone repair strategy. LAP was found to elicit a dose-dependent increase in GelMA compressive modulus and shear-thinning properties. Incorporation of the nanoclay was also found to enhance shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP displayed increased mineralisation capacity (1.41-fold) (p ≤ 0.01) over 14 days. EV release kinetics from these nanocomposite systems were also strongly influenced by LAP concentration with significantly more vesicles being released from GelMA constructs as detected by a CD63 ELISA (p ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), histone acetylation (1.32-fold) and mineralisation (1.87-fold) of human bone marrow stromal cells (hBMSCs) when released from the GelMA-LAP hydrogel compared to the untreated EV gels (p ≤ 0.01). Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs (p ≤ 0.001). Taken together, these findings demonstrate the potential of combining epigenetically enhanced osteoblast-derived EVs with a nanocomposite photocurable hydrogel to promote the therapeutic efficacy of acellular vesicle approaches for bone regeneration.
AUTHOR Wei, Shan and Zhang, Ren-Gang and Wang, Zheng-Yu
Title Deferoxamine/magnesium modified β-tricalcium phosphate promotes the bone regeneration in osteoporotic rats [Abstract]
Year 2022
Journal/Proceedings Journal of Biomaterials Applications
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DOI/URL DOI
Abstract
Recently, Deferoxamine (DFO) and magnesium (Mg) have been identified as critical factors for angiogenesis and bone formation. However, in current research studies, there is a lack of focus on whether DFO plus Mg can affect the regeneration of β-tricalcium phosphate (β-TCP) in osteoporosis and through what biological mechanisms. Therefore, the present work was aimed to preparation and evaluate the effect of Deferoxamine/magnesium modified β-tricalcium phosphate promotes (DFO/Mg-TCP) in ovariectomized rats model and preliminary exploration of possible mechanisms. The MC3T3-E1 cells were co-cultured with the exudate of DFO/Mg-TCP and induced to osteogenesis, and the cell viability, osteogenic activity were observed by Cell Counting Kit-8(CCK-8), Alkaline Phosphatase (ALP) staining, Alizarin Red Staining (RES) and Western Blot. In vitro experiments, CCK-8, ALP and ARS staining results show that the mineralization and osteogenic activity of MC3T3-E1increased significantly after intervention by DFO/Mg-TCP, as well as a higher levels of protein expressions including VEGF, OC, Runx-2 and HIF-1α. In vivo experiment, Micro-CT and Histological analysis evaluation show that DFO/Mg-TCP treatment presented the stronger effect on bone regeneration, bone mineralization and biomaterial degradation, when compared with OVX+Mg-TCP group and OVX+TCP group, as well as a higher VEGF, OC, Runx-2 and HIF-1α gene expression. The present study indicates that treatment with DFO/Mg-TCP was associated with increased regeneration by enhancing the function of osteoblasts in an OVX rat.
AUTHOR Cadle, Rachel and Rogozea, Dan and Moldovan, Leni and Moldovan, Nicanor I.
Title Design and Implementation of Anatomically Inspired Mesenteric and Intestinal Vascular Patterns for Personalized 3D Bioprinting [Abstract]
Year 2022
Journal/Proceedings Applied Sciences
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DOI/URL URL DOI
Abstract
Recent progress in bioprinting has made possible the creation of complex 3D intestinal constructs, including vascularized villi. However, for their integration into functional units useful for experimentation or implantation, the next challenge is to endow them with a larger-scale, anatomically realistic vasculature. In general, the perfusion of bioprinted constructs has remained difficult, and the current solution is to provide them with mostly linear and simply branched channels. To address this limitation, here we demonstrated an image analysis-based workflow leading through computer-assisted design from anatomic images of rodent mesentery and colon to the actual printing of such patterns with paste and hydrogel bioinks. Moreover, we reverse-engineered the 2D intestinal image-derived designs into cylindrical objects, and 3D-printed them in a support hydrogel. These results open the path towards generation of more realistically vascularized tissue constructs for a variety of personalized medicine applications.
AUTHOR Cadle, Rachel and Rogozea, Dan and Moldovan, Leni and Moldovan, Nicanor I.
Title Design and Implementation of Anatomically Inspired Mesenteric and Intestinal Vascular Patterns for Personalized 3D Bioprinting [Abstract]
Year 2022
Journal/Proceedings Applied Sciences
Reftype
DOI/URL URL DOI
Abstract
Recent progress in bioprinting has made possible the creation of complex 3D intestinal constructs, including vascularized villi. However, for their integration into functional units useful for experimentation or implantation, the next challenge is to endow them with a larger-scale, anatomically realistic vasculature. In general, the perfusion of bioprinted constructs has remained difficult, and the current solution is to provide them with mostly linear and simply branched channels. To address this limitation, here we demonstrated an image analysis-based workflow leading through computer-assisted design from anatomic images of rodent mesentery and colon to the actual printing of such patterns with paste and hydrogel bioinks. Moreover, we reverse-engineered the 2D intestinal image-derived designs into cylindrical objects, and 3D-printed them in a support hydrogel. These results open the path towards generation of more realistically vascularized tissue constructs for a variety of personalized medicine applications.
AUTHOR Cadle, Rachel and Rogozea, Dan and Moldovan, Leni and Moldovan, Nicanor I.
Title Design and Implementation of Anatomically Inspired Mesenteric and Intestinal Vascular Patterns for Personalized 3D Bioprinting [Abstract]
Year 2022
Journal/Proceedings Applied Sciences
Reftype
DOI/URL URL DOI
Abstract
Recent progress in bioprinting has made possible the creation of complex 3D intestinal constructs, including vascularized villi. However, for their integration into functional units useful for experimentation or implantation, the next challenge is to endow them with a larger-scale, anatomically realistic vasculature. In general, the perfusion of bioprinted constructs has remained difficult, and the current solution is to provide them with mostly linear and simply branched channels. To address this limitation, here we demonstrated an image analysis-based workflow leading through computer-assisted design from anatomic images of rodent mesentery and colon to the actual printing of such patterns with paste and hydrogel bioinks. Moreover, we reverse-engineered the 2D intestinal image-derived designs into cylindrical objects, and 3D-printed them in a support hydrogel. These results open the path towards generation of more realistically vascularized tissue constructs for a variety of personalized medicine applications.
AUTHOR Curti, Filis and Serafim, Andrada and Olaret, Elena and Dinescu, Sorina and Samoila, Iuliana and Vasile, Bogdan Stefan and Iovu, Horia and Lungu, Adriana and Stancu, Izabela Cristina and Marinescu, Rodica
Title Development of Biocomposite Alginate-Cuttlebone-Gelatin 3D Printing Inks Designed for Scaffolds with Bone Regeneration Potential [Abstract]
Year 2022
Journal/Proceedings Marine Drugs
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DOI/URL URL DOI
Abstract
Fabrication of three-dimensional (3D) scaffolds using natural biomaterials introduces valuable opportunities in bone tissue reconstruction and regeneration. The current study aimed at the development of paste-like 3D printing inks with an extracellular matrix-inspired formulation based on marine materials: sodium alginate (SA), cuttlebone (CB), and fish gelatin (FG). Macroporous scaffolds with microporous biocomposite filaments were obtained by 3D printing combined with post-printing crosslinking. CB fragments were used for their potential to stimulate biomineralization. Alginate enhanced CB embedding within the polymer matrix as confirmed by scanning electron microscopy (ESEM) and micro-computer tomography (micro-CT) and improved the deformation under controlled compression as revealed by micro-CT. SA addition resulted in a modulation of the bulk and surface mechanical behavior, and lead to more elongated cell morphology as imaged by confocal microscopy and ESEM after the adhesion of MC3T3-E1 preosteoblasts at 48 h. Formation of a new mineral phase was detected on the scaffold’s surface after cell cultures. All the results were correlated with the scaffolds’ compositions. Overall, the study reveals the potential of the marine materials-containing inks to deliver 3D scaffolds with potential for bone regeneration applications.
AUTHOR Ramakrishnan, Rashmi and Kasoju, Naresh and Raju, Riya and Geevarghese, Rency and Gauthaman, Ashna and Bhatt, Anugya
Title Exploring the Potential of Alginate-Gelatin-Diethylaminoethyl Cellulose-Fibrinogen based Bioink for 3D Bioprinting of Skin Tissue Constructs [Abstract]
Year 2022
Journal/Proceedings Carbohydrate Polymer Technologies and Applications
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DOI/URL URL DOI
Abstract
Designing printable bioinks for 3D bioprinting capable of supporting cellular viability with post-printing functionality remains challenging. Native ECM offers several physical, chemical, and biological cues that are difficult to restore using only a single component. Herein, we have optimized a multicomponent-based bioink formulation comprising alginate (ALG), gelatin (GEL), diethylaminoethyl cellulose (DCEL) and fibrinogen (FIB), termed as ALG-GEL-DCEL-FIB bioink for potential application in bioprinting and biofabrication of skin tissue equivalents. The designed formulation was extensively studied for its printability, physico-chemical, rheological, and biocompatibility properties. Excellent printability, shape fidelity and cell-laden tissue equivalent printing were established using the RegenHu 3D Discovery Bioprinter. The human primary fibroblast and keratinocyte-laden bioprinted constructs exhibited good cell viability. Long term culture of 4 weeks comprising 5 days of air-liquid-interphase followed by 21 days of submerged culture produced biomimetic tissue histology in the ALG-GEL-DCEL-FIB bioink printed constructs. Specific epidermal-dermal marker expressions proving functionality were evident in immunohistochemical, biochemical and gene expression analysis. The ALG-GEL-DCEL-FIB bioink may be explored further for potential biofabrication and therapeutic applications.
AUTHOR Lee, Hanna and Kim, Soon Hee and Lee, Ji Seung and Lee, Young Jin and Lee, Ok Joo and Ajiteru, Olatunji and Sultan, Md Tipu and Lee, Suk Woo and Park, Chan Hum
Title Functional Skeletal Muscle Regeneration Using Muscle Mimetic Tissue Fabricated by Microvalve-Assisted Coaxial 3D Bioprinting [Abstract]
Year 2022
Journal/Proceedings Advanced Healthcare Materials
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Abstract 3D-printed artificial skeletal muscle, which mimics the structural and functional characteristics of native skeletal muscle, is a promising treatment method for muscle reconstruction. Although various fabrication techniques for skeletal muscle using 3D bio-printers are studied, it is still challenging to build a functional muscle structure. A strategy using microvalve-assisted coaxial 3D bioprinting in consideration of functional skeletal muscle fabrication is reported. The unit (artificial muscle fascicle: AMF) of muscle mimetic tissue is composed of a core filled with medium-based C2C12 myoblast aggregates as a role of muscle fibers and a photo cross-linkable hydrogel-based shell as a role of connective tissue in muscles that enhances printability and cell adhesion and proliferation. Especially, a microvalve system is applied for the core part with even cell distribution and strong cell–cell interaction. This system enhances myotube formation and consequently shows spontaneous contraction. A multi-printed AMF (artificial muscle tissue: AMT) as a piece of muscle is implanted into the anterior tibia (TA) muscle defect site of immunocompromised rats. As a result, the TA-implanted AMT responds to electrical stimulation and represents histologically regenerated muscle tissue. This microvalve-assisted coaxial 3D bioprinting shows a significant step forward to mimicking native skeletal muscle tissue.
AUTHOR Li, Ming-Chia and Chang, Pu-Yuan and Luo, Huai-Rou and Chang, Ling-Yuan and Lin, Chuan-Yi and Yang, Chih-Yu and Lee, Oscar Kuang-Sheng and Lee, Yan-Hwa Wu and Tarng, Der-Cherng
Title Functioning tailor-made 3D-printed vascular graft for hemodialysis [Abstract]
Year 2022
Journal/Proceedings The Journal of Vascular Access
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Abstract
Background:The two ends of arteriovenous graft (AVG) are anastomosed to the upper limb vessels by surgery for hemodialysis therapy. However, the size of upper limb vessels varies to a large extent among different individuals.Methods:According to the shape and size of neck vessels quantified from the preoperative computed tomography angiographic scan, the ethylene-vinyl acetate (EVA)-based AVG was produced in H-shape by the three-dimensional (3D) printer and then sterilized. This study investigated the function of this novel 3D-printed AVG in vitro and in vivo.Results:This 3D-printed AVG can be implanted in the rabbit’s common carotid artery and common jugular vein with ease and functions in vivo. The surgical procedure was quick, and no suture was required. The blood loss was minimal, and no hematoma was noted at least 1 week after the surgery. The blood flow velocity within the implanted AVG was 14.9 ± 3.7 cm/s. Additionally, the in vitro characterization experiments demonstrated that this EVA-based biomaterial is biocompatible and possesses a superior recovery property than ePTFE after hemodialysis needle cannulation.Conclusions:Through the 3D printing technology, the EVA-based AVG can be tailor-made to fit the specific vessel size. This kind of 3D-printed AVG is functioning in vivo, and our results realize personalized vascular implants. Further large-animal studies are warranted to examine the long-term patency.
AUTHOR Mota, Fabio Batista and Braga, Luiza Amara Maciel and Cabral, Bernardo Pereira and Filho, Carlos Gilbert Conte
Title Future of Bioprinted Tissues аnd Organs: A Two-Wave Global Survey [Abstract]
Year 2022
Journal/Proceedings Foresight and STI Governance
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Technologies of 3D and 4D bioprinting make it possible to restore or replace tissues and organs, solving the problem of the lack of donor resources and reducing the risks of implant rejection. This article presents the results of a two-stage global survey of specialists in tissue engineering on the prospects of bioprinting in preclinical studies and clinical practice. A picture of possible tracks and horizons upon which the implementation of the considered solutions is possible is presented. According to the results of the survey, in the next two decades it will be possible to recreate tissues and organs suitable for implantation and drug testing. There will be a market for bioprinted products, the problem of organ shortages and adverse reactions to drugs will be solved. These changes may significantly affect not only the practice of biomedical research, drug testing, and medicine, but also the healthcare sector in general, which implies the need for a preventive review of current policies. A practical and accessible tool for identifying and interviewing a large number of experts around the world is proposed, which may be useful for new Foresight studies.
AUTHOR Mota, Fabio Batista and Braga, Luiza Amara Maciel and Cabral, Bernardo Pereira and Filho, Carlos Gilbert Conte
Title Future of Bioprinted Tissues аnd Organs: A Two-Wave Global Survey [Abstract]
Year 2022
Journal/Proceedings Foresight and STI Governance
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DOI/URL URL
Abstract
Technologies of 3D and 4D bioprinting make it possible to restore or replace tissues and organs, solving the problem of the lack of donor resources and reducing the risks of implant rejection. This article presents the results of a two-stage global survey of specialists in tissue engineering on the prospects of bioprinting in preclinical studies and clinical practice. A picture of possible tracks and horizons upon which the implementation of the considered solutions is possible is presented. According to the results of the survey, in the next two decades it will be possible to recreate tissues and organs suitable for implantation and drug testing. There will be a market for bioprinted products, the problem of organ shortages and adverse reactions to drugs will be solved. These changes may significantly affect not only the practice of biomedical research, drug testing, and medicine, but also the healthcare sector in general, which implies the need for a preventive review of current policies. A practical and accessible tool for identifying and interviewing a large number of experts around the world is proposed, which may be useful for new Foresight studies.
AUTHOR Mota, Fabio Batista and Braga, Luiza Amara Maciel and Cabral, Bernardo Pereira and Filho, Carlos Gilbert Conte
Title Future of Bioprinted Tissues аnd Organs: A Two-Wave Global Survey [Abstract]
Year 2022
Journal/Proceedings Foresight and STI Governance
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DOI/URL URL
Abstract
Technologies of 3D and 4D bioprinting make it possible to restore or replace tissues and organs, solving the problem of the lack of donor resources and reducing the risks of implant rejection. This article presents the results of a two-stage global survey of specialists in tissue engineering on the prospects of bioprinting in preclinical studies and clinical practice. A picture of possible tracks and horizons upon which the implementation of the considered solutions is possible is presented. According to the results of the survey, in the next two decades it will be possible to recreate tissues and organs suitable for implantation and drug testing. There will be a market for bioprinted products, the problem of organ shortages and adverse reactions to drugs will be solved. These changes may significantly affect not only the practice of biomedical research, drug testing, and medicine, but also the healthcare sector in general, which implies the need for a preventive review of current policies. A practical and accessible tool for identifying and interviewing a large number of experts around the world is proposed, which may be useful for new Foresight studies.
AUTHOR Yan Li and Lijing Huang and Guangpin Tai and Feifei Yan and Lin Cai and Chenxing Xin and Shamoon {Al Islam}
Title Graphene Oxide-loaded magnetic nanoparticles within 3D hydrogel form High-performance scaffolds for bone regeneration and tumour treatment [Abstract]
Year 2022
Journal/Proceedings Composites Part A: Applied Science and Manufacturing
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The treatment of tumour-related bone defects should ideally combine bone regeneration with tumour treatment. Additive manufacturing (AM) could feasibly place functional bone-repair materials within composite materials with functional-grade structures, giving them bone repair and anti-tumour effects. Magnetothermal therapy is a promising non-invasive method of tumour treatment that has attracted increasing attention. In this study, we prepared novel hydrogel composite scaffolds of polyvinyl alcohol/sodium alginate/hydroxyapatite (PVA/SA/HA) at low temperature via AM. The scaffolds were loaded with various concentrations of magnetic graphene oxide (MGO) @Fe3O4 nanoparticles. The scaffolds were characterised by fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and thermal gravimetric analysis (TGA), which showed that the scaffolds have good moulding qualities and strong hydrogen bonding between the MGO/PVA/SA/HA components. TGA analysis demonstrated the expected thermal stability of the MGO and scaffolds. Thermal effects can be adjusted by varying the contents of MGO and the strength of an external alternating magnetic field. The prepared MGO hydrogel composite scaffolds enhance biological functions and support bone mesenchymal stem cell differentiation in vitro. The scaffolds also show favourable anti-tumour characteristics with effective magnetothermal conversion in vivo.
AUTHOR Bedell, Matthew L. and Torres, Angelica L. and Hogan, Katie J. and Wang, Ziwen and Wang, Bonnie and Melchiorri, Anthony J. and Grande-Allen, K. Jane and Mikos, Antonios G.
Title Human gelatin-based composite hydrogels for osteochondral tissue engineering and their adaptation into bioinks for extrusion, inkjet, and digital light processing bioprinting [Abstract]
Year 2022
Journal/Proceedings Biofabrication
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The investigation of novel hydrogel systems allows for the study of relationships between biomaterials, cells, and other factors within osteochondral tissue engineering. Three-dimensional (3D) printing is a popular research method that can allow for further interrogation of these questions via the fabrication of 3D hydrogel environments that mimic tissue-specific, complex architectures. However, the adaptation of promising hydrogel biomaterial systems into 3D-printable bioinks remains a challenge. Here, we delineated an approach to that process. First, we characterized a novel methacryloylated gelatin composite hydrogel system and assessed how calcium phosphate and glycosaminoglycan additives upregulated bone- and cartilage-like matrix deposition and certain genetic markers of differentiation within human mesenchymal stem cells (hMSCs), such as RUNX2 and SOX9. Then, new assays were developed and utilized to study the effects of xanthan gum and nanofibrillated cellulose, which allowed for cohesive fiber deposition, reliable droplet formation, and non-fracturing digital light processing (DLP)-printed constructs within extrusion, inkjet, and DLP techniques, respectively. Finally, these bioinks were used to 3D print constructs containing viable encapsulated hMSCs over a 7 d period, where DLP printed constructs facilitated the highest observed increase in cell number over 7 d (∼2.4×). The results presented here describe the promotion of osteochondral phenotypes via these novel composite hydrogel formulations, establish their ability to bioprint viable, cell-encapsulating constructs using three different 3D printing methods on multiple bioprinters, and document how a library of modular bioink additives affected those physicochemical properties important to printability.
AUTHOR Liu, Fengyuan and Quan, Rixiang and Vyas, Cian and Aslan, Enes
Title Hybrid biomanufacturing systems applied in tissue regeneration [Abstract]
Year 2022
Journal/Proceedings International Journal of Bioprinting; Vol 9, No 1 (2023)DO - 10.18063/ijb.v9i1.646
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Scaffold-based approach is a developed strategy in biomanufacturing, which is based on the use of temporary scaffold that performs as a house of implanted cells for their attachment, proliferation, and differentiation. This strategy strongly depends on both materials and manufacturing processes. However, it is very difficult to meet all the requirements, such as biocompatibility, biodegradability, mechanical strength, and promotion of cell-adhesion, using only single material. At present, no single bioprinting technique can meet the requirements for tissue regeneration of all scales. Thus, multi-material and mixing-material scaffolds have been widely investigated. Challenges in terms of resolution, uniform cell distribution, and tissue formation are still the obstacles in the development of bioprinting technique. Hybrid bioprinting techniques have been developed to print scaffolds with improved properties in both mechanical and biological aspects for broad biomedical engineering applications. In this review, we introduce the basic multi-head bioprinters, semi-hybrid and fully-hybrid biomanufacturing systems, highlighting the modifications, the improved properties and the effect on the complex tissue regeneration applications.
AUTHOR Girardeau-Hubert, Sarah and Lynch, Barbara and Zuttion, Francesca and Label, Rabab and Rayee, Chrystelle and Brizion, Sébastien and Ricois, Sylvie and Martinez, Anthony and Park, Eunhye and Kim, Changhwan and Marinho, Paulo André and Shim, Jin-Hyung and Jin, Songwan and Rielland, Maïté and Soeur, Jérémie
Title Impact of microstructure on cell behavior and tissue mechanics in collagen and dermal decellularized extra-cellular matrices [Abstract]
Year 2022
Journal/Proceedings Acta Biomaterialia
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Skin models are used for many applications such as research and development or grafting. Unfortunately, most lack a proper microenvironment producing poor mechanical properties and inaccurate extra-cellular matrix composition and organization. In this report we focused on mechanical properties, extra-cellular matrix organization and cell interactions in human skin samples reconstructed with pure collagen or dermal decellularized extra-cellular matrices (S-dECM) and compared them to native human skin. We found that Full-thickness S-dECM samples presented stiffness two times higher than collagen gel and similar to ex vivo human skin, and proved for the first time that keratinocytes also impact dermal mechanical properties. This was correlated with larger fibers in S-dECM matrices compared to collagen samples and with a differential expression of F-actin, vinculin and tenascin C between S-dECM and collagen samples. This is clear proof of the microenvironment's impact on cell behaviors and mechanical properties. Statement of significance In vitro skin models have been used for a long time for clinical applications or in vitro knowledge and evaluation studies. However, most lack a proper microenvironment producing a poor combination of mechanical properties and appropriate biological outcomes, partly due to inaccurate extra-cellular matrix (ECM) composition and organization. This can lead to limited predictivity and weakness of skin substitutes after grafting. This study shows, for the first time, the importance of a complex and rich microenvironment on cell behaviors, matrix macro- and micro-organization and mechanical properties. The increased composition and organization complexity of dermal skin decellularized extra-cellular matrix populated with differentiated cells produces in vitro skin models closer to native human skin physiology.
AUTHOR Helaehil, Júlia Venturini and Lourenço, Carina Basqueira and Huang, Boyang and Helaehil, Luiza Venturini and de Camargo, Isaque Xavier and Chiarotto, Gabriela Bortolança and Santamaria-Jr, Milton and Bártolo, Paulo and Caetano, Guilherme Ferreira
Title In Vivo Investigation of Polymer-Ceramic PCL/HA and PCL/β-TCP 3D Composite Scaffolds and Electrical Stimulation for Bone Regeneration [Abstract]
Year 2022
Journal/Proceedings Polymers
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Critical bone defects are a major clinical challenge in reconstructive bone surgery. Polycaprolactone (PCL) mixed with bioceramics, such as hydroxyapatite (HA) and tricalcium phosphate (TCP), create composite scaffolds with improved biological recognition and bioactivity. Electrical stimulation (ES) aims to compensate the compromised endogenous electrical signals and to stimulate cell proliferation and differentiation. We investigated the effects of composite scaffolds (PCL with HA; and PCL with β-TCP) and the use of ES on critical bone defects in Wistar rats using eight experimental groups: untreated, ES, PCL, PCL/ES, HA, HA/ES, TCP, and TCP/ES. The investigation was based on histomorphometry, immunohistochemistry, and gene expression analysis. The vascular area was greater in the HA/ES group on days 30 and 60. Tissue mineralization was greater in the HA, HA/ES, and TCP groups at day 30, and TCP/ES at day 60. Bmp-2 gene expression was higher in the HA, TCP, and TCP/ES groups at day 30, and in the TCP/ES and PCL/ES groups at day 60. Runx-2, Osterix, and Osteopontin gene expression were also higher in the TCP/ES group at day 60. These results suggest that scaffolds printed with PCL and TCP, when paired with electrical therapy application, improve bone regeneration.
AUTHOR Lai, Jiahui and Wang, Chong and Liu, Jia and Chen, Shangsi and Liu, Chaoyu and Huang, Xiangxuan and Wu, Jing and Pan, Yue and Xie, Yuancai and Wang, Min
Title Low temperature hybrid 3D printing of hierarchically porous bone tissue engineering scaffolds with in situ delivery of osteogenic peptide and mesenchymal stem cells [Abstract]
Year 2022
Journal/Proceedings Biofabrication
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Compared to other conventional scaffold fabrication techniques, three-dimensional (3D) printing is advantageous in producing bone tissue engineering scaffolds with customized shape, tailored pore size/porosity, required mechanical properties and even desirable biomolecule delivery capability. However, for scaffolds with a large volume, it is highly difficult to get seeded cells to migrate to the central region of the scaffolds, resulting in an inhomogeneous cell distribution and therefore lowering the bone forming ability. To overcome this major obstacle, in this study, cell-laden bone tissue engineering scaffolds consisting of osteogenic peptide (OP) loaded β-tricalcium phosphate (TCP)/poly(lactic-co-glycolic acid) (PLGA) (OP/TCP/PLGA, designated as OTP) nanocomposite struts and rat bone marrow derived mesenchymal stem cell (rBMSC)-laden gelatin/GelMA hydrogel rods were produced through ‘dual-nozzle’ low temperature hybrid 3D printing. The cell-laden scaffolds exhibited a bi-phasic structure and had a mechanical modulus of about 19.6 MPa, which was similar to that of human cancellous bone. OP can be released from the hybrid scaffolds in a sustained manner and achieved a cumulative release level of about 78% after 24 d. rBMSCs encapsulated in the hydrogel rods exhibited a cell viability of about 87.4% right after low temperature hybrid 3D printing and could be released from the hydrogel rods to achieve cell anchorage on the surface of adjacent OTP struts. The OP released from OTP struts enhanced rBMSCs proliferation. Compared to rBMSC-laden hybrid scaffolds without OP incorporation, the rBMSC-laden hybrid scaffolds incorporated with OP significantly up-regulated osteogenic differentiation of rBMSCs by showing a higher level of alkaline phosphatase expression and calcium deposition. This ‘proof-of-concept’ study has provided a facile method to form cell-laden bone tissue engineering scaffolds with not only required mechanical strength, biomimetic structure and sustained biomolecule release profile but also excellent cell delivery capability with uniform cell distribution, which can improve the bone forming ability in the body.
AUTHOR Włodarczyk-Biegun, Małgorzata K. and Villiou, Maria and Koch, Marcus and Muth, Christina and Wang, Peixi and Ott, Jenna and del Campo, Aranzazu
Title Melt Electrowriting of Graded Porous Scaffolds to Mimic the Matrix Structure of the Human Trabecular Meshwork [Abstract]
Year 2022
Journal/Proceedings ACS Biomaterials Science & Engineering
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The permeability of the human trabecular meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases such as glaucoma. Engineered HTMs could help to understand the structure–function relation in natural tissues and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125–500 μm and fiber diameters of 10–12 μm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa and a static compression modulus in the range of 6–360 kPa are obtained by varying the scaffold design, that is, the density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8–14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW for reconstructing complex morphological features of natural tissues.
AUTHOR Bucciarelli, Alessio and Petretta, Mauro and Grigolo, Brunella and Gambari, Laura and Bossi, Alessandra Maria and Grassi, Francesco and Maniglio, Devid
Title Methacrylated Silk Fibroin Additive Manufacturing of Shape Memory Constructs with Possible Application in Bone Regeneration [Abstract]
Year 2022
Journal/Proceedings Gels
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Methacrylated silk (Sil-MA) is a chemically modified silk fibroin specifically designed to be crosslinkable under UV light, which makes this material applicable in additive manufacturing techniques and allows the prototyping and development of patient-specific 2D or 3D constructs. In this study, we produced a thin grid structure based on crosslinked Sil-MA that can be withdrawn and ejected and that can recover its shape after rehydration. A complete chemical and physical characterization of Sil-MA was first conducted. Additionally, we tested Sil-MA biocompatibility according to the International Standard Organization protocols (ISO 10993) ensuring the possibility of using it in future trials. Sil-MA was also tested to verify its ability to support osteogenesis. Overall, Sil-MA was shown to be biocompatible and osteoconductive. Finally, two different additive manufacturing technologies, a Digital Light Processing (DLP) UV projector and a pneumatic extrusion technique, were used to develop a Sil-MA grid construct. A proof-of-concept of its shape-memory property was provided. Together, our data support the hypothesis that Sil-MA grid constructs can be injectable and applicable in bone regeneration applications.
AUTHOR Sarmin, Atiya M. and El Moussaid, Nadia and Suntornnond, Ratima and Tyler, Eleanor J. and Kim, Yang-Hee and Di Cio, Stefania and Megone, William V. and Pearce, Oliver and Gautrot, Julien E. and Dawson, Jonathan and Connelly, John T.
Title Multi-Scale Analysis of the Composition, Structure, and Function of Decellularized Extracellular Matrix for Human Skin and Wound Healing Models [Abstract]
Year 2022
Journal/Proceedings Biomolecules
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The extracellular matrix (ECM) is a complex mixture of structural proteins, proteoglycans, and signaling molecules that are essential for tissue integrity and homeostasis. While a number of recent studies have explored the use of decellularized ECM (dECM) as a biomaterial for tissue engineering, the complete composition, structure, and mechanics of these materials remain incompletely understood. In this study, we performed an in-depth characterization of skin-derived dECM biomaterials for human skin equivalent (HSE) models. The dECM materials were purified from porcine skin, and through mass spectrometry profiling, we quantified the presence of major ECM molecules, including types I, III, and VI collagen, fibrillin, and lumican. Rheological analysis demonstrated the sol-gel and shear-thinning properties of dECM materials, indicating their physical suitability as a tissue scaffold, while electron microscopy revealed a complex, hierarchical structure of nanofibers in dECM hydrogels. The dECM materials were compatible with advanced biofabrication techniques, including 3D printing within a gelatin microparticle support bath, printing with a sacrificial material, or blending with other ECM molecules to achieve more complex compositions and structures. As a proof of concept, we also demonstrate how dECM materials can be fabricated into a 3D skin wound healing model using 3D printing. Skin-derived dECM therefore represents a complex and versatile biomaterial with advantageous properties for the fabrication of next-generation HSEs.
AUTHOR Daskalakis, Evangelos and Huang, Boyang and Vyas, Cian and Acar, Anil Ahmet and Fallah, Ali and Cooper, Glen and Weightman, Andrew and Koc, Bahattin and Blunn, Gordon and Bartolo, Paulo
Title Novel 3D Bioglass Scaffolds for Bone Tissue Regeneration [Abstract]
Year 2022
Journal/Proceedings Polymers
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The design of scaffolds with optimal biomechanical properties for load-bearing applications is an important topic of research. Most studies have addressed this problem by focusing on the material composition and not on the coupled effect between the material composition and the scaffold architecture. Polymer–bioglass scaffolds have been investigated due to the excellent bioactivity properties of bioglass, which release ions that activate osteogenesis. However, material preparation methods usually require the use of organic solvents that induce surface modifications on the bioglass particles, compromising the adhesion with the polymeric material thus compromising mechanical properties. In this paper, we used a simple melt blending approach to produce polycaprolactone/bioglass pellets to construct scaffolds with pore size gradient. The results show that the addition of bioglass particles improved the mechanical properties of the scaffolds and, due to the selected architecture, all scaffolds presented mechanical properties in the cortical bone region. Moreover, the addition of bioglass indicated a positive long-term effect on the biological performance of the scaffolds. The pore size gradient also induced a cell spreading gradient.
AUTHOR Staubli, Flurina and Stoddart, Martin J. and D'Este, Matteo and Schwab, Andrea
Title Pre-culture of human mesenchymal stromal cells in spheroids facilitates chondrogenesis at a low total cell count upon embedding in biomaterials to generate cartilage microtissues [Abstract]
Year 2022
Journal/Proceedings Acta Biomaterialia
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Material-assisted cartilage tissue engineering has limited application in cartilage treatment due to hypertrophic tissue formation and high cell counts required. This study aimed at investigating the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. Pre-cultured (3 days, chondrogenic differentiation media) spheroids (250 cells/spheroid) were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels (four combinations of THA (12.5 vs 16.7 mg/ml) and Col (2.5 vs 1.7 mg/ml) content) at a cell density of 5 × 106 cells/ml (2 × 104 spheroids/ml). Macropellets derived from single hMSCs (2.5 × 105 cells, ScMP) or hMSC spheroids (2.5 × 105 cells, 103 spheroids, SpMP) served as control. hMSC differentiation was analyzed using glycosaminoglycan (GAG) quantification, gene expression analysis and (immuno-)histology. Embedding of hMSC spheroids in THA-Col induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and the production of GAGs . Lower THA led to more pronounced chondrogenic phenotype compared to higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in SpMP culture compared to ScMP group. This study presents hMSC pre-culture in spheroids as promising approach to study chondrogenic differentiation after biomaterial encapsulation at low total cell count (5 × 106/ml) without compromising chondrogenic matrix production. This approach can be applied to assemble microtissues in biomaterials to generate large cartilage construct. Statement of significance In vitro studies investigating the chondrogenic potential of biomaterials are limited due to the low cell-cell contact of encapsulated single cells. Here, we introduce the use of pre-cultured hMSC spheroids to study chondrogenesis upon encapsulation in a biomaterial. The use of spheroids takes advantage of the high cell-cell contact within each spheroid being critical in the early chondrogenesis of hMSCs. At a low seeding density of 5·106 cells/ml (2 × 104 spheroids/ml) we demonstrated hMSC chondrogenesis and cartilaginous matrix deposition. Our results indicate that the pre-culture might have a beneficial effect on hypertrophic gene expression without compromising chondrogenic differentiation. This approach has shown potential to assemble microtissues (here spheroids) in biomaterials to generate large cartilage constructs and to study the effect of biomaterial composition on cell alignment and migration.
AUTHOR Wang, Ruiqi and Deng, Shuai and Wu, Yuping and Wei, Haiying and Jing, Guangping and Zhang, Bosong and Liu, Fengzhen and Tian, Hui and Chen, Xiongbiao and Tian, Weiming
Title Remodelling 3D printed GelMA-HA corneal scaffolds by cornea stromal cells [Abstract]
Year 2022
Journal/Proceedings Colloid and Interface Science Communications
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Engineering scaffolds with a structure mimicking that of native cornea allows for addressing the severe donor shortage for the corneal blindness treatment, which, however, remains challenging. In the light that corneal stromal (CS) cells can play a key role in corneal stroma formation, in this study we incorporated CS cells into three-dimensional (3D) scaffolds printed from hyaluronic acid-modified gelatin-methacrylate (GelMA-HA) scaffolds and characterized the scaffolds in terms of remodeled extracellular matrix (ECM) in vitro. Our results illustrated that the modification of GelMA by HA allowed for 3D printing of corneal scaffolds and further improved the characteristics of primary rabbit-derived corneal stromal cells for remodelling scaffolds. After 60 days, we decellularized the remodeled corneal scaffolds and examined their optical properties; and our results demonstrated that the 3D printed corneal scaffolds provided CS cells with cues that guided them toward the directional and spatial organization and facilitated the ECM remodelling.
AUTHOR Eichholz, Kian and Freeman, Fiona and Pitacco, Pierluca and Nulty, Jessica and Ahern, Daniel and Burdis, Ross and Browe, David and Garcia, Orquidea and Hoey, David and Kelly, Daniel John
Title Scaffold microarchitecture regulates angiogenesis and the regeneration of large bone defects [Abstract]
Year 2022
Journal/Proceedings Biofabrication
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Abstract
Emerging 3D printing technologies can provide exquisite control over the external shape and internal architecture of scaffolds and tissue engineered constructs, enabling systematic studies to explore how geometric design features influence the regenerative process. Here we used fused deposition modelling (FDM) and melt electrowriting (MEW) to investigate how scaffold microarchitecture influences the healing of large bone defects. FDM was used to fabricate scaffolds with relatively large fibre diameters and low porosities, while MEW was used to fabricate scaffolds with smaller fibre diameters and higher porosities, with both scaffolds being designed to have comparable surface areas. Scaffold microarchitecture significantly influenced the healing response following implantation into critically sized femoral defects in rats, with the FDM scaffolds supporting the formation of larger bone spicules through its pores, while the MEW scaffolds supported the formation of a more round bone front during healing. After 12 weeks in vivo, both MEW and FDM scaffolds supported significantly higher levels of defect vascularisation compared to empty controls, while the MEW scaffolds supported higher levels of new bone formation. Somewhat surprisingly, this superior healing in the MEW group did not correlate with higher levels of angiogenesis, with the FDM scaffold supporting greater total vessel formation and the formation of larger vessels, while the MEW scaffold promoted the formation of a dense microvasculature with minimal evidence of larger vessels infiltrating the defect region. To conclude, the small fibre diameter, high porosity and high specific surface area of the MEW scaffold proved beneficial for osteogenesis and bone regeneration, demonstrating that changes in scaffold architecture enabled by this additive manufacturing technique can dramatically modulate angiogenesis and tissue regeneration without the need for complex exogenous growth factors. These results provide a valuable insight into the importance of 3D printed scaffold architecture when developing new bone tissue engineering strategies.
AUTHOR Liu, Jing and Zhou, Zhengtong and Zhang, Min and Song, Feng and Feng, Chong and Liu, Haochen
Title Simple and robust 3D bioprinting of full-thickness human skin tissue [Abstract]
Year 2022
Journal/Proceedings Bioengineered
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ABSTRACTArtificial skins have been used as skin substitutes for wound healing in the clinic, and as in vitro models for safety assessment in cosmetic and pharmaceutical industries. The three-dimensional (3D) bioprinting technique provides a promising strategy in the fabrication of artificial skins. Despite the technological advances, many challenges remain to be conquered, such as the complicated preparation conditions for bio-printed skin and the unavailability of stability and robustness of skin bioprinting. Here, we formulated a novel bio-ink composed of gelatin, sodium alginate and fibrinogen. By optimizing the ratio of components in the bio-ink, the design of the 3D model and the printing conditions, a fibroblasts-containing dermal layer construct was firstly fabricated, on the top of which laminin and keratinocytes were sequentially placed. Through air-liquid interface (ALI) culture by virtue of sterile wire mesh, a full-thickness skin tissue was thus prepared. HE and immunofluorescence staining showed that the bio-printed skin was not only morphologically representative of the human skin, but also expressed the specific markers related to epidermal differentiation and stratum corneum formation. The presented easy and robust preparation of full-thickness skin constructs provides a powerful tool for the establishment of artificial skins, holding critical academic significance and application value.
AUTHOR Burdis, Ross and Chariyev-Prinz, Farhad and Browe, David C. and Freeman, Fiona E. and Nulty, Jessica and McDonnell, Emily E. and Eichholz, Kian F. and Wang, Bin and Brama, Pieter and Kelly, Daniel J.
Title Spatial patterning of phenotypically distinct microtissues to engineer osteochondral grafts for biological joint resurfacing [Abstract]
Year 2022
Journal/Proceedings Biomaterials
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Abstract
Modular biofabrication strategies using microtissues or organoids as biological building blocks have great potential for engineering replacement tissues and organs at scale. Here we describe the development of a biofabrication strategy to engineer osteochondral tissues by spatially localising phenotypically distinct cartilage microtissues within an instructive 3D printed polymer framework. We first demonstrate that immature cartilage microtissues can spontaneously fuse to form homogeneous macrotissues, and that combining less cellular microtissues results in superior fusion and the generation of a more hyaline-like cartilage containing higher levels of sulphated glycosaminoglycans and type II collagen. Furthermore, temporally exposing developing microtissues to transforming growth factor-β accelerates their volumetric growth and subsequent capacity to fuse into larger hyaline cartilage grafts. Next, 3D printed polymeric frameworks are used to further guide microtissue fusion and the subsequent self-organisation process, resulting in the development of a macroscale tissue with zonal collagen organisation analogous to the structure seen in native articular cartilage. To engineer osteochondral grafts, hypertrophic cartilage microtissues are engineered as bone precursor tissues and spatially localised below phenotypically stable cartilage microtissues. Implantation of these engineered grafts into critically-sized caprine osteochondral defects results in effective defect stabilisation and histologically supports the restoration of a more normal articular surface after 6 months in vivo. These findings support the use of such modular biofabrication strategies for biological joint resurfacing.
AUTHOR Hatt, Luan P. and Armiento, Angela R. and Mys, Karen and Thompson, Keith and Hildebrand, Maria and Nehrbass, Dirk and Müller, Werner E. G. and Zeiter, Stephan and Eglin, David and Stoddart, Martin J.
Title Standard in vitro evaluations of engineered bone substitutes are not sufficient to predict in vivo preclinical model outcomes [Abstract]
Year 2022
Journal/Proceedings Acta Biomaterialia
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Abstract
Understanding the optimal conditions required for bone healing can have a substantial impact to target the problem of non–unions and large bone defects. The combination of bioactive factors, regenerative progenitor cells and biomaterials to form a tissue engineered (TE) complex is a promising solution but translation to the clinic has been slow. We hypothesized the typical material testing algorithm used is insufficient and leads to materials being mischaracterized as promising. In the first part of this study, human bone marrow – derived mesenchymal stromal cells (hBM-MSCs) were embedded in three commonly used biomaterials (hyaluronic acid methacrylate, gelatin methacrylate and fibrin) and combined with relevant bioactive osteogenesis factors (dexamethasone microparticles and polyphosphate nanoparticles) to form a TE construct that underwent in vitro osteogenic differentiation for 28 days. Gene expression of relevant transcription factors and osteogenic markers, and von Kossa staining were performed. In the second and third part of this study, the same combination of TE constructs were implanted subcutaneously (cell containing) in T cell-deficient athymic Crl:NIH-Foxn1rnu rats for 8 weeks or cell free in an immunocompetent New Zealand white rabbit calvarial model for 6 weeks, respectively. Osteogenic performance was investigated via MicroCT imaging and histology staining. The in vitro study showed enhanced upregulation of relevant genes and significant mineral deposition within the three biomaterials, generally considered as a positive result. Subcutaneous implantation indicates none to minor ectopic bone formation. No enhanced calvarial bone healing was detected in implanted biomaterials compared to the empty defect. The reasons for the poor correlation of in vitro and in vivo outcomes are unclear and needs further investigation. This study highlights the discrepancy between in vitro and in vivo outcomes, demonstrating that in vitro data should be interpreted with extreme caution. In vitro models with higher complexity are necessary to increase value for translational studies. Statement of significance Preclinical testing of newly developed biomaterials is a crucial element of the development cycle. Despite this, there is still significant discrepancy between in vitro and in vivo test results. Within this study we investigate multiple combinations of materials and osteogenic stimulants and demonstrate a poor correlation between the in vitro and in vivo data. We propose rationale for why this may be the case and suggest a modified testing algorithm.
AUTHOR Ma, Jiayi and Wu, Siyu and Liu, Jun and Liu, Chun and Ni, Su and Dai, Ting and Wu, Xiaoyu and Zhang, Zhenyu and Qu, Jixin and Zhao, Hongbin and Zhou, Dong and Zhao, Xiubo
Title Synergistic effects of nanoattapulgite and hydroxyapatite on vascularization and bone formation in a rabbit tibia bone defect model [Abstract]
Year 2022
Journal/Proceedings Biomater. Sci.
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Abstract
Hydroxyapatite (HA) is a promising scaffold material for the treatment of bone defects. However{,} the lack of angiogenic properties and undesirable mechanical properties (such as fragility) limits the application of HA. Nanoattapulgite (ATP) is a nature-derived clay mineral and has been proven to be a promising bioactive material for bone regeneration due to its ability to induce osteogenesis. In this study{,} polyvinyl alcohol/collagen/ATP/HA (PVA/COL/ATP/HA) scaffolds were printed. Mouse bone marrow mesenchymal stem/stromal cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) were used in vitro to assess the biocompatibility and the osteogenesis and vascularization induction potentials of the scaffolds. Subsequently{,} in vivo micro-CT and histological staining were carried out to evaluate new bone formation in a rabbit tibial defect model. The in vitro results showed that the incorporation of ATP increased the printing fidelity and mechanical properties{,} with values of compressive strengths up to 200% over raw PC-H scaffolds. Simultaneously{,} the expression levels of osteogenic-related genes and vascularization-related genes were significantly increased after the incorporation of ATP. The in vivo results showed that the PVA/COL/ATP/HA scaffolds exhibited synergistic effects on promoting vascularization and bone formation. The combination of ATP and HA provides a promising strategy for vascularized bone tissue engineering.
AUTHOR Salar Amoli, Mehdi and Anand, Resmi and EzEldeen, Mostafa and Amorim, Paulo Alexandre and Geris, Liesbet and Jacobs, Reinhilde and Bloemen, Veerle
Title The development of a 3D printable chitosan-based copolymer with tunable properties for dentoalveolar regeneration [Abstract]
Year 2022
Journal/Proceedings Carbohydrate Polymers
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Abstract
Dentoalveolar tissue engineering is an emerging yet challenging field, considering the lack of suitable materials and difficulty to produce patient-specific hydrogel scaffolds. The present paper aims to produce a 3D printable and tuneable biomaterial by copolymerizing a synthesized water-soluble chitosan derivative called maleic anhydride grafted chitosan (MA-C) with gelatin using genipin, a natural crosslinking agent. Development and testing of this material for 3D printing, degradation, and swelling demonstrated the ability to fabricate scaffolds with controlled physical properties based on pre-determined designs. The MA-C-gelatin copolymer demonstrated excellent biocompatibility, which was verified by analyzing the viability, growth and proliferation of human dental pulp stem cells seeded on MA-C-gelatin constructs through live/dead, alamar blue and DNA quantification assays. Based on the present findings, the proposed material might be a suitable candidate for dentoalveolar tissue engineering, while further research is required to achieve this goal.
AUTHOR Yu, Haiyang and Gong, Wen and Mei, Junhao and Qin, Lihao and Piao, Zeyu and You, Deshu and Gu, Wenxian and Jia, Zhongzhi
Title The efficacy of a paeoniflorin-sodium alginate-gelatin skin scaffold for the treatment of diabetic wound: An in vivo study in a rat model [Abstract]
Year 2022
Journal/Proceedings Biomedicine & Pharmacotherapy
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Abstract
Objective To investigate the efficacy of a paeoniflorin-sodium alginate (SA)-gelatin skin scaffold for treating diabetic wound in a rat model. Methods Bioinks were prepared using various percentages of paeoniflorin in the total weight of a solution containing SA and gelatin. Skin scaffolds containing 0%, 1%, 3%, 5%, and 10% paeoniflorin were printed using 3D bioprinting technology, and scaffold microstructure was observed with scanning electron microscopy. Skin scaffolds were then used in rats with diabetic wounds. H&E staining, Masson staining, and immunohistochemical staining for IL-1β and CD31 were performed on days 7 and 14. Results All skin scaffolds had a mesh-like structure with uniform pore distribution. Wounds healed well in each group, with the 1% and 3% groups demonstrating the most complete healing. H&E staining showed that skin accessory organs had appeared in each group. On day 7, collagen deposition in the 3% group was higher than in the other groups (P<0.05), and IL-1β infiltration was lower in the 10% group than in the 3% group (P = 0.002). On day 14, IL-1β infiltration was not significantly different between the 10% and 3% groups (P = 0.078). The CD31 level was higher in the 3% group than in the other groups on days 7 and 14 (P<0.05). Conclusion A 3% paeoniflorin-SA-gelatin skin scaffold promoted the healing of diabetic wounds in rats. This scaffold promoted collagen deposition and microvascular regeneration and demonstrated anti-inflammatory properties, suggesting that this scaffold type could be used to treat diabetic wounds.
AUTHOR Bácskay, Ildikó and Ujhelyi, Zoltán and Fehér, Pálma and Arany, Petra
Title The Evolution of the 3D-Printed Drug Delivery Systems: A Review [Abstract]
Year 2022
Journal/Proceedings Pharmaceutics
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Since the appearance of the 3D printing in the 1980s it has revolutionized many research fields including the pharmaceutical industry. The main goal is to manufacture complex, personalized products in a low-cost manufacturing process on-demand. In the last few decades, 3D printing has attracted the attention of numerous research groups for the manufacturing of different drug delivery systems. Since the 2015 approval of the first 3D-printed drug product, the number of publications has multiplied. In our review, we focused on summarizing the evolution of the produced drug delivery systems in the last 20 years and especially in the last 5 years. The drug delivery systems are sub-grouped into tablets, capsules, orodispersible films, implants, transdermal delivery systems, microneedles, vaginal drug delivery systems, and micro- and nanoscale dosage forms. Our classification may provide guidance for researchers to more easily examine the publications and to find further research directions.
AUTHOR Anderson, Margaret and Dubey, Nileshkumar and Bogie, Kath and Cao, Chen and Li, Junying and Lerchbacker, Joseph and Mendonça, Gustavo and Kauffmann, Frederic and Bottino, Marco C. and Kaigler, Darnell
Title Three-dimensional printing of clinical scale and personalized calcium phosphate scaffolds for alveolar bone reconstruction [Abstract]
Year 2022
Journal/Proceedings Dental Materials
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Abstract
Objective Alveolar bone defects can be highly variable in their morphology and, as the defect size increases, they become more challenging to treat with currently available therapeutics and biomaterials. This investigation sought to devise a protocol for fabricating customized clinical scale and patient-specific, bioceramic scaffolds for reconstruction of large alveolar bone defects. Methods Two types of calcium phosphate (CaP)-based bioceramic scaffolds (alginate/β-TCP and hydroxyapatite/α-TCP, hereafter referred to as hybrid CaP and Osteoink™, respectively) were designed, 3D printed, and their biocompatibility with alveolar bone marrow stem cells and mechanical properties were determined. Following scaffold optimization, a workflow was developed to use cone beam computed tomographic (CBCT) imaging to design and 3D print, defect-specific bioceramic scaffolds for clinical-scale bone defects. Results Osteoink™ scaffolds had the highest compressive strength when compared to hybrid CaP with different infill orientation. In cell culture medium, hybrid CaP degradation resulted in decreased pH (6.3) and toxicity to stem cells; however, OsteoInk™ scaffolds maintained a stable pH (7.2) in culture and passed the ISO standard for cytotoxicity. Finally, a clinically feasible laboratory workflow was developed and evaluated using CBCT imaging to engineer customized and defect-specific CaP scaffolds using OsteoInk™. It was determined that printed scaffolds had a high degree of accuracy to fit the respective clinical defects for which they were designed (0.27 mm morphological deviation of printed scaffolds from digital design). Significance From patient to patient, large alveolar bone defects are difficult to treat due to high variability in their complex morphologies and architecture. Our findings shows that Osteoink™ is a biocompatible material for 3D printing of clinically acceptable, patient-specific scaffolds with precision-fit for use in alveolar bone reconstructive procedures. Collectively, emerging digital technologies including CBCT imaging, 3D surgical planning, and (bio)printing can be integrated to address this unmet clinical challenge.
AUTHOR Barceló, Xavier and Eichholz, Kian F. and Garcia, Orquidea and Kelly, Daniel J.
Title Tuning the Degradation Rate of Alginate-Based Bioinks for Bioprinting Functional Cartilage Tissue [Abstract]
Year 2022
Journal/Proceedings Biomedicines
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Abstract
Negative foreign body responses following the in vivo implantation of bioprinted implants motivate the development of novel bioinks which can rapidly degrade with the formation of functional tissue, whilst still maintaining desired shapes post-printing. Here, we investigated the oxidation of alginate as a means to modify the degradation rate of alginate-based bioinks for cartilage tissue engineering applications. Raw and partially oxidized alginate (OA) were combined at different ratios (Alginate:OA at 100:0; 75:25; 50:50; 25:75; 0:100) to provide finer control over the rate of bioink degradation. These alginate blends were then combined with a temporary viscosity modifier (gelatin) to produce a range of degradable bioinks with rheological properties suitable for extrusion bioprinting. The rate of degradation was found to be highly dependent on the OA content of the bioink. Despite this high mass loss, the initially printed geometry was maintained throughout a 4 week in vitro culture period for all bioink blends except the 0:100 group. All bioink blends also supported robust chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs), resulting in the development of a hyaline-like tissue that was rich in type II collagen and negative for calcific deposits. Such tuneable inks offer numerous benefits to the field of 3D bioprinting, from providing space in a controllable manner for new extracellular matrix deposition, to alleviating concerns associated with a foreign body response to printed material inks in vivo.
AUTHOR Blume, Cornelia and Kraus, Xenia and Heene, Sebastian and Loewner, Sebastian and Stanislawski, Nils and Cholewa, Fabian and Blume, Holger
Title Vascular implants – new aspects for in situ tissue engineering [Abstract]
Year 2022
Journal/Proceedings Engineering in Life Sciences
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Abstract Conventional synthetic vascular grafts require ongoing anticoagulation, and autologous venous grafts are often not available in elderly patients. This review highlights the development of bioartificial vessels replacing brain-dead donor- or animal-deriving vessels with ongoing immune reactivity. The vision for such bio-hybrids exists in a combination of biodegradable scaffolds and seeding with immune-neutral cells, and here different cells sources such as autologous progenitor cells or stem cells are relevant. This kind of in situ tissue engineering depends on a suitable bioreactor system with elaborate monitoring systems, three-dimensional (3D) visualization and a potential of cell conditioning into the direction of the targeted vascular cell phenotype. Necessary bioreactor tools for dynamic and pulsatile cultivation are described. In addition, a concept for design of vasa vasorum is outlined, that is needed for sustainable nutrition of the wall structure in large caliber vessels. For scaffold design and cell adhesion additives, different materials and technologies are discussed. 3D printing is introduced as a relatively new field with promising prospects, for example, to create complex geometries or micro-structured surfaces for optimal cell adhesion and ingrowth in a standardized and custom designed procedure. Summarizing, a bio-hybrid vascular prosthesis from a controlled biotechnological process is thus coming more and more into view. It has the potential to withstand strict approval requirements applied for advanced therapy medicinal products.
AUTHOR Zhang, Xiao and Liu, Yang and Zuo, Qiang and Wang, Qingyun and Li, Zuxi and Yan, Kai and Yuan, Tao and Zhang, Yi and Shen, Kai and Xie, Rui and Fan, Weimin
Title 3D Bioprinting of Biomimetic Bilayered Scaffold Consisting of Decellularized Extracellular Matrix and Silk Fibroin for Osteochondral Repair [Abstract]
Year 2021
Journal/Proceedings International Journal of Bioprinting; Vol 7, No 4 (2021)
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Abstract
Recently, three-dimensional (3D) bioprinting technology is becoming an appealing approach for osteochondral repair. However, it is challenging to develop a bilayered scaffold with anisotropic structural properties to mimic a native osteochondral tissue. Herein, we developed a bioink consisting of decellularized extracellular matrix and silk fibroin to print the bilayered scaffold. The bilayered scaffold mimics the natural osteochondral tissue by controlling the composition, mechanical properties, and growth factor release in each layer of the scaffold. The in vitro results show that each layer of scaffolds had a suitable mechanical strength and degradation rate. Furthermore, the scaffolds encapsulating transforming growth factor-beta (TGF-β) and bone morphogenetic protein-2 (BMP-2) can act as a controlled release system and promote directed differentiation of bone marrow-derived mesenchymal stem cells. Furthermore, the in vivo experiments suggested that the scaffolds loaded with growth factors promoted osteochondral regeneration in the rabbit knee joint model. Consequently, the biomimetic bilayered scaffold loaded with TGF-β and BMP-2 would be a promising strategy for osteochondral repair.
AUTHOR Nulty, Jessica and Freeman, Fiona E. and Browe, David C. and Burdis, Ross and Ahern, Daniel P. and Pitacco, Pierluca and Lee, Yu Bin and Alsberg, Eben and Kelly, Daniel J.
Title 3D Bioprinting of prevascularised implants for the repair of critically-sized bone defects [Abstract]
Year 2021
Journal/Proceedings Acta Biomaterialia
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Abstract
For 3D bioprinted tissues to be scaled-up to clinically relevant sizes, effective prevascularisation strategies are required to provide the necessary nutrients for normal metabolism and to remove associated waste by-products. The aim of this study was to develop a bioprinting strategy to engineer prevascularised tissues in vitro and to investigate the capacity of such constructs to enhance the vascularisation and regeneration of large bone defects in vivo. From a screen of different bioinks, a fibrin-based hydrogel was found to best support human umbilical vein endothelial cell (HUVEC) sprouting and the establishment of a microvessel network. When this bioink was combined with HUVECs and supporting human bone marrow stem/stromal cells (hBMSCs), these microvessel networks persisted in vitro. Furthermore, only bioprinted tissues containing both HUVECs and hBMSCs, that were first allowed to mature in vitro, supported robust blood vessel development in vivo. To assess the therapeutic utility of this bioprinting strategy, these bioinks were used to prevascularise 3D printed polycaprolactone (PCL) scaffolds, which were subsequently implanted into critically-sized femoral bone defects in rats. Microcomputed tomography (µCT) angiography revealed increased levels of vascularisation in vivo, which correlated with higher levels of new bone formation. Such prevascularised constructs could be used to enhance the vascularisation of a range of large tissue defects, forming the basis of multiple new bioprinted therapeutics. Statement of Significance This paper demonstrates a versatile 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into a bone tissue engineering strategy to improve vascularisation in a rat femoral defect model.
AUTHOR Das,Sanskrita and Nam,Hyoryung and Jang,Jinah
Title 3D bioprinting of stem cell-laden cardiac patch: A promising alternative for myocardial repair
Year 2021
Journal/Proceedings APL Bioengineering
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AUTHOR Zhang, Yi and Wang, Bin and Hu, Junchao and Yin, Tianyuan and Yue, Tao and Liu, Na and Liu, Yuanyuan
Title 3D Composite Bioprinting for Fabrication of Artificial Biological Tissues. [Abstract]
Year 2021
Journal/Proceedings International journal of bioprinting
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Three-dimensional (3D) bioprinting is an important technology for fabricating artificial tissue. To effectively reconstruct the multiscale structure and multi-material gradient of natural tissues and organs, 3D bioprinting has been increasingly developed into multi-process composite mode. The current 3D composite bioprinting is a combination of two or more printing processes, and oftentimes, physical field regulation that can regulate filaments or cells during or after printing may be involved. Correspondingly, both path planning strategy and process control all become more complex. Hence, the computer-aided design and computer-aided manufacturing (CAD/CAM) system that is traditionally used in 3D printing system is now facing challenges. Thus, the scale information that cannot be modeled in the CAD process should be considered in the design of CAM by adding a process management module in the traditional CAD/CAM system and add more information reflecting component gradient in the path planning strategy.
AUTHOR Francesca Cestari and Mauro Petretta and Yuejiao Yang and Antonella Motta and Brunella Grigolo and Vincenzo M. Sglavo
Title 3D printing of PCL/nano-hydroxyapatite scaffolds derived from biogenic sources for bone tissue engineering [Abstract]
Year 2021
Journal/Proceedings Sustainable Materials and Technologies
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Bioactive composites made of ∽85 wt% poly(ε-caprolactone) (PCL) and ∽15 wt% nanometric hydroxyapatite (HA) produced from biogenic sources were 3D printed by an extrusion-based process to obtain porous scaffolds suitable for bone regeneration. Three different composite formulations were considered by using HA synthesized from three distinct natural sources, which were collected as food wastes: cuttlefish bones, mussel shells and chicken eggshells. Composition and thermal properties of the materials were analysed by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and x-ray spectroscopy (XRD), while the morphological and mechanical properties of the 3D scaffolds were studied by means of electron microscopy (SEM) and compression tests. Bioactivity was tested by seeding human osteoblast cell line (MG63) onto the scaffolds which were analysed by confocal microscopy and Alamar Blue and PicoGreen® tests after 1 to 7 culture days. The elastic modulus (177–316 MPa) is found to be within the range reported for typical trabecular bones being increased by the presence of the bio-HA particles. Moreover, cells adhesion, viability and proliferation are largely promoted in the scaffolds containing nanometric HA with respect to pure PCL, the best results being revealed when mussel shell-derived HA is used. Indeed, different biological sources result in different cell proliferation rates, pointing that the biological origin has an impact on the cells-scaffold interaction. In general, the results show that PCL/bio-HA scaffolds possess improved mechanical properties and enhanced bioactivity when compared with pure PCL ones.
AUTHOR Vyas, Cian and Zhang, Jun and Øvrebø, Øystein and Huang, Boyang and Roberts, Iwan and Setty, Mohan and Allardyce, Benjamin and Haugen, Håvard and Rajkhowa, Rangam and Bartolo, Paulo
Title 3D printing of silk microparticle reinforced polycaprolactone scaffolds for tissue engineering applications [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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Abstract
Polycaprolactone (PCL) scaffolds have been widely investigated for tissue engineering applications, however, they exhibit poor cell adhesion and mechanical properties. Subsequently, PCL composites have been produced to improve the material properties. This study utilises a natural material, Bombyx mori silk microparticles (SMP) prepared by milling silk fibre, to produce a composite to enhance the scaffolds properties. Silk is biocompatible and biodegradable with excellent mechanical properties. However, there are no studies using SMPs as a reinforcing agent in a 3D printed thermoplastic polymer scaffold. PCL/SMP (10, 20, 30 wt%) composites were prepared by melt blending. Rheological analysis showed that SMP loading increased the shear thinning and storage modulus of the material. Scaffolds were fabricated using a screw-assisted extrusion-based additive manufacturing system. Scanning electron microscopy and X-ray microtomography was used to determine scaffold morphology. The scaffolds had high interconnectivity with regular printed fibres and pore morphologies within the designed parameters. Compressive mechanical testing showed that the addition of SMP significantly improved the compressive Young's modulus of the scaffolds. The scaffolds were more hydrophobic with the inclusion of SMP which was linked to a decrease in total protein adsorption. Cell behaviour was assessed using human adipose derived mesenchymal stem cells. A cytotoxic effect was observed at higher particle loading (30 wt%) after 7 days of culture. By day 21, 10 wt% loading showed significantly higher cell metabolic activity and proliferation, high cell viability, and cell migration throughout the scaffold. Calcium mineral deposition was observed on the scaffolds during cell culture. Large calcium mineral deposits were observed at 30 wt% and smaller calcium deposits were observed at 10 wt%. This study demonstrates that SMPs incorporated into a PCL scaffold provided effective mechanical reinforcement, improved the rate of degradation, and increased cell proliferation, demonstrating potential suitability for bone tissue engineering applications.
AUTHOR Golafshan, Nasim and Willemsen, Koen and Kadumudi, Firoz Babu and Vorndran, Elke and Dolatshahi-Pirouz, Alireza and Weinans, Harrie and van der Wal, Bart C. H. and Malda, Jos and Castilho, Miguel
Title 3D-Printed Regenerative Magnesium Phosphate Implant Ensures Stability and Restoration of Hip Dysplasia [Abstract]
Year 2021
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract Osteoarthritis of the hip is a painful and debilitating condition commonly occurring in humans and dogs. One of the main causes that leads to hip osteoarthritis is hip dysplasia. Although the current surgical methods to correct dysplasia work satisfactorily in many circumstances, these are associated with serious complications, tissue resorption, and degeneration. In this study, a one-step fabrication of a regenerative hip implant with a patient-specific design and load-bearing properties is reported. The regenerative hip implant is fabricated based on patient imaging files and by an extrusion assisted 3D printing process using a flexible, bone-inducing biomaterial. The novel implant can be fixed with metallic screws to host bone and can be loaded up to physiological loads without signs of critical permanent deformation or failure. Moreover, after exposing the hip implant to accelerated in vitro degradation, it is confirmed that it is still able to support physiological loads even after losing ≈40% of its initial mass. In addition, the osteopromotive properties of the novel hip implant is demonstrated as shown by an increased expression of osteonectin and osteocalcin by cultured human mesenchymal stem cells after 21 days. Overall, the proposed hip implant provides an innovative regenerative and mechanically stable solution for hip dysplasia treatment.
AUTHOR Chelsea Twohig and Mari Helsinga and Amin Mansoorifar and Avathamsa Athirasala and Anthony Tahayeri and Cristiane Miranda França and Silvia Amaya Pajares and Reyan Abdelmoniem and Susanne Scherrer and Stéphane Durual and Jack Ferracane and Luiz E. Bertassoni
Title A dual-ink 3D printing strategy to engineer pre-vascularized bone scaffolds in-vitro [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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Abstract
A functional vascular supply is a key component of any large-scale tissue, providing support for the metabolic needs of tissue-remodeling cells. Although well-studied strategies exist to fabricate biomimetic scaffolds for bone regeneration, success rates for regeneration in larger defects can be improved by engineering microvascular capillaries within the scaffolds to enhance oxygen and nutrient supply to the core of the engineered tissue as it grows. Even though the role of calcium and phosphate has been well understood to enhance osteogenesis, it remains unclear whether calcium and phosphate may have a detrimental effect on the vasculogenic and angiogenic potential of endothelial cells cultured on 3D printed bone scaffolds. In this study, we presented a novel dual-ink bioprinting method to create vasculature interwoven inside CaP bone constructs. In this method, strands of a CaP ink and a sacrificial template material was used to form scaffolds containing CaP fibers and microchannels seeded with vascular endothelial and mesenchymal stem cells (MSCs) within a photo-crosslinkable gelatin methacryloyl (GelMA) hydrogel material. Our results show similar morphology of growing vessels in the presence of CaP bioink, and no significant difference in endothelial cell sprouting was found. Furthermore, our initial results showed the differentiation of hMSCs into pericytes in the presence of CaP ink. These results indicate the feasibility of creating vascularized bone scaffolds, which can be used for enhancing vascular formation in the core of bone scaffolds.
AUTHOR Bin Wang and Pedro J. Díaz-Payno and David C. Browe and Fiona E. Freeman and Jessica Nulty and Ross Burdis and Daniel J. Kelly
Title Affinity-bound growth factor within sulfated interpenetrate network bioinks for bioprinting cartilaginous tissues [Abstract]
Year 2021
Journal/Proceedings Acta Biomaterialia
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3D bioprinting has emerged as a promising technology in the field of tissue engineering and regenerative medicine due to its ability to create anatomically complex tissue substitutes. However, it still remains challenging to develop bioactive bioinks that provide appropriate and permissive environments to instruct and guide the regenerative process in vitro and in vivo. In this study alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize an alginate-gelatin methacryloyl (GelMA) interpenetrating network (IPN) bioink to enable the bioprinting of cartilaginous tissues. The inclusion of alginate sulfate had a limited influence on the viscosity, shear-thinning and thixotropic properties of the IPN bioink, enabling high-fidelity bioprinting and supporting mesenchymal stem cell (MSC) viability post-printing. The stiffness of printed IPN constructs greatly exceeded that achieved by printing alginate or GelMA alone, while maintaining resilience and toughness. Furthermore, given the high affinity of alginate sulfate to heparin-binding growth factors, the sulfated IPN bioink supported the sustained release of transforming growth factor-β3 (TGF-β3), providing an environment that supported robust chondrogenesis in vitro, with little evidence of hypertrophy or mineralization over extended culture periods. Such bioprinted constructs also supported chondrogenesis in vivo, with the controlled release of TGF-β3 promoting significantly higher levels of cartilage-specific extracellular matrix deposition. Altogether, these results demonstrate the potential of bioprinting sulfated bioinks as part of a ‘single-stage’ or ‘point-of-care’ strategy for regenerating cartilaginous tissues. Statement of Significance: This study highlights the potential of using sulfated interpenetrating network (IPN) bioink to support the regeneration of phenotypically stable articular cartilage. Construction of interpenetrate networks in the bioink enables unique high-fidelity bioprinting and unique synergistic mechanical properties. The presence of alginate sulfate provided the capacity of high affinity-binding of TGF-β3, which promoted robust chondrogenesis.
AUTHOR Rachel Cadle and Dan Rogozea and Leni Moldovan and Patricia Parsons-Wingerter and Nicanor I. Moldovan
Title An image analysis-based workflow for 3D bioprinting of anatomically realistic retinal vascular patterns [Abstract]
Year 2021
Journal/Proceedings Bioprinting
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Abstract
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.
AUTHOR Rachel Cadle and Dan Rogozea and Leni Moldovan and Patricia Parsons-Wingerter and Nicanor I. Moldovan
Title An image analysis-based workflow for 3D bioprinting of anatomically realistic retinal vascular patterns [Abstract]
Year 2021
Journal/Proceedings Bioprinting
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DOI/URL URL DOI
Abstract
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.
AUTHOR Rachel Cadle and Dan Rogozea and Leni Moldovan and Patricia Parsons-Wingerter and Nicanor I. Moldovan
Title An image analysis-based workflow for 3D bioprinting of anatomically realistic retinal vascular patterns [Abstract]
Year 2021
Journal/Proceedings Bioprinting
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DOI/URL URL DOI
Abstract
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.
AUTHOR Rachel Cadle and Dan Rogozea and Leni Moldovan and Patricia Parsons-Wingerter and Nicanor I. Moldovan
Title An image analysis-based workflow for 3D bioprinting of anatomically realistic retinal vascular patterns [Abstract]
Year 2021
Journal/Proceedings Bioprinting
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DOI/URL URL DOI
Abstract
There is an enduring need for vascularization of bioprinted constructs with vascular networks optimized for distribution of nutrient-containing fluids, both for in vitro applications and in vivo implantation. However, most of the efforts in this field were directed so far towards generation of simple linear channels, often lined with endothelial cells only, and thus lacking the anatomical details of real vascular networks. To start addressing this need, here we explored the possibility of using actual vascular patterns derived from human ocular fundus for instructing the 3D printing activity. In order to assign to these patterns the organ-specific topology, and eventually vessel branch-defined cellular composition, we describe the use of the branching analysis program VESGEN 2D for planning a workflow that links the primary vascular images with their 3D printing with bioinks. To this end, we show how to process flat vascular images and, for an even more realistic representation, how to retro-engineer concave retinal patterns from flat images and to print them in a supporting hydrogel. This work opens the possibility of bioprinting more anatomically realistic vascular networks, and thus to eventually improve the vascularization of living tissue-engineered constructs.
AUTHOR Yuanhao Wu and Gabriele Maria Fortunato and Babatunde O Okesola and Francesco Luigi Pellerej di Brocchetti and Ratima Suntornnond and John Connelly and Carmelo De Maria and Jose Carlos Rodriguez-Cabello and Giovanni Vozzi and Wen Wang and Alvaro Mata
Title An interfacial self-assembling bioink for the manufacturing of capillary-like structures with tuneable and anisotropic permeability [Abstract]
Year 2021
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
Self-assembling bioinks offer the possibility to biofabricate with molecular precision, hierarchical control, and biofunctionality. For this to become a reality with widespread impact, it is essential to engineer these ink systems ensuring reproducibility and providing suitable standardization. We have reported a self-assembling bioink based on disorder-to-order transitions of an elastin-like recombinamer (ELR) to co-assemble with graphene oxide (GO). Here, we establish reproducible processes, optimize printing parameters for its use as a bioink, describe new advantages that the self-assembling bioink can provide, and demonstrate how to fabricate novel structures with physiological relevance. We fabricate capillary-like structures with resolutions down to ∼10 µm in diameter and ∼2 µm thick tube walls and use both experimental and finite element analysis to characterize the printing conditions, underlying interfacial diffusion-reaction mechanism of assembly, printing fidelity, and material porosity and permeability. We demonstrate the capacity to modulate the pore size and tune the permeability of the resulting structures with and without human umbilical vascular endothelial cells. Finally, the potential of the ELR-GO bioink to enable supramolecular fabrication of biomimetic structures was demonstrated by printing tubes exhibiting walls with progressively different structure and permeability.
AUTHOR Otto, I. A. and Capendale, P. E. and Garcia, J. P. and de Ruijter, M. and van Doremalen, R. F. M. and Castilho, M. and Lawson, T. and Grinstaff, M. W. and Breugem, C. C. and Kon, M. and Levato, R. and Malda, J.
Title Biofabrication of a shape-stable auricular structure for the reconstruction of ear deformities [Abstract]
Year 2021
Journal/Proceedings Materials Today Bio
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Abstract
Bioengineering of the human auricle remains a significant challenge, where the complex and unique shape, the generation of high-quality neocartilage, and shape preservation are key factors. Future regenerative medicine–based approaches for auricular cartilage reconstruction will benefit from a smart combination of various strategies. Our approach to fabrication of an ear-shaped construct uses hybrid bioprinting techniques, a recently identified progenitor cell population, previously validated biomaterials, and a smart scaffold design. Specifically, we generated a 3D-printed polycaprolactone (PCL) scaffold via fused deposition modeling, photocrosslinked a human auricular cartilage progenitor cell–laden gelatin methacryloyl (gelMA) hydrogel within the scaffold, and cultured the bioengineered structure in vitro in chondrogenic media for 30 days. Our results show that the fabrication process maintains the viability and chondrogenic phenotype of the cells, that the compressive properties of the combined PCL and gelMA hybrid auricular constructs are similar to native auricular cartilage, and that biofabricated hybrid auricular structures exhibit excellent shape fidelity compared with the 3D digital model along with deposition of cartilage-like matrix in both peripheral and central areas of the auricular structure. Our strategy affords an anatomically enhanced auricular structure with appropriate mechanical properties, ensures adequate preservation of the auricular shape during a dynamic in vitro culture period, and enables chondrogenically potent progenitor cells to produce abundant cartilage-like matrix throughout the auricular construct. The combination of smart scaffold design with 3D bioprinting and cartilage progenitor cells holds promise for the development of clinically translatable regenerative medicine strategies for auricular reconstruction.
AUTHOR Nulty, Jessica and Burdis, Ross and Kelly, Daniel J.
Title Biofabrication of Prevascularised Hypertrophic Cartilage Microtissues for Bone Tissue Engineering [Abstract]
Year 2021
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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DOI/URL DOI
Abstract
Bone tissue engineering (TE) has the potential to transform the treatment of challenging musculoskeletal pathologies. To date, clinical translation of many traditional TE strategies has been impaired by poor vascularisation of the implant. Addressing such challenges has motivated research into developmentally inspired TE strategies, whereby implants mimicking earlier stages of a tissue’s development are engineered in vitro and then implanted in vivo to fully mature into the adult tissue. The goal of this study was to engineer in vitro tissues mimicking the immediate developmental precursor to long bones, specifically a vascularised hypertrophic cartilage template, and to then assess the capacity of such a construct to support endochondral bone formation in vivo. To this end, we first developed a method for the generation of large numbers of hypertrophic cartilage microtissues using a microwell system, and encapsulated these microtissues into a fibrin-based hydrogel capable of supporting vasculogenesis by human umbilical vein endothelial cells (HUVECs). The microwells supported the formation of bone marrow derived stem/stromal cell (BMSC) aggregates and their differentiation toward a hypertrophic cartilage phenotype over 5 weeks of cultivation, as evident by the development of a matrix rich in sulphated glycosaminoglycan (sGAG), collagen types I, II, and X, and calcium. Prevascularisation of these microtissues, undertaken in vitro 1 week prior to implantation, enhanced their capacity to mineralise, with significantly higher levels of mineralised tissue observed within such implants after 4 weeks in vivo within an ectopic murine model for bone formation. It is also possible to integrate such microtissues into 3D bioprinting systems, thereby enabling the bioprinting of scaled-up, patient-specific prevascularised implants. Taken together, these results demonstrate the development of an effective strategy for prevascularising a tissue engineered construct comprised of multiple individual microtissue “building blocks,” which could potentially be used in the treatment of challenging bone defects.
AUTHOR Falcones, Bryan and Sanz-Fraile, Héctor and Marhuenda, Esther and Mendizábal, Irene and Cabrera-Aguilera, Ignacio and Malandain, Nanthilde and Uriarte, Juan J. and Almendros, Isaac and Navajas, Daniel and Weiss, Daniel J. and Farré, Ramon and Otero, Jorge
Title Bioprintable Lung Extracellular Matrix Hydrogel Scaffolds for 3D Culture of Mesenchymal Stromal Cells [Abstract]
Year 2021
Journal/Proceedings Polymers
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DOI/URL URL DOI
Abstract
Mesenchymal stromal cell (MSC)-based cell therapy in acute respiratory diseases is based on MSC secretion of paracrine factors. Several strategies have proposed to improve this are being explored including pre-conditioning the MSCs prior to administration. We here propose a strategy for improving the therapeutic efficacy of MSCs based on cell preconditioning by growing them in native extracellular matrix (ECM) derived from the lung. To this end, a bioink with tunable stiffness based on decellularized porcine lung ECM hydrogels was developed and characterized. The bioink was suitable for 3D culturing of lung-resident MSCs without the need for additional chemical or physical crosslinking. MSCs showed good viability, and contraction assays showed the existence of cell–matrix interactions in the bioprinted scaffolds. Adhesion capacity and length of the focal adhesions formed were increased for the cells cultured within the lung hydrogel scaffolds. Also, there was more than a 20-fold increase of the expression of the CXCR4 receptor in the 3D-cultured cells compared to the cells cultured in plastic. Secretion of cytokines when cultured in an in vitro model of lung injury showed a decreased secretion of pro-inflammatory mediators for the cells cultured in the 3D scaffolds. Moreover, the morphology of the harvested cells was markedly different with respect to conventionally (2D) cultured MSCs. In conclusion, the developed bioink can be used to bioprint structures aimed to improve preconditioning MSCs for therapeutic purposes.
AUTHOR Burdis, Ross and Chariyev-Prinz, Farhad and Kelly, Daniel J.
Title Bioprinting of biomimetic self-organised cartilage with a supporting joint fixation device [Abstract]
Year 2021
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
Despite sustained efforts, engineering truly biomimetic articular cartilage (AC) via traditional top-down approaches remains challenging. Emerging biofabrication strategies, from 3D bioprinting to scaffold-free approaches that leverage principles of cellular self-organisation, are generating significant interest in the field of cartilage tissue engineering as a means of developing biomimetic tissue analogues in vitro. Although such strategies have advanced the quality of engineered cartilage, recapitulation of many key structural features of native AC, in particular a collagen network mimicking the tissue’s ‘Benninghoff arcade’, remains elusive. Additionally, a complete solution to fixating engineered cartilages in situ within damaged synovial joints has yet to be identified. This study sought to address both of these key challenges by engineering biomimetic AC within a device designed to anchor the tissue within a synovial joint defect. We first designed and fabricated a fixation device capable of anchoring engineered cartilage into the subchondral bone. Next, we developed a strategy for inkjet printing porcine mesenchymal stem/stromal cells (MSCs) into this supporting fixation device, which was also designed to provide instructive cues to direct the self-organisation of MSC condensations towards a stratified engineered AC. We found that a higher starting cell-density supported the development of a more zonally defined collagen network within the engineered tissue. Dynamic culture was implemented to further enhance the quality of this engineered tissue, resulting in an approximate 3 fold increase in glycosaminoglycan and collagen accumulation. Ultimately this strategy supported the development of AC that exhibited near-native levels of glycosaminoglycan accumulation (>5% WW), as well as a biomimetic collagen network organisation with a perpendicular to a parallel fibre arrangement (relative to the tissue surface) from the deep to superficial zones via arcading fibres within the middle zone of the engineered tissue. Collectively, this work demonstrates the successful convergence of novel biofabrication methods, bioprinting strategies and culture regimes to engineer a hybrid implant suited to resurfacing AC defects.
AUTHOR Fisch, Philipp and Broguiere, Nicolas and Finkielsztein, Sergio and Linder, Thomas and Zenobi-Wong, Marcy
Title Bioprinting of Cartilaginous Auricular Constructs Utilizing an Enzymatically Crosslinkable Bioink [Abstract]
Year 2021
Journal/Proceedings Advanced Functional Materials
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DOI/URL DOI
Abstract
Abstract Bioprinting of functional tissues could overcome tissue shortages and allow a more rapid response for treatments. However, despite recent progress in bioprinting, and its outstanding ability to position cells and biomaterials in a precise 3D manner, its success has been limited, due to insufficient maturation of constructs into functional tissue. Here, a novel calcium-triggered enzymatic crosslinking (CTEC) mechanism for bioinks based on the activation cascade of Factor XIII is presented and utilized for the biofabrication of cartilaginous constructs. Hyaluronan transglutaminase (HA-TG), an enzymatically crosslinkable material, has shown excellent characteristics for chondrogenesis and builds the basis of the CTEC bioink. The bioink supports tissue maturation with neocartilage formation and stiffening of constructs up to 400 kPa. Bioprinted constructs remain stable in vivo for 24 weeks and bioprinted auricular constructs transform into cartilaginous grafts. A major limitation of the current study is the deposition of collagen I, indicating the maturation toward fibrocartilage rather than elastic cartilage. Shifting the maturation process toward elastic cartilage will therefore be essential in order for the developed bioinks to offer a novel tissue engineered treatment for microtia patients. CTEC bioprinting furthermore opens up use of enzymatically crosslinkable biopolymers and their modularity to support a multitude of tissues.
AUTHOR Oliveira, H. and Médina, C. and Labrunie, G. and Dusserre, N. and Catros, S. and Magnan, L. and Handschin, C. and Stachowicz, M. L. and Fricain, J.-C. and L’Heureux, N.
Title Cell-assembled extracellular matrix (CAM): a human biopaper for the biofabrication of pre-vascularized tissues able to connect to the host circulation in vivo [Abstract]
Year 2021
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
When considering regenerative approaches, the efficient creation of a functional vasculature, that can support the metabolic needs of bioengineered tissues, is essential for their survival after implantation. However, it is widely recognized that the post-implantation microenvironment of the engineered tissues is often hypoxic due to insufficient vascularization, resulting in ischemia injury and necrosis. This is one of the main limitations of current tissue engineering applications aiming at replacing significant tissue volumes. Here, we have explored the use of a new biomaterial, the cell-assembled extracellular matrix (CAM), as a biopaper to biofabricate a vascular system. CAM sheets are a unique, fully biological and fully human material that has already shown stable long-term implantation in humans. We demonstrated, for the first time, the use of this unprocessed human ECM as a microperforated biopaper. Using microvalve dispensing bioprinting, concentrated human endothelial cells (30 millions ml−1) were deposited in a controlled geometry in CAM sheets and cocultured with HSFs. Following multilayer assembly, thick ECM-based constructs fused and supported the survival and maturation of capillary-like structures for up to 26 d of culture. Following 3 weeks of subcutaneous implantation in a mice model, constructs showed limited degradative response and the pre-formed vasculature successfully connected with the host circulatory system to establish active perfusion.This mechanically resilient tissue equivalent has great potential for the creation of more complex implantable tissues, where rapid anastomosis is sine qua non for cell survival and efficient tissue integration.
AUTHOR Bagnol, Romain and Sprecher, Christoph and Peroglio, Marianna and Chevalier, Jerome and Mahou, Redouan and Büchler, Philippe and Richards, Geoff and Eglin, David
Title Coaxial micro-extrusion of a calcium phosphate ink with aqueous solvents improves printing stability, structure fidelity and mechanical properties [Abstract]
Year 2021
Journal/Proceedings Acta Biomaterialia
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DOI/URL URL DOI
Abstract
Micro-extrusion-based 3D printing of complex geometrical and porous calcium phosphate (CaP) can improve treatment of bone defects through the production of personalized bone substitutes. However, achieving printing and post-printing shape stabilities for the efficient fabrication and application of rapid hardening protocol are still challenging. In this work, the coaxial printing of a self-setting CaP cement with water and ethanol mixtures aiming to increase the ink yield stress upon extrusion and the stability of fabricated structures was explored. Printing height of overhang structure was doubled when aqueous solvents were used and a 2 log increase of the stiffness was achieved post-printing. A standard and fast steam sterilization protocol applied as hardening step on the coaxial printed CaP cement (CPC) ink resulted in constructs with 4 to 5 times higher compressive moduli in comparison to extrusion process in the absence of solvent. This improved mechanical performance is likely due to rapid CPC setting, preventing cracks formation during hardening process. Thus, coaxial micro-extrusion-based 3D printing of a CPC ink with aqueous solvent enhances printability and allows the use of the widespread steam sterilization cycle as a standalone post-processing technique for production of 3D printed personalized CaP bone substitutes. Statement of Significance Coaxial micro-extrusion-based 3D printing of a self-setting CaP cement with water:ethanol mixtures increased the ink yield stress upon extrusion and the stability of fabricated structures. Printing height of overhang structure was doubled when aqueous solvents were used, and a 2 orders of magnitude log increase of the stiffness was achieved post-printing. A fast hardening step consisting of a standard steam sterilization was applied. Four to 5 times higher compressive moduli was obtained for hardened coaxially printed constructs. This improved mechanical performance is likely due to rapid CPC setting in the coaxial printing, preventing cracks formation during hardening process.
AUTHOR Fenelon, Mathilde and Etchebarne, Marion and Siadous, Robin and Grémare, Agathe and Durand, Marlène and Sentilhes, Loic and Catros, Sylvain and Gindraux, Florelle and L'Heureux, Nicolas and Fricain, Jean-Christophe
Title Comparison of amniotic membrane versus the induced membrane for bone regeneration in long bone segmental defects using calcium phosphate cement loaded with BMP-2 [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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DOI/URL URL DOI
Abstract
Thanks to its biological properties, the human amniotic membrane (HAM) combined with a bone substitute could be a single-step surgical alternative to the two-step Masquelet induced membrane (IM) technique for regeneration of critical bone defects. However, no study has directly compared these two membranes. We first designed a 3D-printed scaffold using calcium phosphate cement (CPC). We assessed its suitability in vitro to support human bone marrow mesenchymal stromal cells (hBMSCs) attachment and osteodifferentiation. We then performed a rat femoral critical size defect to compare the two-step IM technique with a single-step approach using the HAM. Five conditions were compared. Group 1 was left empty. Group 2 received the CPC scaffold loaded with rh-BMP2 (CPC/BMP2). Group 3 and 4 received the CPC/BMP2 scaffold covered with lyophilized or decellularized/lyophilized HAM. Group 5 underwent a two- step induced membrane procedure with insertion of a polymethylmethacrylate (PMMA) spacer followed by, after 4 weeks, its replacement with the CPC/BMP2 scaffold wrapped in the IM. Micro-CT and histomorphometric analysis were performed after six weeks. Results showed that the CPC scaffold supported the proliferation and osteodifferentiation of hBMSCs in vitro. In vivo, the CPC/BMP2 scaffold very efficiently induced bone formation and led to satisfactory healing of the femoral defect, in a single-step, without autograft or the need for any membrane covering. In this study, there was no difference between the two-step induced membrane procedure and a single step approach. However, the results indicated that none of the tested membranes further enhanced bone healing compared to the CPC/BMP2 group.
AUTHOR Petretta, Mauro and Gambardella, Alessandro and Boi, Marco and Berni, Matteo and Cavallo, Carola and Marchiori, Gregorio and Maltarello, Maria Cristina and Bellucci, Devis and Fini, Milena and Baldini, Nicola and Grigolo, Brunella and Cannillo, Valeria
Title Composite Scaffolds for Bone Tissue Regeneration Based on PCL and Mg-Containing Bioactive Glasses [Abstract]
Year 2021
Journal/Proceedings Biology
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DOI/URL DOI
Abstract
Polycaprolactone (PCL) is widely used in additive manufacturing for the construction of scaffolds for tissue engineering because of its good bioresorbability, biocompatibility, and processability. Nevertheless, its use is limited by its inadequate mechanical support, slow degradation rate and the lack of bioactivity and ability to induce cell adhesion and, thus, bone tissue regeneration. In this study, we fabricated 3D PCL scaffolds reinforced with a novel Mg-doped bioactive glass (Mg-BG) characterized by good mechanical properties and biological reactivity. An optimization of the printing parameters and scaffold fabrication was performed; furthermore, an extensive microtopography characterization by scanning electron microscopy and atomic force microscopy was carried out. Nano-indentation tests accounted for the mechanical properties of the scaffolds, whereas SBF tests and cytotoxicity tests using human bone-marrow-derived mesenchymal stem cells (BM-MSCs) were performed to evaluate the bioactivity and in vitro viability. Our results showed that a 50/50 wt% of the polymer-to-glass ratio provides scaffolds with a dense and homogeneous distribution of Mg-BG particles at the surface and roughness twice that of pure PCL scaffolds. Compared to pure PCL (hardness H = 35 ± 2 MPa and Young’s elastic modulus E = 0.80 ± 0.05 GPa), the 50/50 wt% formulation showed H = 52 ± 11 MPa and E = 2.0 ± 0.2 GPa, hence, it was close to those of trabecular bone. The high level of biocompatibility, bioactivity, and cell adhesion encourages the use of the composite PCL/Mg-BG scaffolds in promoting cell viability and supporting mechanical loading in the host trabecular bone.
AUTHOR Zhang, Xiao and Liu, Yang and Luo, Chunyang and Zhai, Chenjun and Li, Zuxi and Zhang, Yi and Yuan, Tao and Dong, Shilei and Zhang, Jiyong and Fan, Weimin
Title Crosslinker-free silk/decellularized extracellular matrix porous bioink for 3D bioprinting-based cartilage tissue engineering [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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DOI/URL URL DOI
Abstract
As cartilage tissue lacks the innate ability to mount an adequate regeneration response, damage to it is detrimental to the quality of life of the subject. The emergence of three-dimensional bioprinting (3DBP) technology presents an opportunity to repair articular cartilage defects. However, widespread adoption of this technique has been impeded by difficulty in preparing a suitable bioink and the toxicity inherent in the chemical crosslinking process of most bioinks. Our objective was to develop a crosslinker-free bioink with the same biological activity as the original cartilage extracellular matrix (ECM) and good mechanical strength. We prepared bioinks containing different concentrations of silk fibroin and decellularized extracellular matrix (SF-dECM bioinks) mixed with bone marrow mesenchymal stem cells (BMSCs) for 3D bioprinting. SF and dECM interconnect with each other through physical crosslinking and entanglement. A porous structure was formed by removing the polyethylene glycol from the SF-dECM bioink. The results showed the SF-dECM construct had a suitable mechanical strength and degradation rate, and the expression of chondrogenesis-specific genes was found to be higher than that of the SF control construct group. Finally, we confirmed that a SF-dECM construct that was designed to release TGF-β3 had the ability to promote chondrogenic differentiation of BMSCs and provided a good cartilage repair environment, suggesting it is an ideal scaffold for cartilage tissue engineering.
AUTHOR Puertas-Bartolomé, María and Włodarczyk-Biegun, Małgorzata K. and del Campo, Aránzazu and Vázquez-Lasa, Blanca and San Román, Julio
Title Development of bioactive catechol functionalized nanoparticles applicable for 3D bioprinting [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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DOI/URL URL DOI
Abstract
Efficient wound treatments to target specific events in the healing process of chronic wounds constitute a significant aim in regenerative medicine. In this sense, nanomedicine can offer new opportunities to improve the effectiveness of existing wound therapies. The aim of this study was to develop catechol bearing polymeric nanoparticles (NPs) and to evaluate their potential in the field of wound healing. Thus, NPs wound healing promoting activities, potential for drug encapsulation and controlled release, and further incorporation in a hydrogel bioink formulation to fabricate cell-laden 3D scaffolds are studied. NPs with 2 and 29 M % catechol contents (named NP2 and NP29) were obtained by nanoprecipitation and presented hydrodynamic diameters of 100 and 75 nm respectively. These nanocarriers encapsulated the hydrophobic compound coumarin-6 with 70% encapsulation efficiency values. In cell culture studies, the NPs had a protective effect in RAW 264.7 macrophages against oxidative stress damage induced by radical oxygen species (ROS). They also presented a regulatory effect on the inflammatory response of stimulated macrophages and promoted upregulation of the vascular endothelial growth factor (VEGF) in fibroblasts and endothelial cells. In particular, NP29 were used in a hydrogel bioink formulation using carboxymethyl chitosan and hyaluronic acid as polymeric matrices. Using a reactive mixing bioprinting approach, NP-loaded hydrogel scaffolds with good structural integrity, shape fidelity and homogeneous NPs dispersion, were obtained. The in vitro catechol NPs release profile of the printed scaffolds revealed a sustained delivery. The bioprinted scaffolds supported viability and proliferation of encapsulated L929 fibroblasts over 14 days. We envision that the catechol functionalized NPs and resulting bioactive bioink presented in this work offer promising advantages for wound healing applications, as they: 1) support controlled release of bioactive catechol NPs to the wound site; 2) can incorporate additional therapeutic functions by co-encapsulating drugs; 3) can be printed into 3D scaffolds with tailored geometries based on patient requirements.
AUTHOR Kamdem Tamo, Arnaud and Doench, Ingo and Walter, Lukas and Montembault, Alexandra and Sudre, Guillaume and David, Laurent and Morales-Helguera, Aliuska and Selig, Mischa and Rolauffs, Bernd and Bernstein, Anke and Hoenders, Daniel and Walther, Andreas and Osorio-Madrazo, Anayancy
Title Development of Bioinspired Functional Chitosan/Cellulose Nanofiber 3D Hydrogel Constructs by 3D Printing for Application in the Engineering of Mechanically Demanding Tissues [Abstract]
Year 2021
Journal/Proceedings Polymers
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DOI/URL DOI
Abstract
Soft tissues are commonly fiber-reinforced hydrogel composite structures, distinguishable from hard tissues by their low mineral and high water content. In this work, we proposed the development of 3D printed hydrogel constructs of the biopolymers chitosan (CHI) and cellulose nanofibers (CNFs), both without any chemical modification, which processing did not incorporate any chemical crosslinking. The unique mechanical properties of native cellulose nanofibers offer new strategies for the design of environmentally friendly high mechanical performance composites. In the here proposed 3D printed bioinspired CNF-filled CHI hydrogel biomaterials, the chitosan serves as a biocompatible matrix promoting cell growth with balanced hydrophilic properties, while the CNFs provide mechanical reinforcement to the CHI-based hydrogel. By means of extrusion-based printing (EBB), the design and development of 3D functional hydrogel scaffolds was achieved by using low concentrations of chitosan (2.0–3.0% (w/v)) and cellulose nanofibers (0.2–0.4% (w/v)). CHI/CNF printed hydrogels with good mechanical performance (Young’s modulus 3.0 MPa, stress at break 1.5 MPa, and strain at break 75%), anisotropic microstructure and suitable biological response, were achieved. The CHI/CNF composition and processing parameters were optimized in terms of 3D printability, resolution, and quality of the constructs (microstructure and mechanical properties), resulting in good cell viability. This work allows expanding the library of the so far used biopolymer compositions for 3D printing of mechanically performant hydrogel constructs, purely based in the natural polymers chitosan and cellulose, offering new perspectives in the engineering of mechanically demanding hydrogel tissues like intervertebral disc (IVD), cartilage, meniscus, among others.
AUTHOR Kjar, Andrew and McFarland, Bailey and Mecham, Keetch and Harward, Nathan and Huang, Yu
Title Engineering of tissue constructs using coaxial bioprinting [Abstract]
Year 2021
Journal/Proceedings Bioactive Materials
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DOI/URL URL DOI
Abstract
Bioprinting is a rapidly developing technology for the precise design and manufacture of tissues in various biological systems or organs. Coaxial extrusion bioprinting, an emergent branch, has demonstrated a strong potential to enhance bioprinting's engineering versatility. Coaxial bioprinting assists in the fabrication of complex tissue constructs, by enabling concentric deposition of biomaterials. The fabricated tissue constructs started with simple, tubular vasculature but have been substantially developed to integrate complex cell composition and self-assembly, ECM patterning, controlled release, and multi-material gradient profiles. This review article begins with a brief overview of coaxial printing history, followed by an introduction of crucial engineering components. Afterward, we review the recent progress and untapped potential in each specific organ or biological system, and demonstrate how coaxial bioprinting facilitates the creation of tissue constructs. Ultimately, we conclude that this growing technology will contribute significantly to capabilities in the fields of in vitro modeling, pharmaceutical development, and clinical regenerative medicine.
AUTHOR Firipis, Kate and Nisbet, David R. and Franks, Stephanie J. and Kapsa, Robert M. I. and Pirogova, Elena and Williams, Richard J. and Quigley, Anita
Title Enhancing Peptide Biomaterials for Biofabrication [Abstract]
Year 2021
Journal/Proceedings Polymers
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DOI/URL URL DOI
Abstract
Biofabrication using well-matched cell/materials systems provides unprecedented opportunities for dealing with human health issues where disease or injury overtake the body’s native regenerative abilities. Such opportunities can be enhanced through the development of biomaterials with cues that appropriately influence embedded cells into forming functional tissues and organs. In this context, biomaterials’ reliance on rigid biofabrication techniques needs to support the incorporation of a hierarchical mimicry of local and bulk biological cues that mimic the key functional components of native extracellular matrix. Advances in synthetic self-assembling peptide biomaterials promise to produce reproducible mimics of tissue-specific structures and may go some way in overcoming batch inconsistency issues of naturally sourced materials. Recent work in this area has demonstrated biofabrication with self-assembling peptide biomaterials with unique biofabrication technologies to support structural fidelity upon 3D patterning. The use of synthetic self-assembling peptide biomaterials is a growing field that has demonstrated applicability in dermal, intestinal, muscle, cancer and stem cell tissue engineering.
AUTHOR Oliveira, Hugo and Médina, Chantal and Stachowicz, Marie-Laure and Paiva dos Santos, Bruno and Chagot, Lise and Dusserre, Nathalie and Fricain, Jean-Christophe
Title Extracellular matrix (ECM)-derived bioinks designed to foster vasculogenesis and neurite outgrowth: Characterization and bioprinting [Abstract]
Year 2021
Journal/Proceedings Bioprinting
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DOI/URL URL DOI
Abstract
The field of bioprinting has shown a tremendous development in recent years, focusing on the development of advanced in vitro models and on regeneration approaches. In this scope, the lack of suitable biomaterials that can be efficiently formulated as printable bioinks, while supporting specific cellular events, is currently considered as one of the main limitations in the field. Indeed, extracellular matrix (ECM)-derived biomaterials formulated to enable printability and support cellular response, for instance via integrin binding, are eagerly awaited in the field of bioprinting. Several bioactive laminin sequences, including peptides such as YIGSR and IKVAV, have been identified to promote endothelial cell attachment and/or neurite outgrowth and guidance, respectively. Here, we show the development of two distinct bioinks, designed to foster vasculogenesis or neurogenesis, based on methacrylated collagen and hyaluronic acid (CollMA and HAMA, respectively), both relevant ECM-derived polymers, and on their combination with cysteine-flanked laminin-derived peptides. Using this strategy, it was possible to optimize the bioink printability, by tuning CollMA and HAMA concentration and ratio, and modulate their bioactivity, through adjustments in the cell-active peptide sequence spatial density, without compromising cell viability. We demonstrated that cell-specific bioinks could be customized for the bioprinting of both human umbilical vein cord endothelial cells (HUVECs) or adult rat sensory neurons from the dorsal root ganglia, and could stimulate both vasculogenesis and neurite outgrowth, respectively. This approach holds great potential as it can be tailored to other cellular models, due to its inherent capacity to accommodate different peptide compositions and to generate complex peptide mixtures and/or gradients.
AUTHOR Tarun Agarwal and Marco Costantini and Tapas Kumar Maiti
Title Extrusion 3D printing with Pectin-based ink formulations: Recent trends in tissue engineering and food manufacturing [Abstract]
Year 2021
Journal/Proceedings Biomedical Engineering Advances
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DOI/URL URL DOI
Abstract
3D printing technologies are rapidly revolutionizing all manufacturing sectors due to their ability to create objects with complex geometries in a reproducible and automated manner using material/cell-based formulations, precisely termed printing inks. In this regard, pectin, a naturally occurring plant polysaccharide, has been proposed as a potential component of ink formulations. In this mini-review, we would overview the most recent advances made with pectin-based inks in the fields of tissue engineering and food manufacturing. We also discuss various strategies used to formulate 3D printable pectin inks. Finally, various challenges and prospects for future development are discussed.
AUTHOR Götz, Lisa-Marie and Holeczek, Katharina and Groll, Jürgen and Jüngst, Tomasz and Gbureck, Uwe
Title Extrusion-Based 3D Printing of Calcium Magnesium Phosphate Cement Pastes for Degradable Bone Implants [Abstract]
Year 2021
Journal/Proceedings Materials
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DOI/URL URL DOI
Abstract
This study aimed to develop printable calcium magnesium phosphate pastes that harden by immersion in ammonium phosphate solution post-printing. Besides the main mineral compound, biocompatible ceramic, magnesium oxide and hydroxypropylmethylcellulose (HPMC) were the crucial components. Two pastes with different powder to liquid ratios of 1.35 g/mL and 1.93 g/mL were characterized regarding their rheological properties. Here, ageing over the course of 24 h showed an increase in viscosity and extrusion force, which was attributed to structural changes in HPMC as well as the formation of magnesium hydroxide by hydration of MgO. The pastes enabled printing of porous scaffolds with good dimensional stability and enabled a setting reaction to struvite when immersed in ammonium phosphate solution. Mechanical performance under compression was approx. 8–20 MPa as a monolithic structure and 1.6–3.0 MPa for printed macroporous scaffolds, depending on parameters such as powder to liquid ratio, ageing time, strand thickness and distance.
AUTHOR Paulo Roberto {Lopes Nalesso} and Weiguang Wang and Yanhao Hou and Leonardo Bagne and Amanda Tavares Pereira and Julia Venturini Helaehil and Thiago Antônio {Moretti de Andrade} and Gabriela Bortolança Chiarotto and Paulo Bártolo and Guilherme Ferreira Caetano
Title In vivo investigation of 3D printed polycaprolactone/graphene electro-active bone scaffolds [Abstract]
Year 2021
Journal/Proceedings Bioprinting
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DOI/URL URL DOI
Abstract
Additive manufactured scaffolds are widely used as 3D support structures for tissue engineering. This paper investigates the mechanisms behind bone regeneration due to the combined use of 3D printed poly (ϵ-caprolactone)/graphene (PCL/G) electro-active scaffolds and electrical stimulation. A comprehensive in vivo study was conducted to assess the proposed approach, using a rat model. Results show that the combined use of electro-active scaffolds and electrical stimulation therapy accelerates the bone regeneration process and the formation of more organized new bone, through fast angiogenesis, and a rapid transition to the mineralization and bone remodelling phase. The mechanism is investigated and explained.
AUTHOR e Silva, Edney P. and Huang, Boyang and Helaehil, Júlia V. and Nalesso, Paulo R. L. and Bagne, Leonardo and de Oliveira, Maraiara A. and Albiazetti, Gabriela C. C. and Aldalbahi, Ali and El-Newehy, Mohamed and Santamaria-Jr, Milton and Mendonça, Fernanda A. S. and Bártolo, Paulo and Caetano, Guilherme F.
Title In vivo study of conductive 3D printed PCL/MWCNTs scaffolds with electrical stimulation for bone tissue engineering [Abstract]
Year 2021
Journal/Proceedings Bio-Design and Manufacturing
Reftype e Silva2021
DOI/URL DOI
Abstract
Critical bone defects are considered one of the major clinical challenges in reconstructive bone surgery. The combination of 3D printed conductive scaffolds and exogenous electrical stimulation (ES) is a potential favorable approach for bone tissue repair. In this study, 3D conductive scaffolds made with biocompatible and biodegradable polycaprolactone (PCL) and multi-walled carbon nanotubes (MWCNTs) were produced using the extrusion-based additive manufacturing to treat large calvary bone defects in rats. Histology results show that the use of PCL/MWCNTs scaffolds and ES contributes to thicker and increased bone tissue formation within the bone defect. Angiogenesis and mineralization are also significantly promoted using high concentration of MWCNTs (3 wt%) and ES. Moreover, scaffolds favor the tartrate-resistant acid phosphatase (TRAP) positive cell formation, while the addition of MWCNTs seems to inhibit the osteoclastogenesis but present limited effects on the osteoclast functionalities (receptor activator of nuclear factor κβ ligand (RANKL) and osteoprotegerin (OPG) expressions). The use of ES promotes the osteoclastogenesis and RANKL expressions, showing a dominant effect in the bone remodeling process. These results indicate that the combination of 3D printed conductive PCL/MWCNTs scaffold and ES is a promising strategy to treat critical bone defects and provide a cue to establish an optimal protocol to use conductive scaffolds and ES for bone tissue engineering.
AUTHOR Zamani, Yasaman and Amoabediny, Ghassem and Mohammadi, Javad and Zandieh-Doulabi, Behrouz and Klein-Nulend, Jenneke and Helder, Marco N.
Title Increased Osteogenic Potential of Pre-Osteoblasts on Three-Dimensional Printed Scaffolds Compared to Porous Scaffolds for Bone Regeneration [Abstract]
Year 2021
Journal/Proceedings Iranian Biomedical Journal
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Abstract
Background: One of the main challenges with conventional scaffold fabrication methods is the inability to control scaffold architecture. Recently, scaffolds with controlled shape and architecture have been fabricated using three-dimensional printing (3DP). Herein, we aimed to determine whether the much tighter control of microstructure of 3DP poly(lactic-co-glycolic) acid/β-tricalcium phosphate (PLGA/β-TCP) scaffolds is more effective in promoting osteogenesis than porous scaffolds produced by solvent casting/porogen leaching. Methods: Physical and mechanical properties of porous and 3DP scaffolds were studied. The response of pre-osteoblasts to the scaffolds was analyzed after 14 days. Results: The 3DP scaffolds had a smoother surface (Ra: 22 ± 3 µm) relative to the highly rough surface of porous scaffolds (Ra: 110 ± 15 µm). Water contact angle was 112 ± 4° on porous and 76 ± 6° on 3DP scaffolds. Porous and 3DP scaffolds had the pore size of 408 ± 90 and 315 ± 17 µm and porosity of 85 ± 5% and 39 ± 7%, respectively. Compressive strength of 3DP scaffolds (4.0 ± 0.3 MPa) was higher than porous scaffolds (1.7 ± 0.2 MPa). Collagenous matrix deposition was similar on both scaffolds. Cells proliferated from day 1 to day 14 by fourfold in porous and by 3.8-fold in 3DP scaffolds. Alkaline phosphatase (ALP) activity was 21-fold higher in 3DP scaffolds than porous scaffolds. Conclusion: The 3DP scaffolds show enhanced mechanical properties and ALP activity compared to porous scaffolds in vitro, suggesting that 3DP PLGA/β-TCP scaffolds are possibly more favorable for bone formation.
AUTHOR Daskalakis, Evangelos and Liu, Fengyuan and Huang, Boyang and Acar, Anil A. and Cooper, Glen and Weightman, Andrew and Blunn, Gordon and Koç, Bahattin and Bartolo, Paulo
Title Investigating the Influence of Architecture and Material Composition of 3D Printed Anatomical Design Scaffolds for Large Bone Defects [Abstract]
Year 2021
Journal/Proceedings International Journal of Bioprinting; Vol 7, No 2 (2021)
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Abstract
There is a significant unmet clinical need to prevent amputations due to large bone loss injuries. We are addressing this problem by developing a novel, cost-effective osseointegrated prosthetic solution based on the use of modular pieces, bone bricks, made with biocompatible and biodegradable materials that fit together in a Lego-like way to form the prosthesis. This paper investigates the anatomical designed bone bricks with different architectures, pore size gradients, and material compositions. Polymer and polymer-composite 3D printed bone bricks are extensively morphological, mechanical, and biological characterized. Composite bone bricks were produced by mixing polycaprolactone (PCL) with different levels of hydroxyapatite (HA) and β-tri-calcium phosphate (TCP). Results allowed to establish a correlation between bone bricks architecture and material composition and bone bricks performance. Reinforced bone bricks showed improved mechanical and biological results. Best mechanical properties were obtained with PCL/TCP bone bricks with 38 double zig-zag filaments and 14 spiral-like pattern filaments, while the best biological results were obtained with PCL/HA bone bricks based on 25 double zig-zag filaments and 14 spiral-like pattern filaments.
AUTHOR Wang, Weiguang and Chen, Jun-Xiang and Hou, Yanhao and Bartolo, Paulo and Chiang, Wei-Hung
Title Investigations of Graphene and Nitrogen-Doped Graphene Enhanced Polycaprolactone 3D Scaffolds for Bone Tissue Engineering [Abstract]
Year 2021
Journal/Proceedings Nanomaterials
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Abstract
Scaffolds play a key role in tissue engineering applications. In the case of bone tissue engineering, scaffolds are expected to provide both sufficient mechanical properties to withstand the physiological loads, and appropriate bioactivity to stimulate cell growth. In order to further enhance cell–cell signaling and cell–material interaction, electro-active scaffolds have been developed based on the use of electrically conductive biomaterials or blending electrically conductive fillers to non-conductive biomaterials. Graphene has been widely used as functioning filler for the fabrication of electro-active bone tissue engineering scaffolds, due to its high electrical conductivity and potential to enhance both mechanical and biological properties. Nitrogen-doped graphene, a unique form of graphene-derived nanomaterials, presents significantly higher electrical conductivity than pristine graphene, and better surface hydrophilicity while maintaining a similar mechanical property. This paper investigates the synthesis and use of high-performance nitrogen-doped graphene as a functional filler of poly(ɛ-caprolactone) (PCL) scaffolds enabling to develop the next generation of electro-active scaffolds. Compared to PCL scaffolds and PCL/graphene scaffolds, these novel scaffolds present improved in vitro biological performance.
AUTHOR Trucco, Diego and Sharma, Aarushi and Manferdini, Cristina and Gabusi, Elena and Petretta, Mauro and Desando, Giovanna and Ricotti, Leonardo and Chakraborty, Juhi and Ghosh, Sourabh and Lisignoli, Gina
Title Modeling and Fabrication of Silk Fibroin-Gelatin-Based Constructs Using Extrusion-Based Three-Dimensional Bioprinting [Abstract]
Year 2021
Journal/Proceedings ACS Biomater. Sci. Eng.
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DOI/URL DOI
Abstract
Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure’s fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs’ fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications. Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure’s fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs’ fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications.
AUTHOR Petretta, Mauro and Gambardella, Alessandro and Desando, Giovanna and Cavallo, Carola and Bartolotti, Isabella and Shelyakova, Tatiana and Goranov, Vitaly and Brucale, Marco and Dediu, Valentin Alek and Fini, Milena and Grigolo, Brunella
Title Multifunctional 3D-Printed Magnetic Polycaprolactone/Hydroxyapatite Scaffolds for Bone Tissue Engineering [Abstract]
Year 2021
Journal/Proceedings Polymers
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Abstract
Multifunctional and resistant 3D structures represent a great promise and a great challenge in bone tissue engineering. This study addresses this problem by employing polycaprolactone (PCL)-based scaffolds added with hydroxyapatite (HAp) and superparamagnetic iron oxide nanoparticles (SPION), able to drive on demand the necessary cells and other bioagents for a high healing efficiency. PCL-HAp-SPION scaffolds with different concentrations of the superparamagnetic component were developed through the 3D-printing technology and the specific topographical features were detected by Atomic Force and Magnetic Force Microscopy (AFM-MFM). AFM-MFM measurements confirmed a homogenous distribution of HAp and SPION throughout the surface. The magnetically assisted seeding of cells in the scaffold resulted most efficient for the 1% SPION concentration, providing good cell entrapment and adhesion rates. Mesenchymal Stromal Cells (MSCs) seeded onto PCL-HAp-1% SPION showed a good cell proliferation and intrinsic osteogenic potential, indicating no toxic effects of the employed scaffold materials. The performed characterizations and the collected set of data point on the inherent osteogenic potential of the newly developed PCL-HAp-1% SPION scaffolds, endorsing them towards next steps of in vitro and in vivo studies and validations.
AUTHOR Korpershoek, Jasmijn V. and Ruijter, Mylène de and Terhaard, Bastiaan F. and Hagmeijer, Michella H. and Saris, Daniël B.F. and Castilho, Miguel and Malda, Jos and Vonk, Lucienne A.
Title Potential of Melt Electrowritten Scaffolds Seeded with Meniscus Cells and Mesenchymal Stromal Cells [Abstract]
Year 2021
Journal/Proceedings International Journal of Molecular Sciences
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Abstract
Meniscus injury and meniscectomy are strongly related to osteoarthritis, thus there is a clinical need for meniscus replacement. The purpose of this study is to create a meniscus scaffold with micro-scale circumferential and radial fibres suitable for a one-stage cell-based treatment. Poly-caprolactone-based scaffolds with three different architectures were made using melt electrowriting (MEW) technology and their in vitro performance was compared with scaffolds made using fused-deposition modelling (FDM) and with the clinically used Collagen Meniscus Implants® (CMI®). The scaffolds were seeded with meniscus and mesenchymal stromal cells (MSCs) in fibrin gel and cultured for 28 d. A basal level of proteoglycan production was demonstrated in MEW scaffolds, the CMI®, and fibrin gel control, yet within the FDM scaffolds less proteoglycan production was observed. Compressive properties were assessed under uniaxial confined compression after 1 and 28 d of culture. The MEW scaffolds showed a higher Young’s modulus when compared to the CMI® scaffolds and a higher yield point compared to FDM scaffolds. This study demonstrates the feasibility of creating a wedge-shaped meniscus scaffold with MEW using medical-grade materials and seeding the scaffold with a clinically-feasible cell number and -type for potential translation as a one-stage treatment.
AUTHOR Lotz, Benedict and Bothe, Friederike and Deubel, Anne-Kathrin and Hesse, Eliane and Renz, Yvonne and Werner, Carsten and Schäfer, Simone and Böck, Thomas and Groll, Jürgen and von Rechenberg, Brigitte and Richter, Wiltrud and Hagmann, Sebastien
Title Preclinical Testing of New Hydrogel Materials for Cartilage Repair: Overcoming Fixation Issues in a Large Animal Model [Abstract]
Year 2021
Journal/Proceedings International Journal of Biomaterials
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DOI/URL DOI
Abstract
Reinforced hydrogels represent a promising strategy for tissue engineering of articular cartilage. They can recreate mechanical and biological characteristics of native articular cartilage and promote cartilage regeneration in combination with mesenchymal stromal cells. One of the limitations of in vivo models for testing the outcome of tissue engineering approaches is implant fixation. The high mechanical stress within the knee joint, as well as the concave and convex cartilage surfaces, makes fixation of reinforced hydrogel challenging. Methods. Different fixation methods for full-thickness chondral defects in minipigs such as fibrin glue, BioGlue®, covering, and direct suturing of nonenforced and enforced constructs were compared. Because of insufficient fixation in chondral defects, superficial osteochondral defects in the femoral trochlea, as well as the femoral condyle, were examined using press-fit fixation. Two different hydrogels (starPEG and PAGE) were compared by 3D-micro-CT (μCT) analysis as well as histological analysis. Results. Our results showed fixation of below 50% for all methods in chondral defects. A superficial osteochondral defect of 1 mm depth was necessary for long-term fixation of a polycaprolactone (PCL)-reinforced hydrogel construct. Press-fit fixation seems to be adapted for a reliable fixation of 95% without confounding effects of glue or suture material. Despite the good integration of our constructs, especially in the starPEG group, visible bone lysis was detected in micro-CT analysis. There was no significant difference between the two hydrogels (starPEG and PAGE) and empty control defects regarding regeneration tissue and cell integration. However, in the starPEG group, more cell-containing hydrogel fragments were found within the defect area. Conclusion. Press-fit fixation in a superficial osteochondral defect in the medial trochlear groove of adult minipigs is a promising fixation method for reinforced hydrogels. To avoid bone lysis, future approaches should focus on multilayered constructs recreating the zonal cartilage as well as the calcified cartilage and the subchondral bone plate.
AUTHOR Anh Tong and Quang Long Pham and Paul Abatemarco and Austin Mathew and Dhruv Gupta and Siddharth Iyer and Roman Voronov
Title Review of Low-Cost 3D Bioprinters: State of the Market and Observed Future Trends [Abstract]
Year 2021
Journal/Proceedings SLAS TECHNOLOGY: Translating Life Sciences Innovation
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Three-dimensional (3D) bioprinting has become mainstream for precise and repeatable high-throughput fabrication of complex cell cultures and tissue constructs in drug testing and regenerative medicine, food products, dental and medical implants, biosensors, and so forth. Due to this tremendous growth in demand, an overwhelming amount of hardware manufacturers have recently flooded the market with different types of low-cost bioprinter models—a price segment that is most affordable to typical-sized laboratories. These machines range in sophistication, type of the underlying printing technology, and possible add-ons/features, which makes the selection process rather daunting (especially for a nonexpert customer). Yet, the review articles available in the literature mostly focus on the technical aspects of the printer technologies under development, as opposed to explaining the differences in what is already on the market. In contrast, this paper provides a snapshot of the fast-evolving low-cost bioprinter niche, as well as reputation profiles (relevant to delivery time, part quality, adherence to specifications, warranty, maintenance, etc.) of the companies selling these machines. Specifically, models spanning three dominant technologies—microextrusion, droplet-based/inkjet, and light-based/crosslinking—are reviewed. Additionally, representative examples of high-end competitors (including up-and-coming microfluidics-based bioprinters) are discussed to highlight their major differences and advantages relative to the low-cost models. Finally, forecasts are made based on the trends observed during this survey, as to the anticipated trickling down of the high-end technologies to the low-cost printers. Overall, this paper provides insight for guiding buyers on a limited budget toward making informed purchasing decisions in this fast-paced market.
AUTHOR Iria Seoane-Viaño and Patricija Januskaite and Carmen Alvarez-Lorenzo and Abdul W. Basit and Alvaro Goyanes
Title Semi-solid extrusion 3D printing in drug delivery and biomedicine: Personalised solutions for healthcare challenges [Abstract]
Year 2021
Journal/Proceedings Journal of Controlled Release
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DOI/URL URL DOI
Abstract
Three-dimensional (3D) printing is an innovative additive manufacturing technology, capable of fabricating unique structures in a layer-by-layer manner. Semi-solid extrusion (SSE) is a subset of material extrusion 3D printing, and through the sequential deposition of layers of gel or paste creates objects of any desired size and shape. In comparison to other extrusion-based technologies, SSE 3D printing employs low printing temperatures which makes it suitable for drug delivery and biomedical applications, and the use of disposable syringes provides benefits in meeting critical quality requirements for pharmaceutical use. Besides pharmaceutical manufacturing, SSE 3D printing has attracted increasing attention in the field of bioelectronics, particularly in the manufacture of biosensors capable of measuring physiological parameters or as a means to trigger drug release from medical devices. This review begins by highlighting the major printing process parameters and material properties that influence the feasibility of transforming a 3D design into a 3D object, and follows with a discussion on the current SSE 3D printing developments and their applications in the fields of pharmaceutics, bioprinting and bioelectronics. Finally, the advantages and limitations of this technology are explored, before focusing on its potential clinical applications and suitability for preparing personalised medicines.
AUTHOR Chawla, Shikha and Desando, Giovanna and Gabusi, Elena and Sharma, Aarushi and Trucco, Diego and Chakraborty, Juhi and Manferdini, Cristina and Petretta, Mauro and Lisignoli, Gina and Ghosh, Sourabh
Title The effect of silk-gelatin bioink and TGF-β3 on mesenchymal stromal cells in 3D bioprinted chondrogenic constructs: A proteomic study [Abstract]
Year 2021
Journal/Proceedings Journal of Materials Research
Reftype Chawla2021
DOI/URL DOI
Abstract
Major limitation of 3D bioprinting is the poor understanding of the role of bioink in modulating molecular signaling pathways. Phenotypically stable engineered articular cartilage was fabricated using silk fibroin-gelatin (SF-G) bioink and progenitor cells or mature articular chondrocytes. In the current study, role of SF-G bioink in modulating in vitro chondrogenic signaling pathways in human bone marrow-derived stromal cells (hMSCs) is elucidated. The interaction between SF-G bioink and hMSCs augmented several chondrogenic pathways, including Wnt, HIF-1, and Notch. We explored the debatable role of TGF-β signaling, by assessing the differential protein expression by hMSCs-laden bioprinted constructs in the presence and absence of TGF-β3. hMSCs-laden bioprinted constructs contained a large percentage of collagen type II and Filamin-B, typical to the native articular cartilage. Hypertrophy markers were not identified following TGF-β3 addition. This is first detailed proteomics analysis to identify articular cartilage-specific pathways in SF-G-based 3D bioprinted construct.
AUTHOR Park, Wonbin and Gao, Ge and Cho, Dong-Woo
Title Tissue-Specific Decellularized Extracellular Matrix Bioinks for Musculoskeletal Tissue Regeneration and Modeling Using 3D Bioprinting Technology [Abstract]
Year 2021
Journal/Proceedings International Journal of Molecular Sciences
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Abstract
The musculoskeletal system is a vital body system that protects internal organs, supports locomotion, and maintains homeostatic function. Unfortunately, musculoskeletal disorders are the leading cause of disability worldwide. Although implant surgeries using autografts, allografts, and xenografts have been conducted, several adverse effects, including donor site morbidity and immunoreaction, exist. To overcome these limitations, various biomedical engineering approaches have been proposed based on an understanding of the complexity of human musculoskeletal tissue. In this review, the leading edge of musculoskeletal tissue engineering using 3D bioprinting technology and musculoskeletal tissue-derived decellularized extracellular matrix bioink is described. In particular, studies on in vivo regeneration and in vitro modeling of musculoskeletal tissue have been focused on. Lastly, the current breakthroughs, limitations, and future perspectives are described.
AUTHOR De Moor, Lise and Minne, Mendy and Tytgat, Liesbeth and Vercruysse, Chris and Dubruel, Peter and Van Vlierberghe, Sandra and Declercq, Heidi
Title Tuning the Phenotype of Cartilage Tissue Mimics by Varying Spheroid Maturation and Methacrylamide-Modified Gelatin Hydrogel Characteristics [Abstract]
Year 2021
Journal/Proceedings Macromolecular Bioscience
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DOI/URL DOI
Abstract
Abstract In hybrid bioprinting of cartilage tissue constructs, spheroids are used as cellular building blocks and combined with biomaterials for dispensing. However, biomaterial intrinsic cues can deeply affect cell fate and to date, the influence of hydrogel encapsulation on spheroid viability and phenotype has received limited attention. This study assesses this need and unravels 1) how the phenotype of spheroid-laden constructs can be tuned through adjusting the hydrogel physico–chemical properties and 2) if the spheroid maturation stage prior to encapsulation is a determining factor for the construct phenotype. Articular chondrocyte spheroids with a cartilage specific extracellular matrix (ECM) are generated and different maturation stages, early-, mid-, and late-stage (3, 7, and 14 days, respectively), are harvested and encapsulated in 10, 15, or 20 w/v% methacrylamide-modified gelatin (gelMA) for 14 days. The encapsulation of immature spheroids do not lead to a cartilage-like ECM production but when more mature mid- or late-stage spheroids are combined with a certain concentration of gelMA, a fibrocartilage-like as well as a hyaline cartilage-like phenotype can be induced. As a proof of concept, late-stage spheroids are bioprinted using a 10 w/v% gelMA–Irgacure 2959 solution with the aim to test the processing potential of the spheroid-laden bioink.
AUTHOR Lagatuz, M. and Vyas, R. J. and Predovic, M. and Lim, S. and Jacobs, N. and Martinho, M. and Valizadegan, H. and Kao, D. and Oza, N. and Theriot, C. A. and Zanello, S. B. and Taibbi, G. and Vizzeri, G. and Dupont, M. and Grant, M. B. and Lindner, D. J. and Reinecker, H.-C. and Pinhas, A. and Chui, T. Y. and Rosen, R. B. and Moldovan, N. and Vickerman, M. B. and Radhakrishnan, K. and Parsons-Wingerter, P.
Title Vascular Patterning as Integrative Readout of Complex Molecular and Physiological Signaling by VESsel GENeration Analysis [Abstract]
Year 2021
Journal/Proceedings J Vasc Res
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DOI/URL DOI
Abstract
The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.
AUTHOR Lagatuz, M. and Vyas, R. J. and Predovic, M. and Lim, S. and Jacobs, N. and Martinho, M. and Valizadegan, H. and Kao, D. and Oza, N. and Theriot, C. A. and Zanello, S. B. and Taibbi, G. and Vizzeri, G. and Dupont, M. and Grant, M. B. and Lindner, D. J. and Reinecker, H.-C. and Pinhas, A. and Chui, T. Y. and Rosen, R. B. and Moldovan, N. and Vickerman, M. B. and Radhakrishnan, K. and Parsons-Wingerter, P.
Title Vascular Patterning as Integrative Readout of Complex Molecular and Physiological Signaling by VESsel GENeration Analysis [Abstract]
Year 2021
Journal/Proceedings J Vasc Res
Reftype
DOI/URL DOI
Abstract
The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.
AUTHOR Lagatuz, M. and Vyas, R. J. and Predovic, M. and Lim, S. and Jacobs, N. and Martinho, M. and Valizadegan, H. and Kao, D. and Oza, N. and Theriot, C. A. and Zanello, S. B. and Taibbi, G. and Vizzeri, G. and Dupont, M. and Grant, M. B. and Lindner, D. J. and Reinecker, H.-C. and Pinhas, A. and Chui, T. Y. and Rosen, R. B. and Moldovan, N. and Vickerman, M. B. and Radhakrishnan, K. and Parsons-Wingerter, P.
Title Vascular Patterning as Integrative Readout of Complex Molecular and Physiological Signaling by VESsel GENeration Analysis [Abstract]
Year 2021
Journal/Proceedings J Vasc Res
Reftype
DOI/URL DOI
Abstract
The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.
AUTHOR Devillard, Chloé D. and Marquette, Christophe A.
Title Vascular Tissue Engineering: Challenges and Requirements for an Ideal Large Scale Blood Vessel [Abstract]
Year 2021
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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DOI/URL DOI
Abstract
Since the emergence of regenerative medicine and tissue engineering more than half a century ago, one obstacle has persisted: the in vitro creation of large-scale vascular tissue (>1 cm3) to meet the clinical needs of viable tissue grafts but also for biological research applications. Considerable advancements in biofabrication have been made since Weinberg and Bell, in 1986, created the first blood vessel from collagen, endothelial cells, smooth muscle cells and fibroblasts. The synergistic combination of advances in fabrication methods, availability of cell source, biomaterials formulation and vascular tissue development, promises new strategies for the creation of autologous blood vessels, recapitulating biological functions, structural functions, but also the mechanical functions of a native blood vessel. In this review, the main technological advancements in bio-fabrication are discussed with a particular highlights on 3D bioprinting technologies. The choice of the main biomaterials and cell sources, the use of dynamic maturation systems such as bioreactors and the associated clinical trials will be detailed. The remaining challenges in this complex engineering field will finally be discussed.
AUTHOR Hamid, Omar A. and Eltaher, Hoda M. and Sottile, Virginie and Yang, Jing
Title 3D bioprinting of a stem cell-laden, multi-material tubular composite: An approach for spinal cord repair [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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Abstract
Development of a biomimetic tubular scaffold capable of recreating developmental neurogenesis using pluripotent stem cells offers a novel strategy for the repair of spinal cord tissues. Recent advances in 3D printing technology have facilitated biofabrication of complex biomimetic environments by precisely controlling the 3D arrangement of various acellular and cellular components (biomaterials, cells and growth factors). Here, we present a 3D printing method to fabricate a complex, patterned and embryoid body (EB)-laden tubular scaffold composed of polycaprolactone (PCL) and hydrogel (alginate or gelatine methacrylate (GelMA)). Our results revealed 3D printing of a strong, macro-porous PCL/hydrogel tubular scaffold with a high capacity to control the porosity of the PCL scaffold, wherein the maximum porosity in the PCL wall was 15%. The method was equally employed to create spatiotemporal protein concentration within the scaffold, demonstrating its ability to generate linear and opposite gradients of model molecules (fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) and rhodamine). 3D bioprinting of EBs-laden GelMA was introduced as a novel 3D printing strategy to incorporate EBs in a hydrogel matrix. Cell viability and proliferation were measured post-printing. Following the bioprinting of EBs-laden 5% GelMA hydrogel, neural differentiation of EBs was induced using 1 μM retinoic acid (RA). The differentiated EBs contained βIII-tubulin positive neurons displaying axonal extensions and cells migration. Finally, 3D bioprinting of EBs-laden PCL/GelMA tubular scaffold successfully supported EBs neural differentiation and patterning in response to co-printing with 1 μM RA. 3D printing of a complex heterogeneous tubular scaffold that can encapsulate EBs, spatially controlled protein concentration and promote neuronal patterning will help in developing more biomimetic scaffolds capable of replicating the neural patterning which occurs during neural tube development.
AUTHOR Lin, Che-Wei and Su, Yu-Feng and Lee, Chih-Yun and Kang, Lin and Wang, Yan-Hsiung and Lin, Sung-Yen and Wang, Chih-Kuang
Title 3D printed bioceramics fabricated using negative thermoresponsive hydrogels and silicone oil sealing to promote bone formation in calvarial defects [Abstract]
Year 2020
Journal/Proceedings Ceramics International
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Abstract
The purpose of the present work was to investigate the potential for application and the effectiveness of osteoconductive scaffolds with bicontinuous phases of 3D printed bioceramics (3DP-BCs) based on reverse negative thermoresponsive hydrogels (poly[(N-isopropylacrylamide)-co-(methacrylic acid)]; p(NiPAAm-MAA)). 3DP-BCs have bioceramic objects and microchannel pores when created using robotic deposition additive manufacturing. We evaluated the benefits of silicone oil sealing on the 3DP-BC green body during the sintering process in terms of densification and structural stability. The shrinkage, density, porosity, element composition, phase structure and microstructural analyses and compression strength measurements of sintered 3DP-BC objects are presented and discussed in this study. In addition, the results of cell viability assays and bone healing analyses of the calvarial bone defects in a rabbit model were used to evaluate 3DP-BC performance. The main results indicated that these 3DP-BC scaffolds have optimal continuous pores and adequate compressive strength, which can enable the protection of calvarial defects and provide an environment for cell growth. Therefore, 3DP-BC scaffolds have better new bone regeneration efficiency in rabbit calvarial bone defect models than empty scaffolds and mold-forming bioceramic scaffolds (MF-BCs).
AUTHOR Critchley, Susan and Sheehy, Eamon J. and Cunniffe, Gráinne and Diaz-Payno, Pedro and Carroll, Simon F. and Jeon, Oju and Alsberg, Eben and Brama, Pieter A. J. and Kelly, Daniel J.
Title 3D printing of fibre-reinforced cartilaginous templates for the regeneration of osteochondral defects [Abstract]
Year 2020
Journal/Proceedings Acta Biomaterialia
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Abstract
Successful osteochondral defect repair requires regenerating the subchondral bone whilst simultaneously promoting the development of an overlying layer of articular cartilage that is resistant to vascularization and endochondral ossification. During skeletal development articular cartilage also functions as a surface growth plate, which postnatally is replaced by a more spatially complex bone-cartilage interface. Motivated by this developmental process, the hypothesis of this study is that bi-phasic, fibre-reinforced cartilaginous templates can regenerate both the articular cartilage and subchondral bone within osteochondral defects created in caprine joints. To engineer mechanically competent implants, we first compared a range of 3D printed fibre networks (PCL, PLA and PLGA) for their capacity to mechanically reinforce alginate hydrogels whilst simultaneously supporting mesenchymal stem cell (MSC) chondrogenesis in vitro. These mechanically reinforced, MSC-laden alginate hydrogels were then used to engineer the endochondral bone forming phase of bi-phasic osteochondral constructs, with the overlying chondral phase consisting of cartilage tissue engineered using a co-culture of infrapatellar fat pad derived stem/stromal cells (FPSCs) and chondrocytes. Following chondrogenic priming and subcutaneous implantation in nude mice, these bi-phasic cartilaginous constructs were found to support the development of vascularised endochondral bone overlaid by phenotypically stable cartilage. These fibre-reinforced, bi-phasic cartilaginous templates were then evaluated in clinically relevant, large animal (caprine) model of osteochondral defect repair. Although the quality of repair was variable from animal-to-animal, in general more hyaline-like cartilage repair was observed after 6 months in animals treated with bi-phasic constructs compared to animals treated with commercial control scaffolds. This variability in the quality of repair points to the need for further improvements in the design of 3D bioprinted implants for joint regeneration. Statement of Significance Successful osteochondral defect repair requires regenerating the subchondral bone whilst simultaneously promoting the development of an overlying layer of articular cartilage. In this study, we hypothesised that bi-phasic, fibre-reinforced cartilaginous templates could be leveraged to regenerate both the articular cartilage and subchondral bone within osteochondral defects. To this end we used 3D printed fibre networks to mechanically reinforce engineered transient cartilage, which also contained an overlying layer of phenotypically stable cartilage engineered using a co-culture of chondrocytes and stem cells. When chondrogenically primed and implanted into caprine osteochondral defects, these fibre-reinforced bi-phasic cartilaginous grafts were shown to spatially direct tissue development during joint repair. Such developmentally inspired tissue engineering strategies, enabled by advances in biofabrication and 3D printing, could form the basis of new classes of regenerative implants in orthopaedic medicine.
AUTHOR Wibowo, Arie and Vyas, Cian and Cooper, Glen and Qulub, Fitriyatul and Suratman, Rochim and Mahyuddin, Andi Isra and Dirgantara, Tatacipta and Bartolo, Paulo
Title 3D Printing of Polycaprolactone-Polyaniline Electroactive Scaffolds for Bone Tissue Engineering. [Abstract]
Year 2020
Journal/Proceedings Materials
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Abstract
Electrostimulation and electroactive scaffolds can positively influence and guide cellular behaviour and thus has been garnering interest as a key tissue engineering strategy. The development of conducting polymers such as polyaniline enables the fabrication of conductive polymeric composite scaffolds. In this study, we report on the initial development of a polycaprolactone scaffold incorporating different weight loadings of a polyaniline microparticle filler. The scaffolds are fabricated using screw-assisted extrusion-based 3D printing and are characterised for their morphological, mechanical, conductivity, and preliminary biological properties. The conductivity of the polycaprolactone scaffolds increases with the inclusion of polyaniline. The in vitro cytocompatibility of the scaffolds was assessed using human adipose-derived stem cells to determine cell viability and proliferation up to 21 days. A cytotoxicity threshold was reached at 1% wt. polyaniline loading. Scaffolds with 0.1% wt. polyaniline showed suitable compressive strength (6.45 ± 0.16 MPa) and conductivity (2.46 ± 0.65 × 10(-4) S/cm) for bone tissue engineering applications and demonstrated the highest cell viability at day 1 (88%) with cytocompatibility for up to 21 days in cell culture.
AUTHOR Zamani, Yasaman and Amoabediny, Ghassem and Mohammadi, Javad and Seddiqi, Hadi and Helder, Marco N. and Zandieh-Doulabi, Behrouz and Klein-Nulend, Jenneke and Koolstra, Jan Harm
Title 3D-printed poly(Ɛ-caprolactone) scaffold with gradient mechanical properties according to force distribution in the mandible for mandibular bone tissue engineering [Abstract]
Year 2020
Journal/Proceedings Journal of the Mechanical Behavior of Biomedical Materials
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Abstract
In bone tissue engineering, prediction of forces induced to the native bone during normal functioning is important in the design, fabrication, and integration of a scaffold with the host. The aim of this study was to customize the mechanical properties of a layer-by-layer 3D-printed poly(ϵ-caprolactone) (PCL) scaffold estimated by finite element (FE) modeling in order to match the requirements of the defect, to prevent mechanical failure, and ensure optimal integration with the surrounding tissue. Forces and torques induced on the mandibular symphysis during jaw opening and closing were predicted by FE modeling. Based on the predicted forces, homogeneous-structured PCL scaffolds with 3 different void sizes (0.3, 0.6, and 0.9 mm) were designed and 3D-printed using an extrusion based 3D-bioprinter. In addition, 2 gradient-structured scaffolds were designed and 3D-printed. The first gradient scaffold contained 2 regions (0.3 mm and 0.6 mm void size in the upper and lower half, respectively), whereas the second gradient scaffold contained 3 regions (void sizes of 0.3, 0.6, and 0.9 mm in the upper, middle and lower third, respectively). Scaffolds were tested for their compressive and tensile strength in the upper and lower halves. The actual void size of the homogeneous scaffolds with designed void size of 0.3, 0.6, and 0.9 mm was 0.20, 0.59, and 0.95 mm, respectively. FE modeling showed that during opening and closing of the jaw, the highest force induced on the symphysis was a compressive force in the transverse direction. The compressive force was induced throughout the symphyseal line and reduced from top (362.5 N, compressive force) to bottom (107.5 N, tensile force) of the symphysis. Compressive and tensile strength of homogeneous scaffolds decreased by 1.4-fold to 3-fold with increasing scaffold void size. Both gradient scaffolds had higher compressive strength in the upper half (2 region-gradient scaffold: 4.9 MPa; 3 region-gradient scaffold: 4.1 MPa) compared with the lower half (2 region-gradient scaffold: 2.5 MPa; 3 region-gradient scaffold: 2.7 MPa) of the scaffold. 3D-printed PCL scaffolds had higher compressive strength in the scaffold layer-by-layer building direction compared with the side direction, and a very low tensile strength in the scaffold layer-by-layer building direction. Fluid shear stress and fluid pressure distribution in the gradient scaffolds were more homogeneous than in the 0.3 mm void size scaffold and similar to the 0.6 mm and 0.9 mm void size scaffolds. In conclusion, these data show that the mechanical properties of 3D-printed PCL scaffolds can be tailored based on the predicted forces on the mandibular symphysis. These 3D-printed PCL scaffolds had different mechanical properties in scaffold building direction compared with the side direction, which should be taken into account when placing the scaffold in the defect site. Our findings might have implications for improved performance and integration of scaffolds with native tissue.
AUTHOR Mancini, I. A. D. and Schmidt, S. and Brommer, H. and Pouran, B. and Schäfer, S. and Tessmar, J. and Mensinga, A. and van Rijen, M. H. P. and Groll, J. and Blunk, T. and Levato, R. and Malda, J. and van Weeren, P. R.
Title A composite hydrogel-3D printed thermoplast osteochondral anchor as example for a zonal approach to cartilage repair: in vivo performance in a long-term equine model [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Abstract
Recent research has been focusing on the generation of living personalized osteochondral constructs for joint repair. Native articular cartilage has a zonal structure, which is not reflected in current constructs and which may be a cause of the frequent failure of these repair attempts. Therefore, we investigated the performance of a composite implant that further reflects the zonal distribution of cellular component both in vitro and in vivo in a long-term equine model. Constructs constituted of a 3D-printed poly(ϵ-caprolactone) (PCL) bone anchor from which reinforcing fibers protruded into the chondral part of the construct over which two layers of a thiol-ene cross-linkable hyaluronic acid/poly(glycidol) hybrid hydrogel (HA-SH/P(AGE-co-G)) were fabricated. The top layer contained Articular Cartilage Progenitor Cells (ACPCs) derived from the superficial layer of native cartilage tissue, the bottom layer contained mesenchymal stromal cells (MSCs). The chondral part of control constructs were homogeneously filled with MSCs. After six months in vivo, microtomography revealed significant bone growth into the anchor. Histologically, there was only limited production of cartilage-like tissue (despite persistency of hydrogel) both in zonal and non-zonal constructs. There were no differences in histological scoring; however, the repair tissue was significantly stiffer in defects repaired with zonal constructs. The sub-optimal quality of the repair tissue may be related to several factors, including early loss of implanted cells, or inappropriate degradation rate of the hydrogel. Nonetheless, this approach may be promising and research into further tailoring of biomaterials and of construct characteristics seems warranted.
AUTHOR Wang, Zehao and Hui, Aiping and Zhao, Hongbin and Ye, Xiaohan and Zhang, Chao and Wang, Aiqin and Zhang, Changqing
Title A Novel 3D-bioprinted Porous Nano Attapulgite Scaffolds with Good Performance for Bone Regeneration [Abstract]
Year 2020
Journal/Proceedings International Journal of Nanomedicine
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Abstract
BACKGROUND: Natural clay nanomaterials are an emerging class of biomaterial with great potential for tissue engineering and regenerative medicine applications, most notably for osteogenesis. MATERIALS AND METHODS: Herein, for the first time, novel tissue engineering scaffolds were prepared by 3D bioprinter using nontoxic and bioactive natural attapulgite (ATP) nanorods as starting materials, with polyvinyl alcohol as binder, and then sintered to obtain final scaffolds. The microscopic morphology and structure of ATP particles and scaffolds were observed by transmission electron microscope and scanning electron microscope. In vitro biocompatibility and osteogenesis with osteogenic precursor cell (hBMSCs) were assayed using MTT method, Live/Dead cell staining, alizarin red staining and RT-PCR. In vivo bone regeneration was evaluated with micro-CT and histology analysis in rat cranium defect model. RESULTS: We successfully printed a novel porous nano-ATP scaffold designed with inner channels with a dimension of 500 µm and wall structures with a thickness of 330 µm. The porosity of current 3D-printed scaffolds ranges from 75% to 82% and the longitudinal compressive strength was up to 4.32±0.52 MPa. We found firstly that nano-ATP scaffolds with excellent biocompatibility for hBMSCscould upregulate the expression of osteogenesis-related genes bmp2 and runx2 and calcium deposits in vitro. Interestingly, micro-CT and histology analysis revealed abundant newly formed bone was observed along the defect margin, even above and within the 3D bioprinted porous ATP scaffolds in a rat cranial defect model. Furthermore, histology analysis demonstrated that bone was formed directly following a process similar to membranous ossification without any intermediate cartilage formation and that many newly formed blood vessels are within the pores of 3D-printed scaffolds at four and eight weeks. CONCLUSION: These results suggest that the 3D-printed porous nano-ATP scaffolds are promising candidates for bone tissue engineering by osteogenesis and angiogenesis.
AUTHOR Moxon, Sam and Ferreira, Miguel and Santos, Patricia and Popa, Bogdan and Gloria, Antonio and Katsarava, Ramaz and Tugushi, David and Serra, Armenio and Hooper, Nigel and Kimber, Susan and Fonseca, Ana and Domingos, Marco
Title A Preliminary Evaluation of the Pro-Chondrogenic Potential of 3D-Bioprinted Poly(ester Urea) Scaffolds
Year 2020
Journal/Proceedings Polymers
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AUTHOR Li, Zuxi and Zhang, Xiao and Yuan, Tao and Zhang, Yi and Luo, Chunyang and Zhang, Jiyong and Liu, Yang and Fan, Weimin
Title Addition of Platelet-Rich Plasma to Silk Fibroin Hydrogel Bioprinting for Cartilage Regeneration [Abstract]
Year 2020
Journal/Proceedings Tissue Engineering Part A
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Abstract
The recent advent of 3D bioprinting of biopolymers provides a novel method for fabrication of tissue-engineered scaffolds and also offers a potentially promising avenue in cartilage regeneration. Silk fibroin (SF) is one of the most popular biopolymers used for 3D bioprinting, but further application of SF is hindered by its limited biological activities. Incorporation of growth factors (GFs) has been identified as a solution to improve biological function. Platelet-rich plasma (PRP) is an autologous resource of GFs, which has been widely used in clinic. In this study, we have developed SF-based bioinks incorporated with different concentrations of PRP (12.5%, 25%, and 50%; vol/vol). Release kinetic studies show that SF-PRP bioinks could achieve controlled release of GFs. Subsequently, SF-PRP bioinks were successfully fabricated into scaffolds by bioprinting. Our results revealed that SF-PRP scaffolds possessed proper internal pore structure, good biomechanical properties, and a suitable degradation rate for cartilage regeneration. Live/dead staining showed that 3D, printed SF-PRP scaffolds were biocompatible. Moreover, in vitro studies revealed that tissue-engineered cartilage from the SF-PRP group exhibited improved qualities compared with the pure SF controls, according to histological and immunohistochemical findings. Biochemical evaluations confirmed that SF-PRP (50% PRP, v/v) scaffolds allowed the largest increases in collagen and glycosaminoglycan concentrations, when compared with the pure SF group. These findings suggest that 3D, printed SF-PRP scaffolds could be potential candidates for cartilage tissue engineering.
AUTHOR Huang, Boyang and Vyas, Cian and Byun, Jae Jong and El-Newehy, Mohamed and Huang, Zhucheng and Bártolo, Paulo
Title Aligned multi-walled carbon nanotubes with nanohydroxyapatite in a 3D printed polycaprolactone scaffold stimulates osteogenic differentiation [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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Abstract
The development of highly biomimetic scaffolds in terms of composition and structures, to repair or replace damaged bone tissues, is particularly relevant for tissue engineering. This paper investigates a 3D printed porous scaffold containing aligned multi-walled carbon nanotubes (MWCNTs) and nano-hydroxyapatite (nHA), mimicking the natural bone tissue from the nanoscale to macroscale level. MWCNTs with similar dimensions as collagen fibres are coupled with nHA and mixed within a polycaprolactone (PCL) matrix to produce scaffolds using a screw-assisted extrusion-based additive manufacturing system. Scaffolds with different material compositions were extensively characterised from morphological, mechanical and biological points of views. Transmission electron microscopy and polarised Raman spectroscopy confirm the presence of aligned MWCNTs within the printed filaments. The PCL/HA/MWCNTs scaffold are similar to the nanostructure of native bone and shows overall increased mechanical properties, cell proliferation, osteogenic differentiation and scaffold mineralisation, indicating a promising approach for bone tissue regeneration.
AUTHOR Kamdem Tamo, Arnaud and Doench, Ingo and Morales Helguera, Aliuska and Hoenders, Daniel and Walther, Andreas and Madrazo, Anayancy Osorio
Title Biodegradation of Crystalline Cellulose Nanofibers by Means of Enzyme Immobilized-Alginate Beads and Microparticles [Abstract]
Year 2020
Journal/Proceedings Polymers
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Abstract
Recent advances in nanocellulose technology have revealed the potential of crystalline cellulose nanofibers to reinforce materials which are useful for tissue engineering, among other functions. However, the low biodegradability of nanocellulose can possess some problems in biomedical applications. In this work, alginate particles with encapsulated enzyme cellulase extracted from Trichoderma reesei were prepared for the biodegradation of crystalline cellulose nanofibers, which carrier system could be incorporated in tissue engineering biomaterials to degrade the crystalline cellulose nanoreinforcement in situ and on-demand during tissue regeneration. Both alginate beads and microparticles were processed by extrusion-dropping and inkjet-based methods, respectively. Processing parameters like the alginate concentration, concentration of ionic crosslinker Ca2+, hardening time, and ionic strength of the medium were varied. The hydrolytic activity of the free and encapsulated enzyme was evaluated for unmodified (CNFs) and TEMPO-oxidized cellulose nanofibers (TOCNFs) in suspension (heterogeneous conditions); in comparison to solubilized cellulose derivatives (homogeneous conditions). The enzymatic activity was evaluated for temperatures between 25–75 °C, pH range from 3.5 to 8.0 and incubation times until 21 d. Encapsulated cellulase in general displayed higher activity compared to the free enzyme over wider temperature and pH ranges and for longer incubation times. A statistical design allowed optimizing the processing parameters for the preparation of enzyme-encapsulated alginate particles presenting the highest enzymatic activity and sphericity. The statistical analysis yielded the optimum particles characteristics and properties by using a formulation of 2% (w/v) alginate, a coagulation bath of 0.2 M CaCl2 and a hardening time of 1 h. In homogeneous conditions the highest catalytic activity was obtained at 55 °C and pH 4.8. These temperature and pH values were considered to study the biodegradation of the crystalline cellulose nanofibers in suspension. The encapsulated cellulase preserved its activity for several weeks over that of the free enzyme, which latter considerably decreased and practically showed deactivation after just 10 d. The alginate microparticles with their high surface area-to-volume ratio effectively allowed the controlled release of the encapsulated enzyme and thereby the sustained hydrolysis of the cellulose nanofibers. The relative activity of cellulase encapsulated in the microparticles leveled-off at around 60% after one day and practically remained at that value for three weeks.
AUTHOR Zamani, Yasaman and Mohammadi, Javad and Amoabediny, Ghassem and Helder, Marco N. and Zandieh-Doulabi, Behrouz and Klein-Nulend, Jenneke
Title Bioprinting of Alginate-Encapsulated Pre-osteoblasts in PLGA/β-TCP Scaffolds Enhances Cell Retention but Impairs Osteogenic Differentiation Compared to Cell Seeding after 3D-Printing [Abstract]
Year 2020
Journal/Proceedings Regenerative Engineering and Translational Medicine
Reftype Zamani2020
DOI/URL DOI
Abstract
In tissue engineering, cellularization of scaffolds has typically been performed by seeding the cells after scaffold fabrication. 3D-printing technology now allows bioprinting of cells encapsulated in a hydrogel simultaneously with the scaffold material. Here, we aimed to investigate whether bioprinting or cell seeding post-printing is more effective in enhancing responses of pre-osteoblastic MC3T3-E1 cell line derived from mouse calvaria.
AUTHOR Diloksumpan, Paweena and de Ruijter, Myl{`{e}}ne and Castilho, Miguel and Gbureck, Uwe and Vermonden, Tina and van Weeren, P. Ren{'{e}} and Malda, Jos and Levato, Riccardo
Title Combining multi-scale 3D printing technologies to engineer reinforced hydrogel-ceramic interfaces [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Abstract
Multi-material 3D printing technologies that resolve features at different lengths down to the microscale open new avenues for regenerative medicine, particularly in the engineering of tissue interfaces. Herein, extrusion printing of a bone-biomimetic ceramic ink and melt electrowriting (MEW) of spatially organized polymeric microfibres are integrated for the biofabrication of an osteochondral plug, with a mechanically reinforced bone-to-cartilage interface. A printable physiological temperature-setting bioceramic, based on α-tricalcium phosphate, nanohydroxyapatite and a custom-synthesized biodegradable and crosslinkable poloxamer, was developed as bone support. The mild setting reaction of the bone ink enabled us to print directly within melt electrowritten polycaprolactone meshes, preserving their micro-architecture. Ceramic-integrated MEW meshes protruded into the cartilage region of the composite plug, and were embedded with mechanically soft gelatin-based hydrogels, laden with articular cartilage chondroprogenitor cells. Such interlocking design enhanced the hydrogel-to-ceramic adhesion strength >6.5-fold, compared with non-interlocking fibre architectures, enabling structural stability during handling and surgical implantation in osteochondral defects ex vivo. Furthermore, the MEW meshes endowed the chondral compartment with compressive properties approaching those of native cartilage (20-fold reinforcement versus pristine hydrogel). The osteal and chondral compartment supported osteogenesis and cartilage matrix deposition in vitro, and the neo-synthesized cartilage matrix further contributed to the mechanical reinforcement at the ceramic-hydrogel interface. This multi-material, multi-scale 3D printing approach provides a promising strategy for engineering advanced composite constructs for the regeneration of musculoskeletal and connective tissue interfaces.
AUTHOR Müller, Michael and Fisch, Philipp and Molnar, Marc and Eggert, Sebastian and Binelli, Marco and Maniura-Weber, Katharina and Zenobi-Wong, Marcy
Title Development and thorough characterization of the processing steps of an ink for 3D printing for bone tissue engineering [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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Abstract
Achieving reproducibility in the 3D printing of biomaterials requires a robust polymer synthesis method to reduce batch-to-batch variation as well as methods to assure a thorough characterization throughout the manufacturing process. Particularly biomaterial inks containing large solid fractions such as ceramic particles, often required for bone tissue engineering applications, are prone to inhomogeneity originating from inadequate mixing or particle aggregation which can lead to inconsistent printing results. The production of such an ink for bone tissue engineering consisting of gellan gum methacrylate (GG-MA), hyaluronic acid methacrylate and hydroxyapatite (HAp) particles was therefore optimized in terms of GG-MA synthesis and ink preparation process, and the ink's printability was thoroughly characterized to assure homogeneous and reproducible printing results. A new buffer mediated synthesis method for GG-MA resulted in consistent degrees of substitution which allowed the creation of large 5 g batches. We found that both the new synthesis as well as cryomilling of the polymer components of the ink resulted in a decrease in viscosity from 113 kPa·s to 11.3 kPa·s at a shear rate of 0.1 s−1 but increased ink homogeneity. The ink homogeneity was assessed through thermogravimetric analysis and a newly developed extrusion force measurement setup. The ink displayed strong inter-layer adhesion between two printed ink layers as well as between a layer of ink with and a layer without HAp. The large polymer batch production along with the characterization of the ink during the manufacturing process allows ink production in the gram scale and could be used in applications such as the printing of osteochondral grafts.
AUTHOR Huang, Boyang and Aslan, Enes and Jiang, Zhengyi and Daskalakis, Evangelos and Jiao, Mohan and Aldalbahi, Ali and Vyas, Cian and Bártolo, Paulo
Title Engineered dual-scale poly (ε-caprolactone) scaffolds using 3D printing and rotational electrospinning for bone tissue regeneration [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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Abstract
Large bone defects due to trauma or disease present a significant clinical challenge with limited efficacy of current therapies. A key aim is to develop biomimetic scaffolds that reflect the native tissue structure with 3D printing being an important enabling technology. However, the incorporation of multiple length scales and anisotropic features, mimicking the native architecture, is difficult with current processes. In this study, we propose a simple and versatile hybrid printing process using a screw-assisted additive manufacturing technique combined with rotational electrospinning to fabricate dual-scale anisotropic scaffolds. 3D microscale porous polycaprolactone (PCL) structures with highly aligned nanoscale fibres were successfully produced layer-by-layer. The scaffolds were morphological, mechanical and biological characterised. Human adipose-derived stem cells (hADSCs) were seeded on the hybrid scaffold to evaluate the effects of structural and anisotropic topographic cues on cell attachment, proliferation and osteogenesis differentiation. Results show that the 3D printed microscale structures have uniform and well-defined geometries and the alignment of nanoscale electrospun fibres increases by increasing the electrospinning rotational velocity. Mechanical results show that there is no significant difference between 3D printed scaffolds with or without electrospun meshes. In vitro results show higher cell seeding efficiency and proliferation in dual-scale scaffolds with high density electrospun meshes. A more stretched and elongated cell morphology was observed in aligned nanofibre scaffolds showing higher anisotropic cytoskeletal organization than 3D printed PCL scaffolds without electrospun meshes. The dual-scale scaffolds present improved overall osteogenic markers expressions (COL-1, ALP and OCN). However, no statistical difference between normalised osteogenic marker expressions were observed between dual-scale scaffolds and 3D printed scaffolds. This might be attributed to the poor bioactivity of the substrate material, PCL, suggesting topographical cues might not be sufficient to stimulate cell fate towards to an osteogenic linage. The results suggest that the proposed fabrication strategy is a promising approach for the design of novel bone scaffolds to modulate cell fates by integrating the topographic cue reported in this paper with biochemical cues associated to the use of more bioactive materials.
AUTHOR Song, Jie-Liang and Fu, Xin-Ye and Raza, Ali and Shen, Nai-An and Xue, Ya-Qi and Wang, Hua-Jie and Wang, Jin-Ye
Title Enhancement of mechanical strength of TCP-alginate based bioprinted constructs [Abstract]
Year 2020
Journal/Proceedings Journal of the Mechanical Behavior of Biomedical Materials
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DOI/URL URL DOI
Abstract
To overcome the mechanical drawback of bioink, we proposed a supporter model to enhance the mechanical strength of bioprinted 3D constructs, in which a unit-assembly idea was involved. Based on Computed Tomography images of critical-sized rabbit bone defect, the 3D re-construction was accomplished by a sequenced process using Mimics 17.0, BioCAM and BioCAD software. 3D constructs were bioprinted using polycaprolactone (PCL) ink for the outer supporter under extrusion mode, and cell-laden tricalcium phosphate (TCP)/alginate bioink for the inner filler under air pressure dispensing mode. The relationship of viscosity of bioinks, 3D bioprinting pressure, TCP/alginate ratio and cell survival were investigated by the shear viscosities analysis, live/dead cell test and cell-counting kit 8 measurement. The viscosity of bioinks at 1.0 s−1-shear rate could be adjusted within the range of 1.75 ± 0.29 Pa·s to 155.65 ± 10.86 Pa·s by changing alginate concentration, corresponding to 10 kPa–130 kPa of printing pressure. This design with PCL supporter could significantly enhance the compressive strength and compressive modulus of standardized 3D mechanical testing specimens up to 2.15 ± 0.14 MPa to 2.58 ± 0.09 MPa, and 42.83 ± 4.75 MPa to 53.12 ± 1.19 MPa, respectively. Cells could maintain the high viability (over 80%) under the given printing pressure but cell viability declined with the increase of TCP content. Cell survival after experiencing 7 days of cell culture could be achieved when the ratio of TCP/alginate was 1 : 4. All data supported the feasibility of the supporter and unit-assembly model to enhance mechanical properties of bioprinted 3D constructs.
AUTHOR Steier, Anke and Schmieg, Barbara and Irtel von Brenndorff, Yannic and Meier, Manuel and Nirschl, Hermann and Franzreb, Matthias and Lahann, Joerg
Title Enzyme Scaffolds with Hierarchically Defined Properties via 3D Jet Writing [Abstract]
Year 2020
Journal/Proceedings Macromolecular Bioscience
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DOI/URL DOI
Abstract
Abstract The immobilization of enzymes into polymer hydrogels is a versatile approach to improve their stability and utility in biotechnological and biomedical applications. However, these systems typically show limited enzyme activity, due to unfavorable pore dimensions and low enzyme accessibility. Here, 3D jet writing of water-based bioinks, which contain preloaded enzymes, is used to prepare hydrogel scaffolds with well-defined, tessellated micropores. After 3D jet writing, the scaffolds are chemically modified via photopolymerization to ensure mechanical stability. Enzyme loading and activity in the hydrogel scaffolds is fully retained over 3 d. Important structural parameters of the scaffolds such as pore size, pore geometry, and wall diameter are controlled with micrometer resolution to avoid mass-transport limitations. It is demonstrated that scaffold pore sizes between 120 µm and 1 mm can be created by 3D jet writing approaching the length scales of free diffusion in the hydrogels substrates and resulting in high levels of enzyme activity (21.2% activity relative to free enzyme). With further work, a broad range of applications for enzyme-laden hydrogel scaffolds including diagnostics and enzymatic cascade reactions is anticipated.
AUTHOR Dubey, Nileshkumar and Ferreira, Jessica A. and Malda, Jos and Bhaduri, Sarit B. and Bottino, Marco C.
Title Extracellular Matrix/Amorphous Magnesium Phosphate Bioink for 3D Bioprinting of Craniomaxillofacial Bone Tissue [Abstract]
Year 2020
Journal/Proceedings ACS Applied Materials & Interfaces
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DOI/URL DOI
Abstract
Bioprinting, a promising field in regenerative medicine, holds great potential to create three-dimensional, defect-specific vascularized bones with tremendous opportunities to address unmet craniomaxillofacial reconstructive challenges. A cytocompatible bioink is a critical prerequisite to successfully regenerate functional bone tissue. Synthetic self-assembling peptides have a nanofibrous structure resembling the native extracellular matrix (ECM), making them an excellent bioink component. Amorphous magnesium phosphates (AMPs) have shown greater levels of resorption while maintaining high biocompatibility, osteoinductivity, and low inflammatory response, as compared to their calcium phosphate counterparts. Here, we have established a novel bioink formulation (ECM/AMP) that combines an ECM-based hydrogel containing 2% octapeptide FEFEFKFK and 98% water with AMP particles to realize high cell function with desirable bioprintability. We analyzed the osteogenic differentiation of dental pulp stem cells (DPSCs) encapsulated in the bioink, as well as in vivo bone regeneration, to define the potential of the formulated bioink as a growth factor-free bone-forming strategy. Cell-laden AMP-modified bioprinted constructs showed an improved cell morphology but similar cell viability (∼90%) compared to their AMP-free counterpart. In functional assays, the cell-laden bioprinted constructs modified with AMP exhibited a high level of mineralization and osteogenic gene expression without the use of growth factors, thus suggesting that the presence of AMP-triggered DPSCs’ osteogenic differentiation. Cell-free ECM-based bioprinted constructs were implanted in vivo. In comparison with the ECM group, bone volume per total volume for ECM/1.0AMP was approximately 1.7- and 1.4-fold higher at 4 and 8 weeks, respectively. Further, a significant increase in the bone density was observed in ECM/1.0AMP from 4 to 8 weeks. These results demonstrate that the presence of AMP in the bioink significantly increased bone formation, thus showing promise for in situ bioprinting strategies. We foresee significant potential in translating this innovative bioink toward the regeneration of patient-specific bone tissue for regenerative dentistry. Bioprinting, a promising field in regenerative medicine, holds great potential to create three-dimensional, defect-specific vascularized bones with tremendous opportunities to address unmet craniomaxillofacial reconstructive challenges. A cytocompatible bioink is a critical prerequisite to successfully regenerate functional bone tissue. Synthetic self-assembling peptides have a nanofibrous structure resembling the native extracellular matrix (ECM), making them an excellent bioink component. Amorphous magnesium phosphates (AMPs) have shown greater levels of resorption while maintaining high biocompatibility, osteoinductivity, and low inflammatory response, as compared to their calcium phosphate counterparts. Here, we have established a novel bioink formulation (ECM/AMP) that combines an ECM-based hydrogel containing 2% octapeptide FEFEFKFK and 98% water with AMP particles to realize high cell function with desirable bioprintability. We analyzed the osteogenic differentiation of dental pulp stem cells (DPSCs) encapsulated in the bioink, as well as in vivo bone regeneration, to define the potential of the formulated bioink as a growth factor-free bone-forming strategy. Cell-laden AMP-modified bioprinted constructs showed an improved cell morphology but similar cell viability (∼90%) compared to their AMP-free counterpart. In functional assays, the cell-laden bioprinted constructs modified with AMP exhibited a high level of mineralization and osteogenic gene expression without the use of growth factors, thus suggesting that the presence of AMP-triggered DPSCs’ osteogenic differentiation. Cell-free ECM-based bioprinted constructs were implanted in vivo. In comparison with the ECM group, bone volume per total volume for ECM/1.0AMP was approximately 1.7- and 1.4-fold higher at 4 and 8 weeks, respectively. Further, a significant increase in the bone density was observed in ECM/1.0AMP from 4 to 8 weeks. These results demonstrate that the presence of AMP in the bioink significantly increased bone formation, thus showing promise for in situ bioprinting strategies. We foresee significant potential in translating this innovative bioink toward the regeneration of patient-specific bone tissue for regenerative dentistry.
AUTHOR Abu Awwad, Hosam Al-Deen M. and Thiagarajan, Lalitha and Kanczler, Janos M. and Amer, Mahetab H. and Bruce, Gordon and Lanham, Stuart and Rumney, Robin M. H. and Oreffo, Richard O. C. and Dixon, James E.
Title Genetically-programmed, mesenchymal stromal cell-laden & mechanically strong 3D bioprinted scaffolds for bone repair [Abstract]
Year 2020
Journal/Proceedings Journal of Controlled Release
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Abstract
Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting. Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs), we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation. Importantly we observed osteogenic programming of gene expression by released GET-RUNX2 (8.2-, 3.3- and 3.9-fold increases in OSX, RUNX2 and OPN expression, respectively) and calcification (von Kossa staining) in our scaffolds. The developed biodegradable PLGA/PEG paste formulation augments high-density bone development in a defect model (~2.4-fold increase in high density bone volume) and can be used to rapidly prototype clinically-sized hMSC-laden implants within minutes using mild, cytocompatible extrusion bioprinting. The ability to create mechanically strong 'cancellous bone-like’ printable implants for tissue repair that contain stem cells and controlled-release of programming factors is innovative, and will facilitate the development of novel localized delivery approaches to direct cellular behaviour for many regenerative medicine applications including those for personalized bone repair.
AUTHOR Hauptstein, Julia and Böck, Thomas and Bartolf-Kopp, Michael and Forster, Leonard and Stahlhut, Philipp and Nadernezhad, Ali and Blahetek, Gina and Zernecke-Madsen, Alma and Detsch, Rainer and Jüngst, Tomasz and Groll, Jürgen and Teßmar, Jörg and Blunk, Torsten
Title Hyaluronic Acid-Based Bioink Composition Enabling 3D Bioprinting and Improving Quality of Deposited Cartilaginous Extracellular Matrix [Abstract]
Year 2020
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract In 3D bioprinting, bioinks with high concentrations of polymeric materials are frequently used to enable fabrication of 3D cell-hydrogel constructs with sufficient stability. However, this is often associated with restricted cell bioactivity and an inhomogeneous distribution of newly produced extracellular matrix (ECM). Therefore, this study investigates bioink compositions based on hyaluronic acid (HA), an attractive material for cartilage regeneration, which allow for reduction of polymer content. Thiolated HA and allyl-modified poly(glycidol) in varying concentrations are UV-crosslinked. To adapt bioinks to poly(ε-caprolactone) (PCL)-supported 3D bioprinting, the gels are further supplemented with 1 wt% unmodified high molecular weight HA (hmHA) and chondrogenic differentiation of incorporated human mesenchymal stromal cells is assessed. Strikingly, addition of hmHA to gels with a low polymer content (3 wt%) results in distinct increase of construct quality with a homogeneous ECM distribution throughout the constructs, independent of the printing process. Improved ECM distribution in those constructs is associated with increased construct stiffness after chondrogenic differentiation, as compared to higher concentrated constructs (10 wt%), which only show pericellular matrix deposition. The study contributes to effective bioink development, demonstrating dual function of a supplement enabling PCL-supported bioprinting and at the same time improving biological properties of the resulting constructs.
AUTHOR De Moor, Lise and Fernandez, Sélina and Vercruysse, Chris and Tytgat, Liesbeth and Asadian, Mahtab and De Geyter, Nathalie and Van Vlierberghe, Sandra and Dubruel, Peter and Declercq, Heidi
Title Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids [Abstract]
Year 2020
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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Abstract
To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 μm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.
AUTHOR Hou, Yanhao and Wang, Weiguang and Bartolo, Paulo Jorge Da Silva
Title Investigating the Effect of Carbon Nanomaterials Reinforcing Poly(Ε-Caprolactone) Scaffolds for Bone Repair Applications [Abstract]
Year 2020
Journal/Proceedings International Journal of Bioprinting
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Abstract
Scaffolds, three-dimensional (3D) substrates providing appropriate mechanical support and biological environments for new tissue formation, are the most common approaches in tissue engineering. To improve scaffold properties such as mechanical properties, surface characteristics, biocompatibility and biodegradability, different types of fillers have been used reinforcing biocompatible and biodegradable polymers. This paper investigates and compares the mechanical and biological behaviors of 3D printed poly(ε-caprolactone) scaffolds reinforced with graphene (G) and graphene oxide (GO) at different concentrations. Results show that contrary to G which improves mechanical properties and enhances cell attachment and proliferation, GO seems to show some cytotoxicity, particular at high contents.
AUTHOR Ruiz-Cantu, Laura and Gleadall, Andrew and Faris, Callum and Segal, Joel and Shakesheff, Kevin and Yang, Jing
Title Multi-material 3D bioprinting of porous constructs for cartilage regeneration [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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Abstract
The current gold standard for nasal reconstruction after rhinectomy or severe trauma includes transposition of autologous cartilage grafts in conjunction with coverage using an autologous skin flap. Harvesting autologous cartilage requires a major additional procedure that may create donor site morbidity. Major nasal reconstruction also requires sculpting autologous cartilages to form a cartilage framework, which is complex, highly skill-demanding and very time consuming. These limitations have prompted facial reconstructive surgeons to explore different techniques such as tissue engineered cartilage. This work explores the use of multi-material 3D bioprinting with chondrocyte-laden gelatin methacrylate (GelMA) and polycaprolactone (PCL) to fabricate constructs that can potentially be used for nasal reconstruction. In this study, we have investigated the effect of 3D manufacturing parameters including temperature, needle gauge, UV exposure time, and cell carrier formulation (GelMA) on the viability and functionality of chondrocytes in bioprinted constructs. Furthermore, we printed chondrocyte-laden GelMA and PCL into composite constructs to combine biological and mechanical properties. It was found that 20% w/v GelMA was the best concentration for the 3D bioprinting of the chondrocytes without comprising the scaffold's porous structure and cell functionality. In addition, the 3D bioprinted constructs showed neocartilage formation and similar mechanical properties to nasal alar cartilage after a 50-day culture period. Neocartilage formation was also observed in the composite constructs evidenced by the presence of glycosaminoglycans and collagen type II. This study shows the feasibility of manufacturing neocartilage using chondrocytes/GelMA/PCL 3D bioprinted porous constructs which could be applied as a method for fabricating implants for nose reconstruction.
AUTHOR Lee, Mihyun and Bae, Kraun and Levinson, Clara and Zenobi-Wong, Marcy
Title Nanocomposite bioink exploits dynamic covalent bonds between nanoparticles and polysaccharides for precision bioprinting [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Abstract
The field of bioprinting has made significant recent progress towards engineering tissues with increasing complexity and functionality. It remains challenging, however, to develop bioinks with optimal biocompatibility and good printing fidelity. Here, we demonstrate enhanced printability of a polymer-based bioink based on dynamic covalent linkages between nanoparticles (NPs) and polymers, which retains good biocompatibility. Amine-presenting silica NPs (ca. 45 nm) were added to a polymeric ink containing oxidized alginate (OxA). The formation of reversible imine bonds between amines on the NPs and aldehydes of OxA lead to significantly improved rheological properties and high printing fidelity. In particular, the yield stress increased with increasing amounts of NPs (14.5 Pa without NPs, 79 Pa with 2 wt% NPs). In addition, the presence of dynamic covalent linkages in the gel provided improved mechanical stability over 7 d compared to ionically crosslinked gels. The nanocomposite ink retained high printability and mechanical strength, resulting in generation of centimeter-scale porous constructs and an ear structure with overhangs and high structural fidelity. Furthermore, the nanocomposite ink supported both in vitro and in vivo maturation of bioprinted gels containing chondrocytes. This approach based on simple oxidation can be applied to any polysaccharide, thus the widely applicability of the method is expected to advance the field towards the goal of precision bioprinting.
AUTHOR Hou, Yanhao and Wang, Weiguang and Bártolo, Paulo
Title Novel Poly(ɛ-caprolactone)/Graphene Scaffolds for Bone Cancer Treatment and Bone Regeneration [Abstract]
Year 2020
Journal/Proceedings 3D Printing and Additive Manufacturing
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Abstract
Scaffold-based bone tissue engineering is the most relevant approach for critical-sized bone defects. It is based on the use of three-dimensional substrates to provide the appropriate biomechanical environment for bone regeneration. Despite some successful results previously reported, scaffolds were never designed for disease treatment applications. This article proposes a novel dual-functional scaffold for cancer applications, comprising both treatment and regeneration functions. These functions are achieved by combining a biocompatible and biodegradable polymer and graphene. Results indicate that high concentrations of graphene enhance the mechanical properties of the scaffolds, also increasing the inhibition on cancer cell viability and proliferation.
AUTHOR Lim, Khoon S. and Abinzano, Florencia and Bernal, Paulina Nuñez and Albillos Sanchez, Ane and Atienza-Roca, Pau and Otto, Iris A. and Peiffer, Quentin C. and Matsusaki, Michiya and Woodfield, Tim B. F. and Malda, Jos and Levato, Riccardo
Title One-Step Photoactivation of a Dual-Functionalized Bioink as Cell Carrier and Cartilage-Binding Glue for Chondral Regeneration [Abstract]
Year 2020
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract Cartilage defects can result in pain, disability, and osteoarthritis. Hydrogels providing a chondroregeneration-permissive environment are often mechanically weak and display poor lateral integration into the surrounding cartilage. This study develops a visible-light responsive gelatin ink with enhanced interactions with the native tissue, and potential for intraoperative bioprinting. A dual-functionalized tyramine and methacryloyl gelatin (GelMA-Tyr) is synthesized. Photo-crosslinking of both groups is triggered in a single photoexposure by cell-compatible visible light in presence of tris(2,2′-bipyridyl)dichlororuthenium(II) and sodium persulfate as initiators. Neo-cartilage formation from embedded chondroprogenitor cells is demonstrated in vitro, and the hydrogel is successfully applied as bioink for extrusion-printing. Visible light in situ crosslinking in cartilage defects results in no damage to the surrounding tissue, in contrast to the native chondrocyte death caused by UV light (365–400 nm range), commonly used in biofabrication. Tyramine-binding to proteins in native cartilage leads to a 15-fold increment in the adhesive strength of the bioglue compared to pristine GelMA. Enhanced adhesion is observed also when the ink is extruded as printable filaments into the defect. Visible-light reactive GelMA-Tyr bioinks can act as orthobiologic carriers for in situ cartilage repair, providing a permissive environment for chondrogenesis, and establishing safe lateral integration into chondral defects.
AUTHOR Diloksumpan, Paweena and Bolaños, Rafael Vindas and Cokelaere, Stefan and Pouran, Behdad and de Grauw, Janny and van Rijen, Mattie and van Weeren, René and Levato, Riccardo and Malda, Jos
Title Orthotopic Bone Regeneration within 3D Printed Bioceramic Scaffolds with Region-Dependent Porosity Gradients in an Equine Model [Abstract]
Year 2020
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract The clinical translation of three-dimensionally printed bioceramic scaffolds with tailored architectures holds great promise toward the regeneration of bone to heal critical-size defects. Herein, the long-term in vivo performance of printed hydrogel-ceramic composites made of methacrylated-oligocaprolactone-poloxamer and low-temperature self-setting calcium-phosphates is assessed in a large animal model. Scaffolds printed with different internal architectures, displaying either a designed porosity gradient or a constant pore distribution, are implanted in equine tuber coxae critical size defects. Bone ingrowth is challenged and facilitated only from one direction via encasing the bioceramic in a polycaprolactone shell. After 7 months, total new bone volume and scaffold degradation are significantly greater in structures with constant porosity. Interestingly, gradient scaffolds show lower extent of remodeling and regeneration even in areas having the same porosity as the constant scaffolds. Low regeneration in distal regions from the interface with native bone impairs ossification in proximal regions of the construct, suggesting that anisotropic architectures modulate the cross-talk between distant cells within critical-size defects. The study provides key information on how engineered architectural patterns impact osteoregeneration in vivo, and also indicates the equine tuber coxae as promising orthotopic model for studying materials stimulating bone formation.
AUTHOR Chen, Grona and Xu, Yihua and Chi Lip Kwok, Philip and Kang, Lifeng
Title Pharmaceutical Applications of 3D Printing [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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Abstract
Although 3D printing (3DP) has long been an integral part of industries such as aviation and automotive, its use in healthcare, especially the pharmaceutical industry, is relatively new and currently receiving close attention. At the beginning of 2018, we reviewed the applications of 3DP for drug delivery and drug testing [1]. Due to the rapid development of this field, it is necessary to summarize the latest development in this field after 2 years. In this article, we reviewed the three major areas in pharmaceutical applications. First, drug delivery system is the most studied subject, including controlled release, polypills, gastrofloating, orodispersibles and microneedles. Second, 3DP also helped the development of pharmaceutical devices, including pharmacy dispensing aids and drug eluting devices. Lastly, we reviewed the pharmaceutical models for drug testing, covering acellular and cellular models. We also summarized the materials used in the mentioned articles and their regulatory status for pharmaceutical applications to provide references for future research.
AUTHOR Figueiredo, Lara and Le Visage, Catherine and Weiss, Pierre and Yang, Jing
Title Quantifying Oxygen Levels in 3D Bioprinted Cell-Laden Thick Constructs with Perfusable Microchannel Networks [Abstract]
Year 2020
Journal/Proceedings Polymers
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Abstract
The survival and function of thick tissue engineered implanted constructs depends on pre-existing, embedded, functional, vascular-like structures that are able to integrate with the host vasculature. Bioprinting was employed to build perfusable vascular-like networks within thick constructs. However, the improvement of oxygen transportation facilitated by these vascular-like networks was directly quantified. Using an optical fiber oxygen sensor, we measured the oxygen content at different positions within 3D bioprinted constructs with and without perfusable microchannel networks. Perfusion was found to play an essential role in maintaining relatively high oxygen content in cell-laden constructs and, consequently, high cell viability. The concentration of oxygen changes following switching on and off the perfusion. Oxygen concentration depletes quickly after pausing perfusion but recovers rapidly after resuming the perfusion. The quantification of oxygen levels within cell-laden hydrogel constructs could provide insight into channel network design and cellular responses.
AUTHOR Schipani, Rossana and Scheurer, Stefan and Florentin, Romain and Critchley, Susan E. and Kelly, Daniel John
Title Reinforcing interpenetrating network hydrogels with 3D printed polymer networks to engineer cartilage mimetic composites [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Abstract
Engineering constructs that mimic the complex structure, composition and biomechanics of the articular cartilage represents a promising route to joint regeneration. Such tissue engineering strategies require the development of biomaterials that mimic the mechanical properties of articular cartilage whilst simultaneously providing an environment supportive of chondrogenesis. Here three-dimensional (3D) bioprinting is used to develop polycaprolactone (PCL) fibre networks to mechanically reinforce interpenetrating network (IPN) hydrogels consisting of alginate and gelatin methacryloyl (GelMA). Inspired by the significant tension-compression nonlinearity of the collagen network in articular cartilage, we printed reinforcing PCL networks with different ratios of tensile to compressive modulus. Synergistic increases in compressive modulus were observed when IPN hydrogels were reinforced with PCL networks that were relatively soft in compression and stiff in tension. The resulting composites possessed equilibrium and dynamic mechanical properties that matched or approached that of native articular cartilage. Finite Element (FE) modelling revealed that the reinforcement of IPN hydrogels with specific PCL networks limited radial expansion and increased the hydrostatic pressure generated within the IPN upon the application of compressive loading. Next, multiple-tool biofabrication techniques were used to 3D bioprint PCL reinforced IPN hydrogels laden with a co-culture of bone marrow-derived stromal cells (BMSCs) and chondrocytes (CCs). The bioprinted biomimetic composites were found to support robust chondrogenesis, with encapsulated cells producing hyaline-like cartilage that stained strongly for sGAG and type II collagen deposition, and negatively for type X collagen and calcium deposition. Taken together, these results demonstrate how 3D bioprinting can be used to engineer constructs that are both pro-chondrogenic and biomimetic of the mechanical properties of articular cartilage.
AUTHOR Prasopthum, Aruna and Deng, Zexing and Khan, Ilyas M. and Yin, Zhanhai and Guo, Baolin and Yang, Jing
Title Three dimensional printed degradable and conductive polymer scaffolds promote chondrogenic differentiation of chondroprogenitor cells [Abstract]
Year 2020
Journal/Proceedings Biomaterials Science
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Abstract
Conductive polymers have been used for various biomedical applications including biosensors{,} tissue engineering and regenerative medicine. However{,} the poor processability and brittleness of these polymers hinder the fabrication of three-dimensional structures with desirable geometries. Moreover{,} their application in tissue engineering and regenerative medicine has been so far limited to excitable cells such as neurons and muscle cells. To enable their wider adoption in tissue engineering and regenerative medicine{,} new materials and formulations that overcome current limitations are required. Herein{,} a biodegradable conductive block copolymer{,} tetraaniline-b-polycaprolactone-b-tetraaniline (TPT){,} is synthesised and 3D printed for the first time into porous scaffolds with defined geometries. Inks are formulated by combining TPT with PCL in solutions which are then directly 3D printed to generate porous scaffolds. TPT and PCL are both biodegradable. The combination of TPT with PCL increases the flexibility of the hybrid material compared to pure TPT{,} which is critical for applications that need mechanical robustness of the scaffolds. The highest TPT content shows the lowest tensile failure strain. Moreover{,} the absorption of a cell adhesion-promoting protein (fibronectin) and chondrogenic differentiation of chondroprogenitor cells are found to be dependent on the amount of TPT in the blends. Higher content of TPT in the blends increases both fibronectin adsorption and chondrogenic differentiation{,} though the highest concentration of TPT in the blends is limited by its solubility in the ink. Despite the contradicting effects of TPT concentration on flexibility and chondrogenic differentiation{,} a concentration that strikes a balance between the two factors is still available. It is worth noting that the effect on chondrogenic differentiation is found in scaffolds without external electric stimulation. Our work demonstrates the possibility of 3D printing flexible conductive and biodegradable scaffolds and their potential use in cartilage tissue regeneration{,} and opens up future opportunities in using electric stimulation to control chondrogenesis in these scaffolds.
AUTHOR Vyas, Cian and Ates, Gokhan and Aslan, Enes and Hart, Jack and Huang, Boyang and Bartolo, Paulo
Title Three-Dimensional Printing and Electrospinning Dual-Scale Polycaprolactone Scaffolds with Low-Density and Oriented Fibers to Promote Cell Alignment [Abstract]
Year 2020
Journal/Proceedings 3D Printing and Additive Manufacturing
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Abstract
Complex and hierarchically functionalized scaffolds composed of micro- and nanoscale structures are a key goal in tissue engineering. The combination of three-dimensional (3D) printing and electrospinning enables the fabrication of these multiscale structures. This study presents a polycaprolactone 3D-printed and electrospun scaffold with multiple mesh layers and fiber densities. The results show successful fabrication of a dual-scale scaffold with the 3D-printed scaffold acting as a gap collector with the printed microfibers as the electrodes and the pores a series of insulating gaps resulting in aligned nanofibers. The electrospun fibers are highly aligned perpendicular to the direction of the printed fiber and form aligned meshes within the pores of the scaffold. Mechanical testing showed no significant difference between the number of mesh layers whereas the hydrophobicity of the scaffold increased with increasing fiber density. Biological results indicate that increasing the number of mesh layers improves cell proliferation, migration, and adhesion. The aligned nanofibers within the microscale pores allowed enhanced cell bridging and cell alignment that was not observed in the 3D-printed only scaffold. These results demonstrate a facile method of incorporating low-density and aligned fibers within a 3D-printed scaffold that is a promising development in multiscale hierarchical scaffolds where alignment of cells can be desirable.
AUTHOR Nasim Golafshan and Elke Vorndran and Stefan Zaharievski and Harold Brommer and Firoz Babu Kadumudi and Alireza Dolatshahi-Pirouz and Uwe Gbureck and René {van Weeren} and Miguel Castilho and Jos Malda
Title Tough magnesium phosphate-based 3D-printed implants induce bone regeneration in an equine defect model [Abstract]
Year 2020
Journal/Proceedings Biomaterials
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Abstract
One of the important challenges in bone tissue engineering is the development of biodegradable bone substitutes with appropriate mechanical and biological properties for the treatment of larger defects and those with complex shapes. Recently, magnesium phosphate (MgP) doped with biologically active ions like strontium (Sr2+) have shown to significantly enhance bone formation when compared with the standard calcium phosphate-based ceramics. However, such materials can hardly be shaped into large and complex geometries and more importantly lack the adequate mechanical properties for the treatment of load-bearing bone defects. In this study, we have fabricated bone implants through extrusion assisted three-dimensional (3D) printing of MgP ceramics modified with Sr2+ ions (MgPSr) and a medical grade polycaprolactone (PCL) polymer phase. MgPSr with 30 wt% PCL (MgPSr-PCL30) allowed the printability of relevant size structures (>780 mm3) at room temperature with an interconnected macroporosity of approximately 40%. The printing resulted in implants with a compressive strength of 4.3 MPa, which were able to support up to 50 cycles of loading without plastic deformation. Notably, MgPSr-PCL30 scaffolds were able to promote in vitro bone formation in medium without the supplementation with osteo-inducing components. In addition, long-term in vivo performance of the 3D printed scaffolds was investigated in an equine tuber coxae model over 6 months. The micro-CT and histological analysis showed that implantation of MgPSr-PCL30 induced bone regeneration, while no bone formation was observed in the empty defects. Overall, the novel polymer modified MgP ceramic material and extrusion-based 3D printing process presented here greatly improved the shape ability and load bearing properties of MgP-based ceramics with simultaneously induction of new bone formation.
AUTHOR Iordache, F. and Alexandru, D. and Pisoschi, A. M. and PoP, A.
Title 3D Bioprinting of Blood Vessel Model Using Collagen-Hyaluronic Acid Hydrogel [Abstract]
Year 2019
Journal/Proceedings AgroLife Scientific Journal
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Abstract
3D bioprinting is a technology that supports fabrication of biomimetic tissues with complex architecture. It has application in drug discovery, tissue development, and regenerative medicine. The aim of this study was to create a blood vessel model correlating properties of collagen-hyaluronic acid hydrogel with bioprinter parameters such as speed rate, pressure, number of layers, nozzle diameter, and temperature. The blood vessel model was created using BioCAD software and bioprinted by extrusion technology using collagen-hyaluronic acid hydrogel. We analyzed the water uptake, enzymatic degradation and morphology by scanning electron microscopy and after staining with Hematoxylin and Eosin (H&E) and Trichromic Masson dyes. The results showed that the blood vessel constructs have 2.46 mm (±0.41) mean diameter, 1.4 mm (±0.10) mean thick wall, and 2.8 mm (±0.05) mean height which is appropriate with the model created in the BioCAD software. The optimal parameters for these constructs were: 1.1 bar pressure, 1mm/sec speed rate, 18°C temperature, 0.2 mm nozzle diameter, and 10 numbers of layers. Increasing hydrogel weight by 22% at 2 hours after immersion in PBS suggesting that is hydrophilic. Furthermore, decreasing by up to 47.2% in the presence of collagenase (50 μg/ml) shows that is biodegradable. H&E and Trichromic Masson staining showed that collagen-hyaluronic acid hydrogel organized in a network with pores dimension that could support cells growth and differentiation. In conclusion, our scaffold mimics the blood vessel structure, further experiment will be addressed for study the biocompatibility of these scaffold with mesenchymal stem cells.
AUTHOR Li, J. and Liu, X. and Crook, J. M. and Wallace, G. G.
Title 3D graphene-containing structures for tissue engineering [Abstract]
Year 2019
Journal/Proceedings Materials Today Chemistry
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Abstract
Graphene and its derivatives have been extensively explored in various fields and have shown great promise toward energy harvesting, environmental protection, and health care. 3D graphene-containing structures (3DGCSs) are especially endowed with useable features relating to physicochemical properties within the hierarchical architectures. Thus, 3DGCSs are increasingly being applied for tissue engineering because of their supportability of human cells and functionalization potential. This review focuses on recent progress in tissue engineering utilizing 3DGCSs, providing insights into fabrication, application, and constraints in bionic research.
AUTHOR Mehrotra, Shreya and Moses, Joseph Christakiran and Bandyopadhyay, Ashutosh and Mandal, Biman B.
Title 3D Printing/Bioprinting Based Tailoring of in Vitro Tissue Models: Recent Advances and Challenges [Abstract]
Year 2019
Journal/Proceedings ACS Applied Bio Materials
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Abstract
Prodigious progress in the past decade has pronounced 3D printing as one of the most promising technique for assembling biological materials in a complex layout that mimics native human tissues. With the advent of technology, several improvements in printing techniques have facilitated the development of intricate strategies and designs that were imaginably distant due to the conventional top-down approaches. Most of these advanced strategies generally follow a thorough coordination and an elaborate biomimetic blueprint due to which it is now possible to fabricate in vitro tissue models with ease. However, much remains to be accomplished at several forefronts for utilizing this technology to its full potential. With several printing strategies at the lead, it has now become essential to systematically analyze and learn from several endeavors such that shortcomings can be understood and future efforts can be made toward negating them. Taking account of all the recent tissue specific developments in this field, this review serves as a framework for bringing together in discussion several strategies and constraints in developing small scaled in vitro tissues. Highlighting the growing popularity of the organ and body on chip platforms and their easy scale up using 3D printing, latest advancements, and the challenges in this field are also discussed. Prodigious progress in the past decade has pronounced 3D printing as one of the most promising technique for assembling biological materials in a complex layout that mimics native human tissues. With the advent of technology, several improvements in printing techniques have facilitated the development of intricate strategies and designs that were imaginably distant due to the conventional top-down approaches. Most of these advanced strategies generally follow a thorough coordination and an elaborate biomimetic blueprint due to which it is now possible to fabricate in vitro tissue models with ease. However, much remains to be accomplished at several forefronts for utilizing this technology to its full potential. With several printing strategies at the lead, it has now become essential to systematically analyze and learn from several endeavors such that shortcomings can be understood and future efforts can be made toward negating them. Taking account of all the recent tissue specific developments in this field, this review serves as a framework for bringing together in discussion several strategies and constraints in developing small scaled in vitro tissues. Highlighting the growing popularity of the organ and body on chip platforms and their easy scale up using 3D printing, latest advancements, and the challenges in this field are also discussed.
AUTHOR Shen, Jie and Wang, Wenhao and Zhai, Xinyun and Chen, Bo and Qiao, Wei and Li, Wan and Li, Penghui and Zhao, Ying and Meng, Yuan and Qian, Shi and Liu, Xuanyong and Chu, Paul K. and Yeung, Kelvin W. K.
Title 3D-printed nanocomposite scaffolds with tunable magnesium ionic microenvironment induce in situ bone tissue regeneration [Abstract]
Year 2019
Journal/Proceedings Applied Materials Today
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Abstract
Local tissue microenvironment is able to regulate cell-to-cell interaction that leads to effective tissue repair. This study aims to demonstrate a tunable magnesium ionic (Mg2+) microenvironment in bony tissue that can significantly induce bone defect repair. The concept can be realized by using a newly fabricated nanocomposite comprising of custom-made copolymer polycaprolactone-co-poly(ethylene glycol)-co-polycaprolactone (PCL-PEG-PCL) and surface-modified magnesium oxide (MgO) nanoparticles. In this study, additive manufacturing (AM) technology had been adopted to help design the porous three-dimensional (3D) scaffolds with tunable Mg2+ microenvironment. We found that the wettability and printability of new copolymer had been improved as compared with that of PCL polymer. Additionally, when MgO nanoparticles incorporated into the newly synthesized hydrophilic copolymer matrix, it could lead to increased compressive moduli significantly. In the in vitro studies, the fabricated nanocomposite scaffold with low concentration of Mg2+ microenvironment not only demonstrated better cytocompatibility, but also remarkably enhanced osteogenic differentiation in vitro as compared with the pure PCL and PCL-PEG-PCL co-polymer controls. In the animal studies, we also found that superior and early bone formation and tissue mineralization could be observed in the same 3D printed scaffold. However, the nanocomposite scaffold with high concentration of Mg2+ jeopardized the in situ bony tissue regeneration capability due to excessive magnesium ions in bone tissue microenvironment. Lastly, this study demonstrates that the nanocomposite 3D scaffold with controlled magnesium concentration in bone tissue microenvironment can effectively promote bone defect repair.
AUTHOR Wang, Weiguang and Huang, Boyang and Byun, Jae Jong and Bártolo, Paulo
Title Assessment of PCL/carbon material scaffolds for bone regeneration [Abstract]
Year 2019
Journal/Proceedings Journal of the Mechanical Behavior of Biomedical Materials
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Abstract
Biomanufacturing is a relatively new research domain focusing on the use of additive manufacturing technologies, biomaterials, cells and biomolecular signals to produce tissue constructs for tissue engineering. For bone regeneration, researchers are focusing on the use of polymeric and polymer/ceramic scaffolds seeded with osteoblasts or mesenchymal stem cells. However, the design of high-performance scaffolds in terms of mechanical, cell-stimulation and biological performance is still required. This is the first paper investigating the use of an extrusion additive manufacturing system to produce poly(ε-caprolactone) (PCL), PCL/graphene nanosheet (GNS) and PCL/carbon nanotube (CNT) scaffolds for bone applications. Scaffolds with regular and reproducible architecture were produced and evaluated from chemical, physical and biological points of view. Results suggest that the addition of both graphene and CNT allow the fabrication of scaffolds with improved properties. It also shows that scaffolds containing graphene present better mechanical properties and high cell-affinity improving cell attachment, proliferation and differentiation.
AUTHOR Freeman, F. E. and Browe, D. C. and Nulty, J. and Von Euw, S. and Grayson, W. L. and Kelly, D. J.
Title Biofabrication of multiscale bone extracellular matrix scaffolds for bone tissue engineering. [Abstract]
Year 2019
Journal/Proceedings European Cells and Materials Journal
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Abstract
Interconnected porosity is critical to the design of regenerative scaffolds, as it permits cell migration, vascularisation and diffusion of nutrients and regulatory molecules inside the scaffold. 3D printing is a promising strategy to achieve this as it allows the control over scaffold pore size, porosity and interconnectivity. Thus, the aim of the present study was to integrate distinct biofabrication strategies to develop a multiscale porous scaffold that was not only mechanically functional at the time of implantation, but also facilitated rapid vascularisation and provided stem cells with appropriate cues to enable their differentiation into osteoblasts. To achieve this, polycaprolactone (PCL) was functionalised with decellularised bone extracellular matrix (ECM), to produce osteoinductive filaments for 3D printing. The addition of bone ECM to the PCL not only increased the mechanical properties of the resulting scaffold, but also increased cellular attachment and enhanced osteogenesis of mesenchymal stem cells (MSCs). In vivo, scaffold pore size determined the level of vascularisation, with a larger filament spacing supporting faster vessel in-growth and more new bone formation. By freeze-drying solubilised bone ECM within these 3D-printed scaffolds, it was possible to introduce a matrix network with microscale porosity that further enhanced cellular attachment in vitro and increased vessel infiltration and overall levels of new bone formation in vivo. To conclude, an "off-the-shelf" multiscale bone-ECM-derived scaffold was developed that was mechanically stable and, once implanted in vivo, will drive vascularisation and, ultimately, lead to bone regeneration.
AUTHOR Yilmaz, B. and Tahmasebifar, A. and Baran, E. T.
Title Bioprinting Technologies in Tissue Engineering
Year 2019
Journal/Proceedings Advances in Biochemical Engineering/Biotechnology
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AUTHOR Loai, Sadi and Kingston, Benjamin R. and Wang, Zongjie and Philpott, David N. and Tao, Mingyang and Cheng, Hai-Ling Margaret
Title Clinical Perspectives on 3D Bioprinting Paradigms for Regenerative Medicine
Year 2019
Journal/Proceedings Regenerative Medicine Frontiers
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AUTHOR Marques, C. F. and Diogo, G. S. and Pina, S. and Oliveira, J. M. and Silva, T. H. and Reis, R. L.
Title Collagen-based bioinks for hard tissue engineering applications: a comprehensive review [Abstract]
Year 2019
Journal/Proceedings Journal of Materials Science: Materials in Medicine
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In the last few years, additive manufacturing (AM) has been gaining great interest in the fabrication of complex structures for soft-to-hard tissues regeneration, with tailored porosity, and boosted structural, mechanical, and biological properties. 3D printing is one of the most known AM techniques in the field of biofabrication of tissues and organs. This technique opened up opportunities over the conventional ones, with the capability of creating replicable, customized, and functional structures that can ultimately promote effectively different tissues regeneration. The uppermost component of 3D printing is the bioink, i.e. a mixture of biomaterials that can also been laden with different cell types, and bioactive molecules. Important factors of the fabrication process include printing fidelity, stability, time, shear-thinning properties, mechanical strength and elasticity, as well as cell encapsulation and cell-compatible conditions. Collagen-based materials have been recognized as a promising choice to accomplish an ideal mimetic bioink for regeneration of several tissues with high cell-activating properties. This review presents the state-of-art of the current achievements on 3D printing using collagen-based materials for hard tissue engineering, particularly on the development of scaffolds for bone and cartilage repair/regeneration. The ultimate aim is to shed light on the requirements to successfully print collagen-based inks and the most relevant properties exhibited by the so fabricated scaffolds. In this regard, the adequate bioprinting parameters are addressed, as well as the main materials properties, namely physicochemical and mechanical properties, cell compatibility and commercial availability, covering hydrogels, microcarriers and decellularized matrix components. Furthermore, the fabrication of these bioinks with and without cells used in inkjet printing, laser-assisted printing, and direct in writing technologies are also overviewed. Finally, some future perspectives of novel bioinks are given.
AUTHOR Marchiori, Gregorio and Berni, Matteo and Boi, Marco and Petretta, Mauro and Grigolo, Brunella and Bellucci, Devis and Cannillo, Valeria and Garavelli, Chiara and Bianchi, Michele
Title Design of a novel procedure for the optimization of the mechanical performances of 3D printed scaffolds for bone tissue engineering combining CAD, Taguchi method and FEA [Abstract]
Year 2019
Journal/Proceedings Medical Engineering and Physics
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In order to increase manufacturing and experimental efficiency, a certain degree of control over design performances before realization phase is recommended. In this context, this paper presents an integrated procedure to design 3D scaffolds for bone tissue engineering. The procedure required a combination of Computer Aided Design (CAD), Finite Element Analysis (FEA), and Design methodologies Of Experiments (DOE), firstly to understand the influence of the design parameters, and then to control them. Based on inputs from the literature and limitations imposed by the chosen manufacturing process (Precision Extrusion Deposition), 36 scaffold architectures have been drawn. The porosity of each scaffold has been calculated with CAD. Thereafter, a generic scaffold material was considered and its variable parameters were combined with the geometrical ones according to the Taguchi method, i.e. a DOE method. The compressive response of those principal combinations was simulated by FEA, and the influence of each design parameter on the scaffold compressive behaviour was clarified. Finally, a regression model was obtained correlating the scaffold's mechanical performances to its geometrical and material parameters. This model has been applied to a novel composite material made of polycaprolactone and innovative bioactive glass. By setting specific porosity (50%) and stiffness (0.05 GPa) suitable for trabecular bone substitutes, the model selected 4 of the 36 initial scaffold architectures. Only these 4 more promising geometries will be realized and physically tested for advanced indications on compressive strength and biocompatibility.
AUTHOR Wang, Weiguang and Junior, José Roberto Passarini and Nalesso, Paulo Roberto Lopes and Musson, David and Cornish, Jillian and Mendonça, Fernanda and Caetano, Guilherme Ferreira and Bártolo, Paulo
Title Engineered 3D printed poly(ɛ-caprolactone)/graphene scaffolds for bone tissue engineering [Abstract]
Year 2019
Journal/Proceedings Materials Science and Engineering: C
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Scaffolds are important physical substrates for cell attachment, proliferation and differentiation. Multiple factors could influence the optimal design of scaffolds for a specific tissue, such as the geometry, the materials used to modulate cell proliferation and differentiation, its biodegradability and biocompatibility. The optimal design of a scaffold for a specific tissue strongly depends on both materials and manufacturing processes. Previous studies of human adipose-derived stem cells (hADSCs) seeded on poly(ε-caprolactone) (PCL)/graphene scaffolds have proved that the addition of small concentrations of graphene to PCL scaffolds improves cell proliferation. Based on such results, this paper further investigates, for the first time, both in vitro and in vivo characteristics of 3D printed PCL/graphene scaffolds. Scaffolds were evaluated from morphological, biological and short term immune response points of view. Results show that the produced scaffolds induce an acceptable level of immune response, suggesting high potential for in vivo applications. Finally, the scaffolds were used to treat a rat calvaria critical size defect with and without applying micro electrical stimulation (10 μA). Quantification of connective and new bone tissue formation and the levels of ALP, RANK, RANKL, OPG were considered. Results show that the use of scaffolds containing graphene and electrical stimulation seems to increase cell migration and cell influx, leading to new tissue formation, well-organized tissue deposition and bone remodelling.
AUTHOR Angelopoulos, Ioannis and Allenby, Mark C. and Lim, Mayasari and Zamorano, Mauricio
Title Engineering inkjet bioprinting processes toward translational therapies [Abstract]
Year 2019
Journal/Proceedings Biotechnology and Bioengineering
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Abstract Bioprinting is the assembly of three-dimensional (3D) tissue constructs by layering cell-laden biomaterials using additive manufacturing techniques, offering great potential for tissue engineering and regenerative medicine. Such a process can be performed with high resolution and control by personalized or commercially available inkjet printers. However, bioprinting's clinical translation is significantly limited due to process engineering challenges. Upstream challenges include synthesis, cellular incorporation, and functionalization of “bioinks,” and extrusion of print geometries. Downstream challenges address sterilization, culture, implantation, and degradation. In the long run, bioinks must provide a microenvironment to support cell growth, development, and maturation and must interact and integrate with the surrounding tissues after implantation. Additionally, a robust, scaleable manufacturing process must pass regulatory scrutiny from regulatory bodies such as U.S. Food and Drug Administration, European Medicines Agency, or Australian Therapeutic Goods Administration for bioprinting to have a real clinical impact. In this review, recent advances in inkjet-based 3D bioprinting will be presented, emphasizing on biomaterials available, their properties, and the process to generate bioprinted constructs with application in medicine. Current challenges and the future path of bioprinting and bioinks will be addressed, with emphasis in mass production aspects and the regulatory framework bioink-based products must comply to translate this technology from the bench to the clinic.
AUTHOR Huang, Boyang and Vyas, Cian and Roberts, Iwan and Poutrel, Quentin-Arthur and Chiang, Wei-Hung and Blaker, Jonny J. and Huang, Zhucheng and Bártolo, Paulo
Title Fabrication and characterisation of 3D printed MWCNT composite porous scaffolds for bone regeneration [Abstract]
Year 2019
Journal/Proceedings Materials Science and Engineering: C
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Abstract
Carbon nanotubes (CNTs) with exceptional physical and chemical properties are attracting significant interest in the field of tissue engineering. Several reports investigated CNTs biocompatibility and their impact in terms of cell attachment, proliferation and differentiation mainly using polymer/CNTs membranes. However, these 2D membranes are not able to emulate the complex in vivo environment. In this paper, additive manufacturing (3D printing) is used to create composite 3D porous scaffolds containing different loadings of multi-walled carbon nanotubes (MWCNT) (0.25, 0.75 and 3 wt%) for bone tissue regeneration. Pre-processed and processed materials were extensively characterised in terms of printability, morphological and topographic characteristics and thermal, mechanical and biological properties. Scaffolds with pore sizes ranging between 366 μm and 397 μm were successfully produced and able to sustain early-stage human adipose-derived mesenchymal stem cells attachment and proliferation. Results show that MWCNTs enhances protein adsorption, mechanical and biological properties. Composite scaffolds, particularly the 3 wt% loading of MWCNTs, seem to be good candidates for bone tissue regeneration.
AUTHOR Rathan, Swetha and Dejob, Léa and Schipani, Rossana and Haffner, Benjamin and Möbius, Matthias E. and Kelly, Daniel J.
Title Fiber Reinforced Cartilage ECM Functionalized Bioinks for Functional Cartilage Tissue Engineering [Abstract]
Year 2019
Journal/Proceedings Advanced Healthcare Materials
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Abstract Focal articular cartilage (AC) defects, if left untreated, can lead to debilitating diseases such as osteoarthritis. While several tissue engineering strategies have been developed to promote cartilage regeneration, it is still challenging to generate functional AC capable of sustaining high load-bearing environments. Here, a new class of cartilage extracellular matrix (cECM)-functionalized alginate bioink is developed for the bioprinting of cartilaginous tissues. The bioinks are 3D-printable, support mesenchymal stem cell (MSC) viability postprinting and robust chondrogenesis in vitro, with the highest levels of COLLII and ACAN expression observed in bioinks containing the highest concentration of cECM. Enhanced chondrogenesis in cECM-functionalized bioinks is also associated with progression along an endochondral-like pathway, as evident by increases in RUNX2 expression and calcium deposition in vitro. The bioinks loaded with MSCs and TGF-β3 are also found capable of supporting robust chondrogenesis, opening the possibility of using such bioinks for direct “print-and-implant” cartilage repair strategies. Finally, it is demonstrated that networks of 3D-printed polycaprolactone fibers with compressive modulus comparable to native AC can be used to mechanically reinforce these bioinks, with no loss in cell viability. It is envisioned that combinations of such biomaterials can be used in multiple-tool biofabrication strategies for the bioprinting of biomimetic cartilaginous implants.
AUTHOR Geetha Bai, Renu and Muthoosamy, Kasturi and Manickam, Sivakumar and Hilal-Alnaqbi, Ali
Title Graphene-based 3D scaffolds in tissue engineering: fabrication, applications, and future scope in liver tissue engineering [Abstract]
Year 2019
Journal/Proceedings International journal of nanomedicine
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Tissue engineering embraces the potential of recreating and replacing defective body parts by advancements in the medical field. Being a biocompatible nanomaterial with outstanding physical, chemical, optical, and biological properties, graphene-based materials were successfully employed in creating the perfect scaffold for a range of organs, starting from the skin through to the brain. Investigations on 2D and 3D tissue culture scaffolds incorporated with graphene or its derivatives have revealed the capability of this carbon material in mimicking in vivo environment. The porous morphology, great surface area, selective permeability of gases, excellent mechanical strength, good thermal and electrical conductivity, good optical properties, and biodegradability enable graphene materials to be the best component for scaffold engineering. Along with the apt microenvironment, this material was found to be efficient in differentiating stem cells into specific cell types. Furthermore, the scope of graphene nanomaterials in liver tissue engineering as a promising biomaterial is also discussed. This review critically looks into the unlimited potential of graphene-based nanomaterials in future tissue engineering and regenerative therapy.
AUTHOR Tondera, Christoph and Akbar, Teuku Fawzul and Thomas, Alvin Kuriakose and Lin, Weilin and Werner, Carsten and Busskamp, Volker and Zhang, Yixin and Minev, Ivan R.
Title Highly Conductive, Stretchable, and Cell-Adhesive Hydrogel by Nanoclay Doping [Abstract]
Year 2019
Journal/Proceedings Small
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Abstract Electrically conductive materials that mimic physical and biological properties of tissues are urgently required for seamless brain–machine interfaces. Here, a multinetwork hydrogel combining electrical conductivity of 26 S m−1, stretchability of 800%, and tissue-like elastic modulus of 15 kPa with mimicry of the extracellular matrix is reported. Engineering this unique set of properties is enabled by a novel in-scaffold polymerization approach. Colloidal hydrogels of the nanoclay Laponite are employed as supports for the assembly of secondary polymer networks. Laponite dramatically increases the conductivity of in-scaffold polymerized poly(ethylene-3,4-diethoxy thiophene) in the absence of other dopants, while preserving excellent stretchability. The scaffold is coated with a layer containing adhesive peptide and polysaccharide dextran sulfate supporting the attachment, proliferation, and neuronal differentiation of human induced pluripotent stem cells directly on the surface of conductive hydrogels. Due to its compatibility with simple extrusion printing, this material promises to enable tissue-mimetic neurostimulating electrodes.
AUTHOR Apelgren, Peter and Karabulut, Erdem and Amoroso, Matteo and Mantas, Athanasios and Martínez Ávila, Héctor and Kölby, Lars and Kondo, Tetsuo and Toriz, Guillermo and Gatenholm, Paul
Title In Vivo Human Cartilage Formation in Three-Dimensional Bioprinted Constructs with a Novel Bacterial Nanocellulose Bioink [Abstract]
Year 2019
Journal/Proceedings ACS Biomaterials Science & Engineering
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Bacterial nanocellulose (BNC) is a 3D network of nanofibrils exhibiting excellent biocompatibility. Here, we present the aqueous counter collision (ACC) method of BNC disassembly to create bioink with suitable properties for cartilage-specific 3D-bioprinting. BNC was disentangled by ACC, and fibril characteristics were analyzed. Bioink printing fidelity and shear-thinning properties were evaluated. Cell-laden bioprinted grid constructs (5 × 5 × 1 mm3) containing human nasal chondrocytes (10 M mL-1) were implanted in nude mice and explanted after 30 and 60 days. Both ACC and hydrolysis resulted in significantly reduced fiber lengths, with ACC resulting in longer fibrils and fewer negative charges relative to hydrolysis. Moreover, ACC-BNC bioink showed outstanding printability, postprinting mechanical stability, and structural integrity. In vivo, cell-laden structures were rapidly integrated, maintained structural integrity, and showed chondrocyte proliferation, with 32.8 ± 13.8 cells per mm2 observed after 30 days and 85.6 ± 30.0 cells per mm2 at day 60 (p = 0.002). Furthermore, a full-thickness skin graft was attached and integrated completely on top of the 3D-bioprinted construct. The novel ACC disentanglement technique makes BNC biomaterial highly suitable for 3D-bioprinting and clinical translation, suggesting cell-laden 3D-bioprinted ACC-BNC as a promising solution for cartilage repair. Bacterial nanocellulose (BNC) is a 3D network of nanofibrils exhibiting excellent biocompatibility. Here, we present the aqueous counter collision (ACC) method of BNC disassembly to create bioink with suitable properties for cartilage-specific 3D-bioprinting. BNC was disentangled by ACC, and fibril characteristics were analyzed. Bioink printing fidelity and shear-thinning properties were evaluated. Cell-laden bioprinted grid constructs (5 × 5 × 1 mm3) containing human nasal chondrocytes (10 M mL-1) were implanted in nude mice and explanted after 30 and 60 days. Both ACC and hydrolysis resulted in significantly reduced fiber lengths, with ACC resulting in longer fibrils and fewer negative charges relative to hydrolysis. Moreover, ACC-BNC bioink showed outstanding printability, postprinting mechanical stability, and structural integrity. In vivo, cell-laden structures were rapidly integrated, maintained structural integrity, and showed chondrocyte proliferation, with 32.8 ± 13.8 cells per mm2 observed after 30 days and 85.6 ± 30.0 cells per mm2 at day 60 (p = 0.002). Furthermore, a full-thickness skin graft was attached and integrated completely on top of the 3D-bioprinted construct. The novel ACC disentanglement technique makes BNC biomaterial highly suitable for 3D-bioprinting and clinical translation, suggesting cell-laden 3D-bioprinted ACC-BNC as a promising solution for cartilage repair.
AUTHOR Schipani, Rossana and Nolan, David R. and Lally, Caitrίona and Kelly, Daniel J.
Title Integrating finite element modelling and 3D printing to engineer biomimetic polymeric scaffolds for tissue engineering [Abstract]
Year 2019
Journal/Proceedings Connective Tissue Research
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Abstract
ABSTRACTThe suitability of a scaffold for tissue engineering is determined by a number of interrelated factors. The biomaterial should be biocompatible and cell instructive, with a porosity and pore interconnectivity that facilitates cellular migration and the transport of nutrients and waste products into and out of the scaffolds. For the engineering of load bearing tissues, the scaffold may also be required to possess specific mechanical properties and/or ensure the transfer of mechanical stimuli to cells to direct their differentiation. Achieving these design goals is challenging, but could potentially be realised by integrating computational tools such as finite element (FE) modelling with three-dimensional (3D) printing techniques to assess how scaffold architecture and material properties influence the performance of the implant. In this study we first use Fused Deposition Modelling (FDM) to modulate the architecture of polycaprolactone (PCL) scaffolds, exploring the influence of varying fibre diameter, spacing and laydown pattern on the structural and mechanical properties of such scaffolds. We next demonstrate that a simple FE modelling strategy, which captures key aspects of the printed scaffold’s actual geometry and material behaviour, can be used to accurately model the mechanical characteristics of such scaffolds. We then show the utility of this strategy by using FE modelling to help design 3D printed scaffolds with mechanical properties mimicking that of articular cartilage. In conclusion, this study demonstrates that a relatively simple FE modelling approach can be used to inform the design of 3D printed scaffolds to ensure their bulk mechanical properties mimic specific target tissues.
AUTHOR Sharma, Aarushi and Desando, Giovanna and Petretta, Mauro and Chawla, Shikha and Bartolotti, Isabella and Manferdini, Cristina and Paolella, Francesca and Gabusi, Elena and Trucco, Diego and Ghosh, Sourabh and Lisignoli, Gina
Title Investigating the Role of Sustained Calcium Release in Silk-Gelatin-Based Three-Dimensional Bioprinted Constructs for Enhancing the Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stromal Cells
Year 2019
Journal/Proceedings ACS Biomaterials Science & Engineering
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AUTHOR Xu, Yichi and Peng, Jiang and Richards, Geoff and Lu, Shibi and Eglin, David
Title Optimization of electrospray fabrication of stem cell–embedded alginate–gelatin microspheres and their assembly in 3D-printed poly(ε-caprolactone) scaffold for cartilage tissue engineering [Abstract]
Year 2019
Journal/Proceedings Journal of Orthopaedic Translation
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Abstract
Objective Our study reports the optimization of electrospray human bone marrow stromal cell (hBMSCs)–embedded alginate–gelatin (Alg-Gel, same as following) microspheres for the purpose of their assembly in 3D-printed poly(ε-caprolactone) (PCL) scaffold for the fabrication of a mechanically stable and biological supportive tissue engineering cartilage construct. Methods The fabrication of the Alg-Gel microspheres using an electrospray technique was optimized in terms of polydispersity, yield of microspheres and circularity and varying fabrication conditions. PCL scaffolds were designed and printed by melt extrusion. Then, four groups were set: Alg-hBMSC microspheres cultured in the 2D well plate (Alg-hBMSCs+2D) group, Alg-Gel-hBMSC microspheres cultured in the 2D well plate (Alg-Gel-hBMSCs+2D) group, Alg-Gel-hBMSC microspheres embedded in PCL scaffold cultured in the 2D well plate (Alg-Gel-hBMSCs+2D) group and Alg-Gel-hBMSCs microspheres cultured in the 3D bioreactor (Alg-Gel-hBMSCs+3D) group. Cell viability, proliferation and chondrogenic differentiation were evaluated, and mechanical test was performed. Results Nonaggregated, low polydispersity and almost spherical microspheres of average diameter of 200–300 μm were produced with alginate 1.5 w: v%, gelatin (Type B) concentration of 0.5 w: v % and CaCl2 coagulating bath concentration of 3.0 w: v %, using 30G needle size and 8 kV and 0.6 bar voltage and air pressure, respectively. Alginate with gelatin hydrogel improved viability and promoted hBMSC proliferation better than alginate microspheres. Interestingly, hBMSCs embedded in microspheres assembled in 3D-printed PCL scaffold and cultured in a 3D bioreactor were more proliferative in comparison to the previous two groups (p < 0.05). Similarly, the GAG content, GAG/DNA ratio as well as Coll 2 and Aggr gene expression were increased in the last two groups. Conclusion Optimization of hBMSC-embedded Alg-Gel microspheres produced by electrospray has been performed. The Alg-Gel composition selected allows conservation of hBMSC viability and supports proliferation and matrix deposition. The possibility to seed and assemble microspheres in designed 3D-printed PCL scaffolds for the fabrication of a mechanically stable and biological supportive tissue engineering cartilage construct was demonstrated. Translational potential of this article We optimize and demonstrate that electrospray microsphere fabrication is a cytocompatible and facile process to produce the hBMSC-embedded microsize tissue-like particles that can easily be assembled into a stable construct. This finding could have application in the development of mechanically competent stem cell–based tissue engineering of cartilage regeneration.
AUTHOR Fenton, Owen S. and Paolini, Marion and Andresen, Jason L. and Müller, Florence J. and Langer, Robert
Title Outlooks on Three-Dimensional Printing for Ocular Biomaterials Research [Abstract]
Year 2019
Journal/Proceedings Journal of Ocular Pharmacology and Therapeutics
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Abstract Given its potential for high-resolution, customizable, and waste-free fabrication of medical devices and in vitro biological models, 3-dimensional (3D) bioprinting has broad utility within the biomaterials field. Indeed, 3D bioprinting has to date been successfully used for the development of drug delivery systems, the recapitulation of hard biological tissues, and the fabrication of cellularized organ and tissue-mimics, among other applications. In this study, we highlight convergent efforts within engineering, cell biology, soft matter, and chemistry in an overview of the 3D bioprinting field, and we then conclude our work with outlooks toward the application of 3D bioprinting for ocular research in vitro and in vivo.
AUTHOR Filardo, G. and Petretta, M. and Cavallo, C. and Roseti, L. and Durante, S. and Albisinni, U. and Grigolo, B.
Title Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold [Abstract]
Year 2019
Journal/Proceedings Bone and Joint Research
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Abstract
Objectives Meniscal injuries are often associated with an active lifestyle. The damage of meniscal tissue puts young patients at higher risk of undergoing meniscal surgery and, therefore, at higher risk of osteoarthritis. In this study, we undertook proof-of-concept research to develop a cellularized human meniscus by using 3D bioprinting technology. Methods A 3D model of bioengineered medial meniscus tissue was created, based on MRI scans of a human volunteer. The Digital Imaging and Communications in Medicine (DICOM) data from these MRI scans were processed using dedicated software, in order to obtain an STL model of the structure. The chosen 3D Discovery printing tool was a microvalve-based inkjet printhead. Primary mesenchymal stem cells (MSCs) were isolated from bone marrow and embedded in a collagen-based bio-ink before printing. LIVE/DEAD assay was performed on realized cell-laden constructs carrying MSCs in order to evaluate cell distribution and viability. Results This study involved the realization of a human cell-laden collagen meniscus using 3D bioprinting. The meniscus prototype showed the biological potential of this technology to provide an anatomically shaped, patient-specific construct with viable cells on a biocompatible material. Conclusion This paper reports the preliminary findings of the production of a custom-made, cell-laden, collagen-based human meniscus. The prototype described could act as the starting point for future developments of this collagen-based, tissue-engineered structure, which could aid the optimization of implants designed to replace damaged menisci. Cite this article: G. Filardo, M. Petretta, C. Cavallo, L. Roseti, S. Durante, U. Albisinni, B. Grigolo. Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold. Bone Joint Res 2019;8:101–106. DOI: 10.1302/2046-3758.82.BJR-2018-0134.R1.
AUTHOR Prendergast, Margaret E. and Burdick, Jason A.
Title Recent Advances in Enabling Technologies in 3D Printing for Precision Medicine [Abstract]
Year 2019
Journal/Proceedings Advanced Materials
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Abstract
Abstract Advances in areas such as data analytics, genomics, and imaging have revealed individual patient complexities and exposed the inherent limitations of generic therapies for patient treatment. These observations have also fueled the development of precision medicine approaches, where therapies are tailored for the individual rather than the broad patient population. 3D printing is a field that intersects with precision medicine through the design of precision implants with patient-directed shapes, structures, and materials or for the development of patient-specific in vitro models that can be used for screening precision therapeutics. Toward their success, advances in 3D printing and biofabrication technologies are needed with enhanced resolution, complexity, reproducibility, and speed and that encompass a broad range of cells and materials. The overall goal of this progress report is to highlight recent advances in 3D printing technologies that are helping to enable advances important in precision medicine.
AUTHOR Dooley, Max and Prasopthum, Aruna and Liao, Zhiyu and Sinjab, Faris and McLaren, Jane and Rose, Felicity R. A. J. and Yang, Jing and Notingher, Ioan
Title Spatially-offset Raman spectroscopy for monitoring mineralization of bone tissue engineering scaffolds: feasibility study based on phantom samples [Abstract]
Year 2019
Journal/Proceedings Biomedical Optics Express
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Abstract
Using phantom samples, we investigated the feasibility of spatially-offset Raman spectroscopy (SORS) as a tool for monitoring non-invasively the mineralization of bone tissue engineering scaffold in-vivo. The phantom samples consisted of 3D-printed scaffolds of poly-caprolactone (PCL) and hydroxyapatite (HA) blends, with varying concentrations of HA, to mimic the mineralisation process. The scaffolds were covered by a 4 mm layer of skin to simulate the real in-vivo measurement conditions. At a concentration of HA approximately 1/3 that of bone (~0.6 g/cm3), the characteristic Raman band of HA (960 cm−1) was detectable when the PCL:HA layer was located at 4 mm depth within the scaffold (i.e. 8 mm below the skin surface). For the layers of the PCL:HA immediately under the skin (i.e. top of the scaffold), the detection limit of HA was 0.18 g/cm3, which is approximately one order of magnitude lower than that of bone. Similar results were also found for the phantoms simulating uniform and inward gradual mineralisation of the scaffold, indicating the suitability of SORS to detect early stages of mineralisation. Nevertheless, the results also show that the contribution of the materials surrounding the scaffold can be significant and methods for subtraction need to be investigated in the future. In conclusion, these results indicate that spatially-offset Raman spectroscopy is a promising technique for in-vivo longitudinal monitoring scaffold mineralization and bone re-growth.
AUTHOR Pedrotty, Dawn M. and Volodymyr, Kuzmenko and Erdem, Karabulut and Sugrue Alan, M. and Christopher, Livia and Vaidya Vaibhav, R. and McLeod Christopher, J. and Asirvatham Samuel, J. and Paul, Gatenholm and Suraj, Kapa
Title Three-Dimensional Printed Biopatches With Conductive Ink Facilitate Cardiac Conduction When Applied to Disrupted Myocardium
Year 2019
Journal/Proceedings Circulation: Arrhythmia and Electrophysiology
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AUTHOR Caetano, Guilherme and Wang, Weiguang and Murashima, Adriana and Passarini, José Roberto and Bagne, Leonardo and Leite, Marcel and Hyppolito, Miguel and Al-Deyab, Salem and El-Newehy, Mohamed and Bártolo, Paulo and Frade, Marco Andrey Cipriani
Title Tissue Constructs with Human Adipose-Derived Mesenchymal Stem Cells to Treat Bone Defects in Rats [Abstract]
Year 2019
Journal/Proceedings Materials
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Abstract
The use of porous scaffolds created by additive manufacturing is considered a viable approach for the regeneration of critical-size bone defects. This paper investigates the xenotransplantation of polycaprolactone (PCL) tissue constructs seeded with differentiated and undifferentiated human adipose-derived mesenchymal stem cells (hADSCs) to treat calvarial critical-sized defect in Wistar rats. PCL scaffolds without cells were also considered. In vitro and in vivo biological evaluations were performed to assess the feasibility of these different approaches. In the case of cell seeded scaffolds, it was possible to observe the presence of hADSCs in the rat tissue contributing directly (osteoblasts) and indirectly (stimulation by paracrine factors) to tissue formation, organization and mineralization. The presence of bone morphogenetic protein-2 (BMP-2) in the rat tissue treated with cell-seeded PCL scaffolds suggests that the paracrine factors of undifferentiated hADSC cells could stimulate BMP-2 production by surrounding cells, leading to osteogenesis. Moreover, BMP-2 acts synergistically with growth factors to induce angiogenesis, leading to higher numbers of blood vessels in the groups containing undifferentiated and differentiated hADSCs.
AUTHOR Kang, Yuan and Wang, Chaoli and Qiao, Youbei and Gu, Junwei and Zhang, Han and Peijs, Ton and Kong, Jie and Zhang, Guangcheng and Shi, Xuetao
Title Tissue-Engineered Trachea Consisting of Electrospun Patterned sc-PLA/GO-g-IL Fibrous Membranes with Antibacterial Property and 3D-Printed Skeletons with Elasticity [Abstract]
Year 2019
Journal/Proceedings Biomacromolecules
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Abstract
In this study, a tissue-engineered trachea, consisting of multilevel structural electrospun polylactide (PLA) membranes enveloping 3D-printed thermoplastic polyurethane (TPU) skeletons, was developed to create a mechanically robust, antibacterial and bioresorbable graft for the tracheal reconstruction. The study design incorporated two distinct uses of stereocomplex PLA: patterned electrospun fibers to enhance tissue integration compared to the random layered fibers, meanwhile possessing good antibacterial property; and 3D-printed TPU scaffold with elasticity to provide external support and protection. Herein, ionic liquid (IL)-functioned graphene oxide (GO) was synthesized and presented enhanced mechanical and hydrophilicity properties. More interesting, antibacterial activity of the GO-g-IL modified PLA membranes were proved by Escherichia coli and Staphylococcus aureus, showing superior antibacterial effect compared to single GO or IL. The synergistic antibacterial effect could be related to that GO break cytomembrane of bacteria by its extremely sharp edges, while IL works by electrostatic interaction between its cationic structures and electronegative phosphate groups of bacteria membranes, leading to the loss of cell electrolyte and cell death. Hence, after L929 fibroblast cells were seeded on patterned fibrous membranes with phenotypic shape, further effective cell infiltration, cell proliferation and attachment were observed. In addition, the tissue-engineered trachea scaffolds were implanted into rabbit models. The in vivo result confirmed that the scaffolds with patterned membranes manifested favorable biocompatibility and promoted tissue regeneration. In this study, a tissue-engineered trachea, consisting of multilevel structural electrospun polylactide (PLA) membranes enveloping 3D-printed thermoplastic polyurethane (TPU) skeletons, was developed to create a mechanically robust, antibacterial and bioresorbable graft for the tracheal reconstruction. The study design incorporated two distinct uses of stereocomplex PLA: patterned electrospun fibers to enhance tissue integration compared to the random layered fibers, meanwhile possessing good antibacterial property; and 3D-printed TPU scaffold with elasticity to provide external support and protection. Herein, ionic liquid (IL)-functioned graphene oxide (GO) was synthesized and presented enhanced mechanical and hydrophilicity properties. More interesting, antibacterial activity of the GO-g-IL modified PLA membranes were proved by Escherichia coli and Staphylococcus aureus, showing superior antibacterial effect compared to single GO or IL. The synergistic antibacterial effect could be related to that GO break cytomembrane of bacteria by its extremely sharp edges, while IL works by electrostatic interaction between its cationic structures and electronegative phosphate groups of bacteria membranes, leading to the loss of cell electrolyte and cell death. Hence, after L929 fibroblast cells were seeded on patterned fibrous membranes with phenotypic shape, further effective cell infiltration, cell proliferation and attachment were observed. In addition, the tissue-engineered trachea scaffolds were implanted into rabbit models. The in vivo result confirmed that the scaffolds with patterned membranes manifested favorable biocompatibility and promoted tissue regeneration.
AUTHOR Lim, Seng Han and Kathuria, Himanshu and Tan, Justin Jia Yao and Kang, Lifeng
Title 3D printed drug delivery and testing systems — a passing fad or the future? [Abstract]
Year 2018
Journal/Proceedings Advanced Drug Delivery Reviews
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Abstract
The US Food and Drug Administration approval of the first 3D printed tablet in 2015 has ignited growing interest in 3D printing, or additive manufacturing (AM), for drug delivery and testing systems. Beyond just a novel method for rapid prototyping, AM provides key advantages over traditional manufacturing of drug delivery and testing systems. These includes the ability to fabricate complex geometries to achieve variable drug release kinetics; ease of personalising pharmacotherapy for patient and lowering the cost for fabricating personalised dosages. Furthermore, AM allows fabrication of complex and micron-sized tissue scaffolds and models for drug testing systems that closely resemble in vivo conditions. However, there are several limitations such as regulatory concerns that may impede the progression to market. Here, we provide an overview of the advantages of AM drug delivery and testing, as compared to traditional manufacturing techniques. Also, we discuss the key challenges and future directions for AM enabled pharmaceutical applications.
AUTHOR Caetano, Guilherme Ferreira and Wang, Weiguang and Chiang, Wei-Hung and Cooper, Glen and Diver, Carl and Blaker, Jonny James and Frade, Marco Andrey and Bártolo, Paulo
Title 3D-Printed Poly(ɛ-caprolactone)/Graphene Scaffolds Activated with P1-Latex Protein for Bone Regeneration [Abstract]
Year 2018
Journal/Proceedings 3D Printing and Additive Manufacturing
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Abstract Biomanufacturing is a relatively new research domain focusing on the use of additive manufacturing technologies, biomaterials, cells, and biomolecular signals to produce tissue constructs for tissue engineering. For bone regeneration, researchers are focusing on the use of polymeric and polymer/ceramic scaffolds seeded with osteoblasts or mesenchymal stem cells. However, high-performance scaffolds in terms of mechanical, cell stimulation, and biological performance are still required. This article investigates the use of an extrusion additive manufacturing system to produce poly(ɛ-caprolactone) (PCL) and PCL/graphene nanosheet scaffolds for bone applications. Scaffolds with regular and reproducible architecture and uniform dispersion of graphene were produced and coated with P1-latex protein extracted from the Hevea brasiliensis rubber tree. Results show that the obtained scaffolds cultivated with human adipose-derived stem cells present no toxicity effects. The presence of graphene nanosheet and P1-latex protein in the scaffolds increased cell proliferation compared with PCL scaffolds. Moreover, the presence of P1-latex protein promotes earlier osteogenic differentiation, suggesting that PCL/graphene/P1-latex protein scaffolds are suitable for bone regeneration applications.
AUTHOR Wang, Hanxiao and das Neves Domingos, Marco Andre and Scenini, Fabio
Title Advanced mechanical and thermal characterization of 3D bioextruded poly(ε-caprolactone)-based composites [Abstract]
Year 2018
Journal/Proceedings Rapid Prototyping Journal
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Abstract
Purpose The main purpose of the present work is to study the effect of nano hydroxyapatite (HA) and graphene oxide (GO) particles on thermal and mechanical performances of 3D printed poly(ε-caprolactone) (PCL) filaments used in Bone Tissue Engineering (BTE). Design/methodology/approach Raw materials were prepared by melt blending, followed by 3D printing via 3D Discovery (regenHU Ltd., CH) with all fabricating parameters kept constant. Filaments, including pure PCL, PCL/HA, and PCL/GO, were tested under the same conditions. Several techniques were used to mechanically, thermally, and microstructurally evaluate properties of these filaments, including Differential Scanning Calorimetry (DSC), tensile test, nano indentation, and Scanning Electron Microscope (SEM). Findings Results show that both HA and GO nano particles are capable of improving mechanical performance of PCL. Enhanced mechanical properties of PCL/HA result from reinforcing effect of HA, while a different mechanism is observed in PCL/GO, where degree of crystallinity plays an important role. In addition, GO is more efficient at enhancing mechanical performance of PCL compared with HA. Originality/value For the first time, a systematic study about effects of nano HA and GO particles on bioactive scaffolds produced by Additive Manufacturing (AM) for bone tissue engineering applications is conducted in this work. Mechanical and thermal behaviors of each sample, pure PCL, PCL/HA and PCL/GO, are reported, correlated, and compared with literature.
AUTHOR Fortunato, Gabriele Maria and Maria, Carmelo De and Eglin, David and Serra, Tiziano and Vozzi, Giovanni
Title An ink-jet printed electrical stimulation platform for muscle tissue regeneration [Abstract]
Year 2018
Journal/Proceedings Bioprinting
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Abstract
Conducting polymeric materials have been used to modulate response of cells seeded on their surfaces. However, there is still major improvement to be made related to their biocompatibility, conductivity, stability in biological milieu, and processability toward truly tissue engineered functional device. In this work, conductive polymer, poly(3,4-ethylene-dioxythiophene):polystyrene-sulfonate (PEDOT:PSS), and its possible applications in tissue engineering were explored. In particular PEDOT:PSS solution was inkjet printed onto a gelatin substrate for obtaining a conductive structure. Mechanical and electrical characterizations, structural stability by swelling and degradation tests were carried out on different PEDOT-based samples obtained by varying the number of printed PEDOT layers from 5 to 50 on gelatin substrate. Biocompatibility of substrates was investigated on C2C12 myoblasts, through metabolic activity assay and imaging analysis during a 7-days culture period, to assess cell morphology, differentiation and alignment. The results of this first part allowed to proceed with the second part of the study in which these substrates were used for the design of an electrical stimulation device, with the aim of providing the external stimulus (3 V amplitude square wave at 1 and 2 Hz frequency) to guide myotubes alignment and enhance differentiation, having in this way promising applications in the field of muscle tissue engineering.
AUTHOR Park, Hae Sang and Lee, Ji Seung and Jung, Harry and Kim, Do Yeon and Kim, Sang Wook and Sultan, Md. Tipu and Park, Chan Hum
Title An omentum-cultured 3D-printed artificial trachea: in vivo bioreactor [Abstract]
Year 2018
Journal/Proceedings Artificial Cells, Nanomedicine, and Biotechnology
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Abstract
AbstractThe purpose of this study was to evaluate whether the prior implantation of a 3D-printed polycaprolactone (PCL) artificial trachea in the omentum is beneficial for revascularization of the scaffold and reduces associated complications in the reconstruction of a circumferential tracheal defect. Ten New Zealand rabbits were divided into 2 groups: (1) PCL-OC group (PCL scaffold cultured in omentum for 2 weeks before transplantation) and (2) PCL group. In the PCL-OC group, newly formed connective tissue completely covered the luminal surface of the scaffold with mild inflammation at 2 weeks postoperatively; a minor degree of stenosis was noted at 8 weeks postoperatively. The PCL group showed scaffold exposure without any tissue regeneration at 2 weeks postoperatively, and a moderate degree of luminal stenosis 6 weeks after implantation. Histology revealed highly organized regenerated tissue composed of ciliated respiratory epithelium, and submucosal layer in the PCL-OC group. Neo-cartilage regeneration was noted in part of the regenerated tissue. The PCL group demonstrated severe inflammation and an unorganized structure compared to that of the PCL-OC group. In vivo omentum culture of the tracheal scaffold before transplantation is beneficial for rapid re-epithelialization and revascularization of the scaffold. It also prevents postoperative luminal stenosis.
AUTHOR Mouser, Vivian H. M. and Levato, Riccardo and Mensinga, Anneloes and Dhert, Wouter J. A. and Gawlitta, Debby and Malda, Jos
Title Bio-ink development for three-dimensional bioprinting of hetero-cellular cartilage constructs [Abstract]
Year 2018
Journal/Proceedings Connective Tissue Research
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Abstract
ABSTRACTBioprinting is a promising tool to fabricate organized cartilage. This study aimed to investigate the printability of gelatin-methacryloyl/gellan gum (gelMA/gellan) hydrogels with and without methacrylated hyaluronic acid (HAMA), and to explore (zone-specific) chondrogenesis of chondrocytes, articular cartilage progenitor cells (ACPCs), and multipotent mesenchymal stromal cells (MSCs) embedded in these bio-inks.The incorporating of HAMA in gelMA/gellan bio-ink increased filament stability, as measured using a filament collapse assay, but did not influence (zone-specific) chondrogenesis of any of the cell types. Highest chondrogenic potential was observed for MSCs, followed by ACPCs, which displayed relatively high proteoglycan IV mRNA levels. Therefore, two-zone constructs were printed with gelMA/gellan/HAMA containing ACPCs in the superficial region and MSCs in the middle/deep region. Chondrogenic differentiation was confirmed, however, printing influence cellular differentiation.ACPC- and MSC-laden gelMA/gellan/HAMA hydrogels are of interest for the fabrication of cartilage constructs. Nevertheless, this study underscores the need for careful evaluation of the effects of printing on cellular differentiation.
AUTHOR García-Lizarribar, Andrea and Fernández-Garibay, Xiomara and Velasco-Mallorquí, Ferran and G. Castaño, Albert and Samitier, Josep and Ramón-Azcón, Javier
Title Composite Biomaterials as Long-Lasting Scaffolds for 3D Bioprinting of Highly Aligned Muscle Tissue
Year 2018
Journal/Proceedings Macromolecular Bioscience
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AUTHOR Visscher, D. O. and Gleadall, A. and Buskermolen, J. K. and Burla, F. and Segal, J. and Koenderink, G. H. and Helder, M. N. and van Zuijlen, P. P. M.
Title Design and fabrication of a hybrid alginate hydrogel/poly(ε-caprolactone) mold for auricular cartilage reconstruction [Abstract]
Year 2018
Journal/Proceedings Journal of Biomedical Materials Research Part B: Applied Biomaterials
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Abstract
Abstract The aim of this study was to design and manufacture an easily assembled cartilage implant model for auricular reconstruction. First, the printing accuracy and mechanical properties of 3D-printed poly-ε-caprolactone (PCL) scaffolds with varying porosities were determined to assess overall material properties. Next, the applicability of alginate as cell carrier for the cartilage implant model was determined. Using the optimal outcomes of both experiments (in terms of (bio)mechanical properties, cell survival, neocartilage formation, and printing accuracy), a hybrid auricular implant model was developed. PCL scaffolds with 600 μm distances between strands exhibited the best mechanical properties and most optimal printing quality for further exploration. In alginate, chondrocytes displayed high cell survival (~83% after 21 days) and produced cartilage-like matrix in vitro. Alginate beads cultured in proliferation medium exhibited slightly higher compressive moduli (6 kPa) compared to beads cultured in chondrogenic medium (3.5 kPa, p > .05). The final auricular mold could be printed with 300 μm pores and high fidelity, and the injected chondrocytes survived the culture period of 21 days. The presented hybrid auricular mold appears to be an adequate model for cartilage tissue engineering and may provide a novel approach to auricular cartilage regeneration for facial reconstruction. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res B Part B: Appl Biomater, 2018.
AUTHOR Schmieg, Barbara and Schimek, Adrian and Franzreb, Matthias
Title Development and performance of a 3D‐printable Polyethylenglycol‐Diacrylate hydrogel suitable for enzyme entrapment and long‐term biocatalytic applications [Abstract]
Year 2018
Journal/Proceedings Engineering in Life Sciences
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Abstract
Physical entrapment of enzymes within a porous matrix is a fast and gentle process to immobilize biocatalysts to enable their recycling and long‐term use. This study introduces the development of a biocompatible 3D‐printing material suitable for enzyme entrapment, while having good rheological and UV‐hardening properties. Three different viscosity‐enhancing additives have been tested in combination with a polyethylenglycol‐diacrylate‐based hydrogel system. The addition of polyxanthan or hectorite clay particles results in hydrogels that degrade over hours or days, releasing entrapped compounds. In contrast, the addition of nanometer‐sized silicate particles ensures processability while preventing disintegration of the hydrogel. Lattice structures with a total height of 6 mm consisting of 40 layers were 3D‐printed with all materials and characterized by image analysis. Rheological measurements identified a shear stress window of 200 < τ < 500 Pa at shear rates of 25 s−1 and 25°C for well‐defined geometries with an extrusion‐based printhead. Enzymes immobilized in these long‐term stable hydrogel structures retained an effective activity of approximately 10% compared to the free enzyme in solution. It could be shown that the reduction of effective activity isn't caused by a significant reduction of the intrinsic enzyme activity but by mass transfer limitations within the printed hydrogel structures. This article is protected by copyright. All rights reserved
AUTHOR Prasopthum, Aruna and Shakesheff, Kevin M. and Yang, Jing
Title Direct three-dimensional printing of polymeric scaffolds with nanofibrous topography [Abstract]
Year 2018
Journal/Proceedings Biofabrication
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Abstract
Three-dimensional (3D) printing is a powerful manufacturing tool for making 3D structures with well-defined architectures for a wide range of applications. The field of tissue engineering has also adopted this technology to fabricate scaffolds for tissue regeneration. The ability to control architecture of scaffolds, e.g. matching anatomical shapes and having defined pore size, has since been improved significantly. However, the material surface of these scaffolds is smooth and does not resemble that found in natural extracellular matrix (ECM), in particular, the nanofibrous morphology of collagen. This natural nanoscale morphology plays a critical role in cell behaviour. Here, we have developed a new approach to directly fabricate polymeric scaffolds with an ECM-like nanofibrous topography and defined architectures using extrusion-based 3D printing. 3D printed tall scaffolds with interconnected pores were created with disparate features spanning from nanometres to centimetres. Our approach removes the need for a sacrificial mould and subsequent mould removal compared to previous methods. Moreover, the nanofibrous topography of the 3D printed scaffolds significantly enhanced protein absorption, cell adhesion and differentiation of human mesenchymal stem cells when compared to those with smooth material surfaces. These 3D printed scaffolds with both defined architectures and nanoscale ECM-mimicking morphologies have potential applications in cartilage and bone regeneration.
AUTHOR Zamani, Yasaman and Mohammadi, Javad and Amoabediny, Ghassem and Visscher, Dafydd O. and Helder, Marco N. and Zandieh-Doulabi, Behrouz and Klein-Nulend, Jenneke
Title Enhanced osteogenic activity by {MC}3T3-E1 pre-osteoblasts on chemically surface-modified poly($upepsilon$-caprolactone) 3D-printed scaffolds compared to {RGD} immobilized scaffolds [Abstract]
Year 2018
Journal/Proceedings Biomedical Materials
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Abstract
In bone tissue engineering, the intrinsic hydrophobicity and surface smoothness of three-dimensional (3D)-printed poly(ε-caprolactone) scaffolds hamper cell attachment, proliferation and differentiation. This intrinsic hydrophobicity of poly(ε-caprolactone) can be overcome by surface modifications, such as surface chemical modification or immobilization of biologically active molecules on the surface. Moreover, surface chemical modification may alter surface smoothness. Whether surface chemical modification or immobilization of a biologically active molecule on the surface is more effective to enhance pre-osteoblast proliferation and differentiation is currently unknown. Therefore, we aimed to investigate the osteogenic response of MC3T3-E1 pre-osteoblasts to chemically surface-modified and RGD-immobilized 3D-printed poly(ε-caprolactone) scaffolds. Poly(ε-caprolactone) scaffolds were 3D-printed consisting of strands deposited layer by layer with alternating 0°/90° lay-down pattern. 3D-printed poly(ε-caprolactone) scaffolds were surface-modified by either chemical modification using 3 M sodium hydroxide (NaOH) for 24 or 72 h, or by RGD-immobilization. Strands were visualized by scanning electron microscopy. MC3T3-E1 pre-osteoblasts were seeded onto the scaffolds and cultured up to 14 d. The strands of the unmodified poly(ε-caprolactone) scaffold had a smooth surface. NaOH treatment changed the scaffold surface topography from smooth to a honeycomb-like surface pattern, while RGD immobilization did not alter the surface topography. MC3T3-E1 pre-osteoblast seeding efficiency was similar (44%–54%) on all scaffolds after 12 h. Cell proliferation increased from day 1 to day 14 in unmodified controls (1.9-fold), 24 h NaOH-treated scaffolds (3-fold), 72 h NaOH-treated scaffolds (2.2-fold), and RGD-immobilized scaffolds (4.5-fold). At day 14, increased collagenous matrix deposition was achieved only on 24 h NaOH-treated (1.8-fold) and RGD-immobilized (2.2-fold) scaffolds compared to unmodified controls. Moreover, 24 h, but not 72 h, NaOH-treated scaffolds, increased alkaline phosphatase activity by 5-fold, while the increase by RGD immobilization was only 2.5-fold. Only 24 h NaOH-treated scaffolds enhanced mineralization (2.0-fold) compared to unmodified controls. In conclusion, RGD immobilization (0.011 μg mg−1 scaffold) on the surface and 24 h NaOH treatment of the surface of 3D-printed PCL scaffold both enhance pre-osteoblast proliferation and matrix deposition while only 24 h NaOH treatment results in increased osteogenic activity, making it the treatment of choice to promote bone formation by osteogenic cells.
AUTHOR Monz{'o}n, Mario and Liu, Chaozong and Ajami, Sara and Oliveira, Miguel and Donate, Ricardo and Ribeiro, Viviana and Reis, Rui L.
Title Functionally graded additive manufacturing to achieve functionality specifications of osteochondral scaffolds
Year 2018
Journal/Proceedings Bio-Design and Manufacturing
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AUTHOR Romanazzo, S. and Vedicherla, S. and Moran, C. and Kelly, D. J.
Title Meniscus ECM‐functionalised hydrogels containing infrapatellar fat pad‐derived stem cells for bioprinting of regionally defined meniscal tissue [Abstract]
Year 2018
Journal/Proceedings Journal of Tissue Engineering and Regenerative Medicine
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Abstract
Abstract Injuries to the meniscus of the knee commonly lead to osteoarthritis. Current therapies for meniscus regeneration, including meniscectomies and scaffold implantation, fail to achieve complete functional regeneration of the tissue. This has led to increased interest in cell and gene therapies and tissue engineering approaches to meniscus regeneration. The implantation of a biomimetic implant, incorporating cells, growth factors, and extracellular matrix (ECM)‐derived proteins, represents a promising approach to functional meniscus regeneration. The objective of this study was to develop a range of ECM‐functionalised bioinks suitable for 3D bioprinting of meniscal tissue. To this end, alginate hydrogels were functionalised with ECM derived from the inner and outer regions of the meniscus and loaded with infrapatellar fat pad‐derived stem cells. In the absence of exogenously supplied growth factors, inner meniscus ECM promoted chondrogenesis of fat pad‐derived stem cells, whereas outer meniscus ECM promoted a more elongated cell morphology and the development of a more fibroblastic phenotype. With exogenous growth factors supplementation, a more fibrogenic phenotype was observed in outer ECM‐functionalised hydrogels supplemented with connective tissue growth factor, whereas inner ECM‐functionalised hydrogels supplemented with TGFβ3 supported the highest levels of Sox‐9 and type II collagen gene expression and sulfated glycosaminoglycans (sGAG) deposition. The final phase of the study demonstrated the printability of these ECM‐functionalised hydrogels, demonstrating that their codeposition with polycaprolactone microfibres dramatically improved the mechanical properties of the 3D bioprinted constructs with no noticeable loss in cell viability. These bioprinted constructs represent an exciting new approach to tissue engineering of functional meniscal grafts.
AUTHOR Gill, Elisabeth L. and Li, Xia and Birch, Mark A. and Huang, Yan Yan Shery
Title Multi-length scale bioprinting towards simulating microenvironmental cues [Abstract]
Year 2018
Journal/Proceedings Bio-Design and Manufacturing
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Abstract
It is envisaged that the creation of cellular environments at multiple length scales, that recapitulate in vivo bioactive and structural roles, may hold the key to creating functional, complex tissues in the laboratory. This review considers recent advances in biofabrication and bioprinting techniques across different length scales. Particular focus is placed on 3D printing of hydrogels and fabrication of biomaterial fibres that could extend the feature resolution and material functionality of soft tissue constructs. The outlook from this review discusses how one might create and simulate microenvironmental cues in vitro. A fabrication platform that integrates the competencies of different biofabrication technologies is proposed. Such a multi-process, multiscale fabrication strategy may ultimately translate engineering capability into an accessible life sciences toolkit, fulfilling its potential to deliver in vitro disease models and engineered tissue implants.
AUTHOR Huang, Yun-An and Ho, Chris T. and Lin, Yu-Hsuan and Lee, Chen-Ju and Ho, Szu-Mo and Li, Ming-Chia and Hwang, Eric
Title Nanoimprinted Anisotropic Topography Preferentially Guides Axons and Enhances Nerve Regeneration [Abstract]
Year 2018
Journal/Proceedings Macromolecular Bioscience
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Abstract
Abstract Surface topography has a profound effect on the development of the nervous system, such as neuronal differentiation and morphogenesis. While the interaction of neurons and the surface topography of their local environment is well characterized, the neuron–topography interaction during the regeneration process remains largely unknown. To address this question, an anisotropic surface topography resembling linear grooves made from poly(ethylene-vinyl acetate) (EVA), a soft and biocompatible polymer, using nanoimprinting, is established. It is found that neurons from both the central and peripheral nervous system can survive and grow on this grooved surface. Additionally, it is observed that axons but not dendrites specifically align with these grooves. Furthermore, it is demonstrated that neurons on the grooved surface are capable of regeneration after an on-site injury. More importantly, these injured neurons have an accelerated and enhanced regeneration. Together, the data demonstrate that this anisotropic topography guides axon growth and improves axon regeneration. This opens up the possibility to study the effect of surface topography on regenerating axons and has the potential to be developed into a medical device for treating peripheral nerve injuries.
AUTHOR Kumari, Sushma and Bargel, Hendrik and Anby, Mette U. and Lafargue, David and Scheibel, Thomas
Title Recombinant Spider Silk Hydrogels for Sustained Release of Biologicals [Abstract]
Year 2018
Journal/Proceedings ACS Biomaterials Science and Engineering
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Abstract
Therapeutic biologics (i.e., proteins) have been widely recognized for the treatment, prevention, and cure of a variety of human diseases and syndromes. However, design of novel protein-delivery systems to achieve a nontoxic, constant, and efficient delivery with minimal doses of therapeutic biologics is still challenging. Here, recombinant spider silk-based materials are employed as a delivery system for the administration of therapeutic biologicals. Hydrogels made of the recombinant spider silk protein eADF4(C16) were used to encapsulate the model biologicals BSA, HRP, and LYS by direct loading or through diffusion, and their release was studied. Release of model biologicals from eADF4(C16) hydrogels is in part dependent on the electrostatic interaction between the biological and the recombinant spider silk protein variant used. In addition, tailoring the pore sizes of eADF4(C16) hydrogels strongly influenced the release kinetics. In a second approach, a particles-in-hydrogel system was used, showing a prolonged release in comparison with that of plain hydrogels (from days to week). The particle-enforced spider silk hydrogels are injectable and can be 3D printed. These initial studies indicate the potential of recombinant spider silk proteins to design novel injectable hydrogels that are suitable for delivering therapeutic biologics.
AUTHOR Gleadall, Andrew and Visscher, Dafydd and Yang, Jing and Thomas, Daniel and Segal, Joel
Title Review of additive manufactured tissue engineering scaffolds: relationship between geometry and performance [Abstract]
Year 2018
Journal/Proceedings Burns and Trauma
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DOI/URL DOI
Abstract
Material extrusion additive manufacturing has rapidly grown in use for tissue engineering research since its adoption in the year 2000. It has enabled researchers to produce scaffolds with intricate porous geometries that were not feasible with traditional manufacturing processes. Researchers can control the structural geometry through a wide range of customisable printing parameters and design choices including material, print path, temperature, and many other process parameters. Currently, the impact of these choices is not fully understood. This review focuses on how the position and orientation of extruded filaments, which sometimes referred to as the print path, lay-down pattern, or simply ``scaffold design'', affect scaffold properties and biological performance. By analysing trends across multiple studies, new understanding was developed on how filament position affects mechanical properties. Biological performance was also found to be affected by filament position, but a lack of consensus between studies indicates a need for further research and understanding. In most research studies, scaffold design was dictated by capabilities of additive manufacturing software rather than free-form design of structural geometry optimised for biological requirements. There is scope for much greater application of engineering innovation to additive manufacture novel geometries. To achieve this, better understanding of biological requirements is needed to enable the effective specification of ideal scaffold geometries.
AUTHOR Kuzmenko, Volodymyr and Karabulut, Erdem and Pernevik, Elin and Enoksson, Peter and Gatenholm, Paul
Title Tailor-made conductive inks from cellulose nanofibrils for 3D printing of neural guidelines [Abstract]
Year 2018
Journal/Proceedings Carbohydrate Polymers
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DOI/URL URL DOI
Abstract
Neural tissue engineering (TE), an innovative biomedical method of brain study, is very dependent on scaffolds that support cell development into a functional tissue. Recently, 3D patterned scaffolds for neural TE have shown significant positive effects on cells by a more realistic mimicking of actual neural tissue. In this work, we present a conductive nanocellulose-based ink for 3D printing of neural TE scaffolds. It is demonstrated that by using cellulose nanofibrils and carbon nanotubes as ink constituents, it is possible to print guidelines with a diameter below 1 mm and electrical conductivity of 3.8 × 10−1 S cm−1. The cell culture studies reveal that neural cells prefer to attach, proliferate, and differentiate on the 3D printed conductive guidelines. To our knowledge, this is the first research effort devoted to using cost-effective cellulosic 3D printed structures in neural TE, and we suppose that much more will arise in the near future.
AUTHOR Huang, Ying and Zhang, Xiao-Fei and Gao, Guifang and Yonezawa, Tomo and Cui, Xiaofeng
Title 3D bioprinting and the current applications in tissue engineering [Abstract]
Year 2017
Journal/Proceedings Biotechnology Journal
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Abstract
Bioprinting as an enabling technology for tissue engineering possesses the promises to fabricate highly mimicked tissue or organs with digital control. As one of the biofabrication approaches, bioprinting has the advantages of high throughput and precise control of both scaffold and cells. Therefore, this technology is not only ideal for translational medicine but also for basic research applications. Bioprinting has already been widely applied to construct functional tissues such as vasculature, muscle, cartilage, and bone. In this review, the authors introduce the most popular techniques currently applied in bioprinting, as well as the various bioprinting processes. In addition, the composition of bioink including scaffolds and cells are described. Furthermore, the most current applications in organ and tissue bioprinting are introduced. The authors also discuss the challenges we are currently facing and the great potential of bioprinting. This technology has the capacity not only in complex tissue structure fabrication based on the converted medical images, but also as an efficient tool for drug discovery and preclinical testing. One of the most promising future advances of bioprinting is to develop a standard medical device with the capacity of treating patients directly on the repairing site, which requires the development of automation and robotic technology, as well as our further understanding of biomaterials and stem cell biology to integrate various printing mechanisms for multi-phasic tissue engineering.
AUTHOR Sommer, Marianne R. and Alison, Lauriane and Minas, Clara and Tervoort, Elena and Ruhs, Patrick A. and Studart, Andre R.
Title 3D printing of concentrated emulsions into multiphase biocompatible soft materials [Abstract]
Year 2017
Journal/Proceedings Soft Matter
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Abstract
3D printing via direct ink writing (DIW) is a versatile additive manufacturing approach applicable to a variety of materials ranging from ceramics over composites to hydrogels. Due to the mild processing conditions compared to other additive manufacturing methods{,} DIW enables the incorporation of sensitive compounds such as proteins or drugs into the printed structure. Although emulsified oil-in-water systems are commonly used vehicles for such compounds in biomedical{,} pharmaceutical{,} and cosmetic applications{,} printing of such emulsions into architectured soft materials has not been fully exploited and would open new possibilities for the controlled delivery of sensitive compounds. Here{,} we 3D print concentrated emulsions into soft materials{,} whose multiphase architecture allows for site-specific incorporation of both hydrophobic and hydrophilic compounds into the same structure. As a model ink{,} concentrated emulsions stabilized by chitosan-modified silica nanoparticles are studied{,} because they are sufficiently stable against coalescence during the centrifugation step needed to create a bridging network of droplets. The resulting ink is ideal for 3D printing as it displays high yield stress{,} storage modulus and elastic recovery{,} through the formation of networks of droplets as well as of gelled silica nanoparticles in the presence of chitosan. To demonstrate possible architectures{,} we print biocompatible soft materials with tunable hierarchical porosity containing an encapsulated hydrophobic compound positioned in specific locations of the structure. The proposed emulsion-based ink system offers great flexibility in terms of 3D shaping and local compositional control{,} and can potentially help address current challenges involving the delivery of incompatible compounds in biomedical applications.
AUTHOR Suntornnond, R. and Tan, E. Y. S. and An, J. and Chua, C. K.
Title A highly printable and biocompatible hydrogel composite for direct printing of soft and perfusable vasculature-like structures [Abstract]
Year 2017
Journal/Proceedings Scientific Reports
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Abstract
Vascularization is one major obstacle in bioprinting and tissue engineering. In order to create thick tissues or organs that can function like original body parts, the presence of a perfusable vascular system is essential. However, it is challenging to bioprint a hydrogel-based three-dimensional vasculature-like structure in a single step. In this paper, we report a new hydrogel-based composite that offers impressive printability, shape integrity, and biocompatibility for 3D bioprinting of a perfusable complex vasculature-like structure. The hydrogel composite can be used on a non-liquid platform and is printable at human body temperature. Moreover, the hydrogel composite supports both cell proliferation and cell differentiation. Our results represent a potentially new vascularization strategy for 3D bioprinting and tissue engineering.
AUTHOR Charbe, Nitin B. and McCarron, Paul A. and Lane, Majella E. and Tambuwala, Murtaza M.
Title Application of three-dimensional printing for colon targeted drug delivery systems [Abstract]
Year 2017
Journal/Proceedings International Journal of Pharmaceutical Investigation
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Abstract
Orally administered solid dosage forms currently dominate over all other dosage forms and routes of administrations. However, human gastrointestinal tract (GIT) poses a number of obstacles to delivery of the drugs to the site of interest and absorption in the GIT. Pharmaceutical scientists worldwide have been interested in colon drug delivery for several decades, not only for the delivery of the drugs for the treatment of colonic diseases such as ulcerative colitis and colon cancer but also for delivery of therapeutic proteins and peptides for systemic absorption. Despite extensive research in the area of colon targeted drug delivery, we have not been able to come up with an effective way of delivering drugs to the colon. The current tablets designed for colon drug release depend on either pH-dependent or time-delayed release formulations. During ulcerative colitis the gastric transit time and colon pH-levels is constantly changing depending on whether the patient is having a relapse or under remission. Hence, the current drug delivery system to the colon is based on one-size-fits-all. Fails to effectively deliver the drugs locally to the colon for colonic diseases and delivery of therapeutic proteins and peptides for systemic absorption from the colon. Hence, to overcome the current issues associated with colon drug delivery, we need to provide the patients with personalized tablets which are specifically designed to match the individual's gastric transit time depending on the disease state. Three-dimensional (3D) printing (3DP) technology is getting cheaper by the day and bespoke manufacturing of 3D-printed tablets could provide the solutions in the form of personalized colon drug delivery system. This review provides a bird's eye view of applications and current advances in pharmaceutical 3DP with emphasis on the development of colon targeted drug delivery systems.
AUTHOR Aljohani, Waeljumah and Ullah, Muhammad Wajid and Zhang, Xianglin and Yang, Guang
Title Bioprinting and its applications in tissue engineering and regenerative medicine [Abstract]
Year 2017
Journal/Proceedings International Journal of Biological Macromolecules
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Abstract
Abstract Bioprinting of three-dimensional constructs mimicking natural-like extracellular matrix has revolutionized biomedical technology. Bioprinting technology circumvents various discrepancies associated with current tissue engineering strategies by providing an automated and advanced platform to fabricate various biomaterials through precise deposition of cells and polymers in a premeditated fashion. However, few obstacles associated with development of 3D scaffolds including varied properties of polymers used and viability, controlled distribution, and vascularization, etc. of cells hinder bioprinting of complex structures. Therefore, extensive efforts have been made to explore the potential of various natural polymers (e.g. cellulose, gelatin, alginate, and chitosan, etc.) and synthetic polymers in bioprinting by tuning their printability and cross-linking features, mechanical and thermal properties, biocompatibility, and biodegradability, etc. This review describes the potential of these polymers to support adhesion and proliferation of viable cells to bioprint cell laden constructs, bone, cartilage, skin, and neural tissues, and blood vessels, etc. for various applications in tissue engineering and regenerative medicines. Further, it describes various challenges associated with current bioprinting technology and suggests possible solutions. Although at early stage of development, the potential benefits of bioprinting technology are quite clear and expected to open new gateways in biomedical, pharmaceutics and several other fields in near future.
AUTHOR Nguyen, Duong and Hägg, Daniel and Forsman, Alma and Ekholm, Josefine and Nimkingratana, Puwapong and Brantsing, Camilla and Kalogeropoulos, Theodoros and Zaunz, Samantha and Concaro, Sebastian and Brittberg, Mats and Lindahl, Anders and Gatenholm, Paul and Enejder, Annika and Simonsson, Stina
Title Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink [Abstract]
Year 2017
Journal/Proceedings Scientific Reports
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Abstract
Cartilage lesions can progress into secondary osteoarthritis and cause severe clinical problems in numerous patients. As a prospective treatment of such lesions, human-derived induced pluripotent stem cells (iPSCs) were shown to be 3D bioprinted into cartilage mimics using a nanofibrillated cellulose (NFC) composite bioink when co-printed with irradiated human chondrocytes. Two bioinks were investigated: NFC with alginate (NFC/A) or hyaluronic acid (NFC/HA). Low proliferation and phenotypic changes away from pluripotency were seen in the case of NFC/HA. However, in the case of the 3D-bioprinted NFC/A (60/40, dry weight % ratio) constructs, pluripotency was initially maintained, and after five weeks, hyaline-like cartilaginous tissue with collagen type II expression and lacking tumorigenic Oct4 expression was observed in 3D -bioprinted NFC/A (60/40, dry weight % relation) constructs. Moreover, a marked increase in cell number within the cartilaginous tissue was detected by 2-photon fluorescence microscopy, indicating the importance of high cell densities in the pursuit of achieving good survival after printing. We conclude that NFC/A bioink is suitable for bioprinting iPSCs to support cartilage production in co-cultures with irradiated chondrocytes.
AUTHOR D'Amora, Ugo and D'Este, Matteo and Eglin, David and Safari, Fatemeh and Sprecher, Christoph and Gloria, Antonio and De Santis, Roberto and Alini, Mauro and Ambrosio, Luigi
Title Collagen Density Gradient on 3D Printed Poly(ε-Caprolactone) Scaffolds for Interface Tissue Engineering
Year 2017
Journal/Proceedings Journal of tissue engineering and regenerative medicine
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AUTHOR Baumann, Bernhard and Jungst, Tomasz and Stichler, Simone and Feineis, Susanne and Wiltschka, Oliver and Kuhlmann, Matthias and Lindén, Mika and Groll, Jürgen
Title Control of Nanoparticle Release Kinetics from 3D Printed Hydrogel Scaffolds [Abstract]
Year 2017
Journal/Proceedings Angewandte Chemie International Edition
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Abstract
The convergence of biofabrication with nanotechnology is largely unexplored but enables geometrical control of cell-biomaterial arrangement combined with controlled drug delivery and release. As a step towards integration of these two fields of research, this study demonstrates that modulation of electrostatic nanoparticle–polymer and nanoparticle–nanoparticle interactions can be used for tuning nanoparticle release kinetics from 3D printed hydrogel scaffolds. This generic strategy can be used for spatiotemporal control of the release kinetics of nanoparticulate drug vectors in biofabricated constructs.
AUTHOR Mouser, V. H. M. and Abbadessa, A. and Levato, R. and Hennink, W. E. and Vermonden, T. and Gawlitta, D. and Malda, J.
Title Development of a thermosensitive HAMA-containing bio-ink for the fabrication of composite cartilage repair constructs [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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Abstract
Fine-tuning of bio-ink composition and material processing parameters is crucial for the development of biomechanically relevant cartilage constructs. This study aims to design and develop cartilage constructs with tunable internal architectures and relevant mechanical properties. More specifically, the potential of methacrylated hyaluronic acid (HAMA) added to thermosensitive hydrogels composed of methacrylated poly[ N -(2-hydroxypropyl)methacrylamide mono/dilactate] (pHPMA-lac)/polyethylene glycol (PEG) triblock copolymers, to optimize cartilage-like tissue formation by embedded chondrocytes, and enhance printability was explored. Additionally, co-printing with polycaprolactone (PCL) was performed for mechanical reinforcement. Chondrocyte-laden hydrogels composed of pHPMA-lac-PEG and different concentrations of HAMA (0%–1% w/w) were cultured for 28 d in vitro and subsequently evaluated for the presence of cartilage-like matrix. Young’s moduli were determined for hydrogels with the different HAMA concentrations. Additionally, hydrogel/PCL constructs with different internal architectures were co-printed and analyzed for their mechanical properties. The results of this study demonstrated a dose-dependent effect of HAMA concentration on cartilage matrix synthesis by chondrocytes. Glycosaminoglycan (GAG) and collagen type II content increased with intermediate HAMA concentrations (0.25%–0.5%) compared to HAMA-free controls, while a relatively high HAMA concentration (1%) resulted in increased fibrocartilage formation. Young’s moduli of generated hydrogel constructs ranged from 14 to 31 kPa and increased with increasing HAMA concentration. The pHPMA-lac-PEG hydrogels with 0.5% HAMA were found to be optimal for cartilage-like tissue formation. Therefore, this hydrogel system was co-printed with PCL to generate porous or solid constructs with different mesh sizes. Young’s moduli of these composite constructs were in the range of native cartilage (3.5–4.6 MPa). Interestingly, the co-printing procedure influenced the mechanical properties of the final constructs. These findings are relevant for future bio-ink development, as they demonstrate the importance of selecting proper HAMA concentrations, as well as appropriate print settings and construct designs for optimal cartilage matrix deposition and final mechanical properties of constructs, respectively.
AUTHOR Stichler, Simone and Böck, Thomas and Paxton, Naomi Claire and Bertlein, Sarah and Levato, Riccardo and Schill, Verena and Smolan, Willi and Malda, Jos and Tessmar, Joerg and Blunk, Torsten and Groll, Juergen
Title Double printing of hyaluronic acid / poly(glycidol) hybrid hydrogels with poly(ε-caprolactone) for MSC chondrogenesis [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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Abstract
Abstract This study investigates the use of allyl-functionalized poly(glycidol)s (P(AGE-co-G)) as cytocompatible cross-linker for thiol-functionalized hyaluronic acid (HA-SH) and the optimization of this hybrid hydrogel as bioink for 3D bioprinting. Chemical cross-linking of gels with 10 wt.% overall polymer concentration was achieved by UV-induced radical thiol-ene coupling between the thiol and allyl groups. Addition of unmodified high molecular weight HA (1.36 MDa) allowed tuning of the rheology for extrusion based bioprinting. Incorporation of additional HA resulted in hydrogels with lower Young’s modulus and higher swelling ratio especially in the first 24 h, but a comparable equilibrium swelling for all gels after 24 h. Embedding of human and equine mesenchymal stem cells (MSCs) in the gels and subsequent in vitro culture showed promising chondrogenic differentiation after 21 d for cells from both origins. Moreover, cells could be printed with these gels, and embedded hMSCs showed good cell survival for at least 21 d in culture. To achieve mechanical stable and robust constructs for the envisioned application in articular cartilage, the formulations were adjusted for double printing with the thermoplastic poly--caprolactone (PCL).
AUTHOR Mancini, Irina and Vindas Bolaños, Rafael and Brommer, Harold and Castilho, Miguel and Ribeiro, Alexandro and van Loon, Johannes and Mensinga, Anneloes and Rijen, Mattie and Malda, Jos and van Weeren, Paul
Title Fixation of hydrogel constructs for cartilage repair in the equine model: a challenging issue [Abstract]
Year 2017
Journal/Proceedings Tissue Engineering Part C: Methods
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Abstract
u> Objective To evaluate the use of commercial and autologous fibrin glue and of an alternative method based on a 3D-printed polycaprolactone (PCL) anchor for the fixation of hydrogel-based scaffolds in an equine model for cartilage repair. Methods In a first study, three different hydrogel-based materials were orthotopically implanted in nine horses for 1-4 weeks in 6mm diameter full thickness cartilage defects in the medial femoral trochlear ridge and fixated with commercially available fibrin glue (CFG). One defect was filled with CFG only as a control. In a second study, CFG and autologous fibrin glue (AFG) were compared in an ectopic equine model. The third study compared the efficacy of AFG and a 3D-printed PCL-based osteal anchor for fixation of PCL-reinforced hydrogels in 3 horses for 2 weeks, with a 4 week follow-up to evaluate integration of bone with the PCL anchor. Short-term scaffold integration and cell infiltration were evaluated by micro-CT and histology as outcome parameters. Results The first study showed signs of subchondral bone resorption in all defects, including the controls filled with CFG only, with significant infiltration of neutrophils. Ectopically, CFG induced clear inflammation with strong neutrophil accumulation, AFG was less reactive, showing fibroblast infiltration only. In the third study the fixation potential for PCL-reinforced hydrogels of AFG was inferior to the PCL anchor. PCL-reinforcement had disappeared from two defects and showed signs of dislodging in the remaining four. All 6 constructs fixated with the PCL anchor were still in place after 2 weeks. At 4 weeks, the PCL anchor showed good integration and signs of new bone formation. Conclusions The use of AFG should be preferred to xenogeneic products in the horse, but AFG is subject to individual variations and laborious to make. The PCL anchor provide the best fixation, however this technique involves the whole osteochondral unit, which entails a different conceptual approach to cartilage repair.
AUTHOR Levato, Riccardo and Webb, William R. and Otto, Iris A. and Mensinga, Anneloes and Zhang, Yadan and van Rijen, Mattie and van Weeren, René and Khan, Ilyas M. and Malda, Jos
Title The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells
Year 2017
Journal/Proceedings Acta Biomaterialia
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AUTHOR Freeman, Fiona E. and Kelly, Daniel J.
Title Tuning Alginate Bioink Stiffness and Composition for Controlled Growth Factor Delivery and to Spatially Direct MSC Fate within Bioprinted Tissues [Abstract]
Year 2017
Journal/Proceedings Scientific Reports
Reftype Freeman2017
DOI/URL DOI
Abstract
Alginate is a commonly used bioink in 3D bioprinting. Matrix stiffness is a key determinant of mesenchymal stem cell (MSC) differentiation, suggesting that modulation of alginate bioink mechanical properties represents a promising strategy to spatially regulate MSC fate within bioprinted tissues. In this study, we define a printability window for alginate of differing molecular weight (MW) by systematically varying the ratio of alginate to ionic crosslinker within the bioink. We demonstrate that the MW of such alginate bioinks, as well as the choice of ionic crosslinker, can be tuned to control the mechanical properties (Young’s Modulus, Degradation Rate) of 3D printed constructs. These same factors are also shown to influence growth factor release from the bioinks. We next explored if spatially modulating the stiffness of 3D bioprinted hydrogels could be used to direct MSC fate inside printed tissues. Using the same alginate and crosslinker, but varying the crosslinking ratio, it is possible to bioprint constructs with spatially varying mechanical microenvironments. Moreover, these spatially varying microenvironments were found to have a significant effect on the fate of MSCs within the alginate bioinks, with stiffer regions of the bioprinted construct preferentially supporting osteogenesis over adipogenesis.
AUTHOR Abbadessa, Anna and Landín, Mariana and Oude Blenke, Erik and Hennink, Wim E. and Vermonden, Tina
Title Two-component thermosensitive hydrogels: Phase separation affecting rheological behavior [Abstract]
Year 2017
Journal/Proceedings European Polymer Journal
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Abstract
Abstract Extracellular matrices are mainly composed of a mixture of different biopolymers and therefore the use of two or more building blocks for the development of tissue-mimicking hydrogels is nowadays an attractive strategy in tissue-engineering. Multi-component hydrogel systems may undergo phase separation, which in turn can lead to new, unexpected material properties. The aim of this study was to understand the role of phase separation on the mechanical properties and 3D printability of hydrogels composed of triblock copolymers of polyethylene glycol (PEG) and methacrylated poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate) (pHPMAlac) blended with methacrylated hyaluronic acid (HAMA). To this end, hydrogels composed of different concentrations of PEG/pHPMAlac and HAMA, were analyzed for phase behavior and rheological properties. Subsequently, phase separation and rheological behavior as function of the two polymer concentrations were mathematically processed to generate a predictive model. Results showed that PEG/pHPMAlac/HAMA hydrogels were characterized by hydrophilic, HAMA-richer internal domains dispersed in a more hydrophobic continuous phase, composed of PEG/pHPMAlac, and that the volume fraction of the dispersed phase increased by increasing HAMA concentration. Storage modulus, yield stress and viscosity increased with increasing HAMA concentration for low/medium HAMA contents (≤0.75% w/w), while a further increase of HAMA resulted in a decrease of the mentioned properties. On the other hand, by increasing the concentration of PEG/pHPMAlac these rheological properties were enhanced. The generated models showed a good fitting with experimental data, and were used to identify an exemplary 3D printability window for PEG/pHPMAlac/HAMA hydrogels, which was verified by rheological characterization and preparation of 3D printed scaffolds. In conclusion, a clear relationship between phase separation and rheological behavior in these two-component hydrogels can be described by complex functions of the two polymer concentrations. The predictive model generated in this study can be used as a valid tool for the identification of hydrogel compositions with desired, selected characteristics.
AUTHOR Gu, Bon Kang and Choi, Dong Jin and Park, Sang Jun and Kim, Min Sup and Kang, Chang Mo and Kim, Chun-Ho
Title 3-dimensional bioprinting for tissue engineering applications [Abstract]
Year 2016
Journal/Proceedings Biomaterials Research
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Abstract
The 3-dimensional (3D) printing technologies, referred to as additive manufacturing (AM) or rapid prototyping (RP), have acquired reputation over the past few years for art, architectural modeling, lightweight machines, and tissue engineering applications. Among these applications, tissue engineering field using 3D printing has attracted the attention from many researchers. 3D bioprinting has an advantage in the manufacture of a scaffold for tissue engineering applications, because of rapid-fabrication, high-precision, and customized-production, etc. In this review, we will introduce the principles and the current state of the 3D bioprinting methods. Focusing on some of studies that are being current application for biomedical and tissue engineering fields using printed 3D scaffolds.
AUTHOR Daly, Andrew C. and Cunniffe, Gr{'{a}}inne M. and Sathy, Binulal N. and Jeon, Oju and Alsberg, Eben and Kelly, Daniel J.
Title 3D Bioprinting of Developmentally Inspired Templates for Whole Bone Organ Engineering [Abstract]
Year 2016
Journal/Proceedings Advanced Healthcare Materials
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Abstract
The ability to print defined patterns of cells and extracellular-matrix components in three dimensions has enabled the engineering of simple biological tissues; however, bioprinting functional solid organs is beyond the capabilities of current biofabrication technologies. An alternative approach would be to bioprint the developmental precursor to an adult organ, using this engineered rudiment as a template for subsequent organogenesis in vivo. This study demonstrates that developmentally inspired hypertrophic cartilage templates can be engineered in vitro using stem cells within a supporting gamma-irradiated alginate bioink incorporating Arg-Gly-Asp adhesion peptides. Furthermore, these soft tissue templates can be reinforced with a network of printed polycaprolactone fibers, resulting in a ≈350 fold increase in construct compressive modulus providing the necessary stiffness to implant such immature cartilaginous rudiments into load bearing locations. As a proof-of-principal, multiple-tool biofabrication is used to engineer a mechanically reinforced cartilaginous template mimicking the geometry of a vertebral body, which in vivo supported the development of a vascularized bone organ containing trabecular-like endochondral bone with a supporting marrow structure. Such developmental engineering approaches could be applied to the biofabrication of other solid organs by bioprinting precursors that have the capacity to mature into their adult counterparts over time in vivo.
AUTHOR {{'{A}}}vila, H{'{e}}ctor Mart{'{i}}nez and Schwarz, Silke and Rotter, Nicole and Gatenholm, Paul
Title 3D bioprinting of human chondrocyte-laden nanocellulose hydrogels for patient-specific auricular cartilage regeneration [Abstract]
Year 2016
Journal/Proceedings Bioprinting
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Abstract
Abstract Auricular cartilage tissue engineering (TE) aims to provide an effective treatment for patients with acquired or congenital auricular defects. Bioprinting has gained attention in several {TE} strategies for its ability to spatially control the placement of cells, biomaterials and biological molecules. Although considerable advances have been made to bioprint complex 3D tissue analogues, the development of hydrogel bioinks with good printability and bioactive properties must improve in order to advance the translation of 3D bioprinting into the clinic. In this study, the biological functionality of a bioink composed of nanofibrillated cellulose and alginate (NFC-A) is extensively evaluated for auricular cartilage TE. 3D bioprinted auricular constructs laden with human nasal chondrocytes (hNC) are cultured for up to 28 days and the redifferentiation capacity of hNCs in NFC-A is studied on gene expression as well as on protein levels. 3D bioprinting with NFC-A bioink facilitates the biofabrication of cell-laden, patient-specific auricular constructs with an open inner structure, high cell density and homogenous cell distribution. The cell-laden NFC-A constructs exhibit an excellent shape and size stability as well as an increase in cell viability and proliferation during in vitro culture. Furthermore, NFC-A bioink supports the redifferentiation of hNCs and neo-synthesis of cartilage-specific extracellular matrix components. This demonstrated that NFC-A bioink supports redifferentiation of hNCs while offering proper printability in a biologically relevant aqueous 3D environment, making it a promising tool for auricular cartilage {TE} and many other biomedical applications.
AUTHOR Daly, Andrew C. and Critchley, Susan E. and Rencsok, Emily M. and Kelly, Daniel J.
Title A comparison of different bioinks for 3D bioprinting of fibrocartilage and hyaline cartilage [Abstract]
Year 2016
Journal/Proceedings Biofabrication
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Abstract
Cartilage is a dense connective tissue with limited self-repair capabilities. Mesenchymal stem cell (MSC) laden hydrogels are commonly used for fibrocartilage and articular cartilage tissue engineering, however they typically lack the mechanical integrity for implantation into high load bearing environments. This has led to increased interested in 3D bioprinting of cell laden hydrogel bioinks reinforced with stiffer polymer fibres. The objective of this study was to compare a range of commonly used hydrogel bioinks (agarose, alginate, GelMA and BioINK™) for their printing properties and capacity to support the development of either hyaline cartilage or fibrocartilage in vitro . Each hydrogel was seeded with MSCs, cultured for 28 days in the presence of TGF- β 3 and then analysed for markers indicative of differentiation towards either a fibrocartilaginous or hyaline cartilage-like phenotype. Alginate and agarose hydrogels best supported the development of hyaline-like cartilage, as evident by the development of a tissue staining predominantly for type II collagen. In contrast, GelMA and BioINK ™ (a PEGMA based hydrogel) supported the development of a more fibrocartilage-like tissue, as evident by the development of a tissue containing both type I and type II collagen. GelMA demonstrated superior printability, generating structures with greater fidelity, followed by the alginate and agarose bioinks. High levels of MSC viability were observed in all bioinks post-printing (∼80%). Finally we demonstrate that it is possible to engineer mechanically reinforced hydrogels with high cell viability by co-depositing a hydrogel bioink with polycaprolactone filaments, generating composites with bulk compressive moduli comparable to articular cartilage. This study demonstrates the importance of the choice of bioink when bioprinting different cartilaginous tissues for musculoskeletal applications.
AUTHOR Sears, Nick A. and Seshadri, Dhruv R. and Dhavalikar, Prachi S. and Cosgriff-Hernandez, Elizabeth
Title A Review of Three-Dimensional Printing in Tissue Engineering [Abstract]
Year 2016
Journal/Proceedings Tissue Engineering Part B: Reviews
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Abstract
Recent advances in three-dimensional (3D) printing technologies have led to a rapid expansion of applications from the creation of anatomical training models for complex surgical procedures to the printing of tissue engineering constructs. In addition to achieving the macroscale geometry of organs and tissues, a print layer thickness as small as 20 mm allows for reproduction of the microarchitectures of bone and other tissues. Techniques with even higher precision are currently being investigated to enable reproduction of smaller tissue features such as hepatic lobules. Current research in tissue engineering focuses on the development of compatible methods (printers) and materials (bioinks) that are capable of producing biomimetic scaffolds. In this review, an overview of current 3D printing techniques used in tissue engineering is provided with an emphasis on the printing mechanism and the resultant scaffold characteristics. Current practical challenges and technical limitations are emphasized and future trends of bioprinting are discussed.
AUTHOR Abbadessa, Anna and Mouser, Vivian H. M. and Blokzijl, Maarten M. and Gawlitta, Debby and Dhert, Wouter J. A. and Hennink, Wim E. and Malda, Jos and Vermonden, Tina
Title A Synthetic Thermosensitive Hydrogel for Cartilage Bioprinting and Its Biofunctionalization with Polysaccharides [Abstract]
Year 2016
Journal/Proceedings Biomacromolecules
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Abstract
Hydrogels based on triblock copolymers of polyethylene glycol and partially methacrylated poly[N-(2-hydroxypropyl) methacrylamide mono/dilactate] make up an attractive class of biomaterials because of their biodegradability, cytocompatibility, and tunable thermoresponsive and mechanical properties. If these properties are fine-tuned, the hydrogels can be three-dimensionally bioprinted, to generate, for instance, constructs for cartilage repair. This study investigated whether hydrogels based on the polymer mentioned above with a 10% degree of methacrylation (M10P10) support cartilage formation by chondrocytes and whether the incorporation of methacrylated chondroitin sulfate (CSMA) or methacrylated hyaluronic acid (HAMA) can improve the mechanical properties, long-term stability, and printability. Chondrocyte-laden M10P10 hydrogels were cultured for 42 days to evaluate chondrogenesis. M10P10 hydrogels with or without polysaccharides were evaluated for their mechanical properties (before and after UV photo-cross-linking), degradation kinetics, and printability. Extensive cartilage matrix production occurred in M10P10 hydrogels, highlighting their potential for cartilage repair strategies. The incorporation of polysaccharides increased the storage modulus of polymer mixtures and decreased the degradation kinetics in cross-linked hydrogels. Addition of HAMA to M10P10 hydrogels improved printability and resulted in three-dimensional constructs with excellent cell viability. Hence, this novel combination of M10P10 with HAMA forms an interesting class of hydrogels for cartilage bioprinting.
AUTHOR Abbadessa, A. and Blokzijl, M. M. and Mouser, V. H. M. and Marica, P. and Malda, J. and Hennink, W. E. and Vermonden, T.
Title A thermo-responsive and photo-polymerizable chondroitin sulfate-based hydrogel for 3D printing applications [Abstract]
Year 2016
Journal/Proceedings Carbohydrate Polymers
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Abstract
Abstract The aim of this study was to design a hydrogel system based on methacrylated chondroitin sulfate (CSMA) and a thermo-sensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate)-polyethylene glycol triblock copolymer (M15P10) as a suitable material for additive manufacturing of scaffolds. {CSMA} was synthesized by reaction of chondroitin sulfate with glycidyl methacrylate (GMA) in dimethylsulfoxide at 50 °C and its degree of methacrylation was tunable up to 48.5%, by changing reaction time and {GMA} feed. Unlike polymer solutions composed of {CSMA} alone (20% w/w), mixtures based on 2% w/w of {CSMA} and 18% of {M15P10} showed strain-softening, thermo-sensitive and shear-thinning properties more pronounced than those found for polymer solutions based on {M15P10} alone. Additionally, they displayed a yield stress of 19.2 ± 7.0 Pa. The 3D printing of this hydrogel resulted in the generation of constructs with tailorable porosity and good handling properties. Finally, embedded chondrogenic cells remained viable and proliferating over a culture period of 6 days. The hydrogel described herein represents a promising biomaterial for cartilage 3D printing applications.
AUTHOR Visscher, Dafydd O. and Farré-Guasch, Elisabet and Helder, Marco N. and Gibbs, Susan and Forouzanfar, Tymour and van Zuijlen, Paul P. and Wolff, Jan
Title Advances in Bioprinting Technologies for Craniofacial Reconstruction [Abstract]
Year 2016
Journal/Proceedings Trends in Biotechnology
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Abstract
Recent developments in craniofacial reconstruction have shown important advances in both the materials and methods used. While autogenous tissue is still considered to be the gold standard for these reconstructions, the harvesting procedure remains tedious and in many cases causes significant donor site morbidity. These limitations have subsequently led to the development of less invasive techniques such as 3D bioprinting that could offer possibilities to manufacture patient-tailored bioactive tissue constructs for craniofacial reconstruction. Here, we discuss the current technological and (pre)clinical advances of 3D bioprinting for use in craniofacial reconstruction and highlight the challenges that need to be addressed in the coming years. Recent developments in craniofacial reconstruction have shown important advances in both the materials and methods used. While autogenous tissue is still considered to be the gold standard for these reconstructions, the harvesting procedure remains tedious and in many cases causes significant donor site morbidity. These limitations have subsequently led to the development of less invasive techniques such as 3D bioprinting that could offer possibilities to manufacture patient-tailored bioactive tissue constructs for craniofacial reconstruction. Here, we discuss the current technological and (pre)clinical advances of 3D bioprinting for use in craniofacial reconstruction and highlight the challenges that need to be addressed in the coming years.
AUTHOR M{"u}ller, Michael and {"O}zt{"u}rk, Ece and Arlov, {O}ystein and Gatenholm, Paul and Zenobi-Wong, Marcy
Title Alginate Sulfate--Nanocellulose Bioinks for Cartilage Bioprinting Applications [Abstract]
Year 2016
Journal/Proceedings Annals of Biomedical Engineering
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Abstract
One of the challenges of bioprinting is to identify bioinks which support cell growth, tissue maturation, and ultimately the formation of functional grafts for use in regenerative medicine. The influence of this new biofabrication technology on biology of living cells, however, is still being evaluated. Recently we have identified a mitogenic hydrogel system based on alginate sulfate which potently supports chondrocyte phenotype, but is not printable due to its rheological properties (no yield point). To convert alginate sulfate to a printable bioink, it was combined with nanocellulose, which has been shown to possess very good printability. The alginate sulfate/nanocellulose ink showed good printing properties and the non-printed bioink material promoted cell spreading, proliferation, and collagen II synthesis by the encapsulated cells. When the bioink was printed, the biological performance of the cells was highly dependent on the nozzle geometry. Cell spreading properties were maintained with the lowest extrusion pressure and shear stress. However, extruding the alginate sulfate/nanocellulose bioink and chondrocytes significantly compromised cell proliferation, particularly when using small diameter nozzles and valves.
AUTHOR Visscher, Dafydd O. and Bos, Ernst J. and Peeters, Mirte and Kuzmin, Nikolay V. and Groot, Marie Louise and Helder, Marco N. and van Zuijlen, Paul P. M.
Title Cartilage Tissue Engineering: Preventing Tissue Scaffold Contraction Using a 3D-Printed Polymeric Cage. [Abstract]
Year 2016
Journal/Proceedings Tissue engineering Part C: Methods
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Abstract
Scaffold contraction is a common but underestimated problem in the field of tissue engineering. It becomes particularly problematic when creating anatomically complex shapes such as the ear. The aim of this study was to develop a contraction-free biocompatible scaffold construct for ear cartilage tissue engineering. To address this aim, we used three constructs: (i) a fibrin/hyaluronic acid (FB/HA) hydrogel, (ii) a FB/HA hydrogel combined with a collagen I/III scaffold, and (iii) a cage construct containing (ii) surrounded by a 3D-printed poly-varepsilon-caprolactone mold. A wide range of different cell types were tested within these constructs, including chondrocytes, perichondrocytes, adipose-derived mesenchymal stem cells, and their combinations. After in vitro culturing for 1, 14, and 28 days, all constructs were analyzed. Macroscopic observation showed severe contraction of the cell-seeded hydrogel (i). This could be prevented, in part, by combining the hydrogel with the collagen scaffold (ii) and prevented in total using the 3D-printed cage construct (iii). (Immuno)histological analysis, multiphoton laser scanning microscopy, and biomechanical analysis showed extracellular matrix deposition and increased Young's modulus and thereby the feasibility of ear cartilage engineering. These results demonstrated that the 3D-printed cage construct is an adequate model for contraction-free ear cartilage engineering using a range of cell combinations.
AUTHOR Caetano, Guilherme and Violante, Ricardo and Sant{'{}}Ana, Ana Beatriz and Murashima, Adriana Batista and Domingos, Marco and Gibson, Andrew and B{'{a}}rtolo, Paulo and Frade, Marco Andrey
Title Cellularized versus decellularized scaffolds for bone regeneration [Abstract]
Year 2016
Journal/Proceedings Materials Letters
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Abstract
Abstract An optimal scaffold based strategy for in vivo repair of large bone defects and its associated problems is presented in this work. Three polymeric scaffolds produced by using an extrusion-based additive manufacturing system were examined in a rat critical bone defect model: scaffolds without cells, with undifferentiated Adipose-derived mesenchymal stem cells (ADSCs) and differentiated {ADSCs} (osteoblasts). Scaffolds with undifferentiated cells seem to be the best strategy as they exhibited around 22% more bone formation than natural bone healing, and around 15% more than the two other cases. Authors observed that scaffolds enabled cell migration and tissue formation. Results suggest that undifferentiated {ADSCs} strongly contribute to new bone formation with no rejection if scaffolds are used to support cell migration, proliferation and differentiation. Our long-term goal is to engineer high-quality cell seeded-scaffolds (autograft and allograft) for bone regeneration, mainly in elderly patients.
AUTHOR Ruiz-Cantu, Laura and Gleadall, Andrew and Faris, Callum and Segal, Joel and Shakesheff, Kevin and Yang, Jing
Title Characterisation of the surface structure of 3D printed scaffolds for cell infiltration and surgical suturing [Abstract]
Year 2016
Journal/Proceedings Biofabrication
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Abstract
3D printing is of great interest for tissue engineering scaffolds due to the ability to form complex geometries and control internal structures, including porosity and pore size. The porous structure of scaffolds plays an important role in cell ingrowth and nutrition infusion. Although the internal porosity and pore size of 3D printed scaffolds have been frequently studied, the surface porosity and pore size, which are critical for cell infiltration and mass transport, have not been investigated. The surface geometry can differ considerably from the internal scaffold structure depending on the 3D printing process. It is vital to be able to control the surface geometry of scaffolds as well as the internal structure to fabricate optimal architectures. This work presents a method to control the surface porosity and pore size of 3D printed scaffolds. Six scaffold designs have been printed with surface porosities ranging from 3% to 21%. We have characterised the overall scaffold porosity and surface porosity using optical microscopy and microCT. It has been found that surface porosity has a significant impact on cell infiltration and proliferation. In addition, the porosity of the surface has been found to have an effect on mechanical properties and on the forces required to penetrate the scaffold with a surgical suturing needle. To the authors’ knowledge, this study is the first to investigate the surface geometry of extrusion-based 3D printed scaffolds and demonstrates the importance of surface geometry in cell infiltration and clinical manipulation.
AUTHOR Melchels, Ferry P. W. and Blokzijl, Maarten M. and Levato, Riccardo and Peiffer, Quentin C. and de Ruijter, Myl{`{e}}ne and Hennink, Wim E. and Vermonden, Tina and Malda, Jos
Title Hydrogel-based reinforcement of 3D bioprinted constructs [Abstract]
Year 2016
Journal/Proceedings Biofabrication
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Abstract
Progress within the field of biofabrication is hindered by a lack of suitable hydrogel formulations. Here, we present a novel approach based on a hybrid printing technique to create cellularized 3D printed constructs. The hybrid bioprinting strategy combines a reinforcing gel for mechanical support with a bioink to provide a cytocompatible environment. In comparison with thermoplastics such as IMG [http://ej.iop.org/images/1758-5090/8/3/035004/bfaa2f97ieqn1.gif] {$epsilon $} -polycaprolactone, the hydrogel-based reinforcing gel platform enables printing at cell-friendly temperatures, targets the bioprinting of softer tissues and allows for improved control over degradation kinetics. We prepared amphiphilic macromonomers based on poloxamer that form hydrolysable, covalently cross-linked polymer networks. Dissolved at a concentration of 28.6%w/w in water, it functions as reinforcing gel, while a 5%w/w gelatin-methacryloyl based gel is utilized as bioink. This strategy allows for the creation of complex structures, where the bioink provides a cytocompatible environment for encapsulated cells. Cell viability of equine chondrocytes encapsulated within printed constructs remained largely unaffected by the printing process. The versatility of the system is further demonstrated by the ability to tune the stiffness of printed constructs between 138 and 263 kPa, as well as to tailor the degradation kinetics of the reinforcing gel from several weeks up to more than a year.
AUTHOR Carrel, Jean‐Pierre and Wiskott, Anselm and Scherrer, Susanne and Durual, Stéphane
Title Large Bone Vertical Augmentation Using a Three‐Dimensional Printed TCP/HA Bone Graft: A Pilot Study in Dog Mandible [Abstract]
Year 2016
Journal/Proceedings Clinical Implant Dentistry and Related Research
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Abstract Background Osteoflux is a three‐dimensional printed calcium phosphate porous structure for oral bone augmentation. It is a mechanically stable scaffold with a well‐defined interconnectivity and can be readily shaped to conform to the bone bed's morphology. Purpose An animal experiment is reported whose aim was to assess the performance and safety of the scaffold in promoting vertical growth of cortical bone in the mandible. Materials and methods Four three‐dimensional blocks (10 mm length, 5 mm width, 5 mm height) were affixed to edentulous segments of the dog's mandible and covered by a collagen membrane. During bone bed preparation, particular attention was paid not to create defects 0.5 mm or more so that the real potential of the three‐dimensional block in driving vertical bone growth can be assessed. Histomorphometric analyses were performed after 8 weeks. Results At 8 weeks, the three‐dimensional blocks led to substantial vertical bone growth up to 4.5 mm from the bone bed. Between 0 and 1 mm in height, 44% of the surface was filled with new bone, at 1 to 3 mm it was 20% to 35%, 18% at 3 to 4, and ca. 6% beyond 4 mm. New bone was evenly distributed along in mesio‐distal direction and formed a new crest contour in harmony with the natural mandibular shape. Conclusions After two months of healing, the three‐dimensional printed blocks conducted new bone growth above its natural bed, up to 4.5 mm in a canine mandibular model. Furthermore, the new bone was evenly distributed in height and density along the block. These results are very promising and need to be further evaluated by a complete powerful study using the same model.
AUTHOR Hou, Xiaochun and Liu, Shiying and Wang, Min and Wiraja, Christian and Huang, Wei and Chan, Peggy and Tan, Timothy and Xu, Chenjie
Title Layer-by-Layer 3D Constructs of Fibroblasts in Hydrogel for Examining Transdermal Penetration Capability of Nanoparticles [Abstract]
Year 2016
Journal/Proceedings Journal of Laboratory Automation
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Abstract
Nanoparticles are emerging transdermal delivery systems. Their size and surface properties determine their efficacy and efficiency to penetrate through the skin layers. This work utilizes three-dimensional (3D) bioprinting technology to generate a simplified artificial skin model to rapidly screen nanoparticles for their transdermal penetration ability. Specifically, this model is built through layer-by-layer alternate printing of blank collagen hydrogel and fibroblasts. Through controlling valve on-time, the spacing between printing lines could be accurately tuned, which could enable modulation of cell infiltration in the future. To confirm the effectiveness of this platform, a 3D construct with one layer of fibroblasts sandwiched between two layers of collagen hydrogel is used to screen silica nanoparticles with different surface charges for their penetration ability, with positively charged nanoparticles demonstrating deeper penetration, consistent with the observation from an existing study involving living skin tissue.
AUTHOR Wang, Weiguang and Caetano, Guilherme and Chiang, Wei-Hung and Sousa, Ana Leticia and Blaker, Jonny and Frade, M. A. R. C. O. and Frade, Cipriani and Jorge Bártolo, Paulo
Title Morphological, mechanical and biological assessment of PCL/pristine graphene scaffolds for bone regeneration [Abstract]
Year 2016
Journal/Proceedings International Journal of Bioprinting
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Scaffolds are physical substrates for cell attachment, proliferation, and differentiation, ultimately leading to the regeneration of tissues. They must be designed according to specific biomechanical requirements such as mechanical properties, surface characteristics, biodegradability, biocompatibility, and porosity. The optimal design of a scaffold for a specific tissue strongly depends on both materials and manufacturing processes. Polymeric scaffolds reinforced with electro-active particles could play a key role in tissue engineering by modulating cell proliferation and differentiation. This paper investigates the use of an extrusion additive manufacturing system to produce PCL/pristine graphene scaffolds for bone tissue applications. PCL/pristine graphene blends were prepared using a melt blending process. Scaffolds with regular and reproducible architecture were produced with different concentrations of pristine graphene. Scaffolds were evaluated from morphological, mechanical, and biological view. The results suggest that the addition of pristine graphene improves the mechanical performance of the scaffolds, reduces the hydrophobicity, and improves cell viability and proliferation.
AUTHOR Ng, Wei Long and Yeong, Wai Yee and Naing, May Win
Title Polyelectrolyte gelatin-chitosan hydrogel optimized for 3D bioprinting in skin tissue engineering [Abstract]
Year 2016
Journal/Proceedings International Journal of Bioprinting
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Abstract
Bioprinting is a promising automated platform that enables the simultaneous deposition of multiple types of cells and biomaterials to fabricate complex three-dimensional (3D) tissue constructs. Most of the previous bioprinting works focused on collagen-based biomaterial, which has poor printability and long crosslinking time. This posed a immerse challenge to create a 3D construct with pre-determined shape and configuration. There is a need for a functional material with good printability in order to fabricate a 3D skin construct. Recently, the use of chitosan for wound healing applications has attracted huge attention due to its attractive traits such as its antimicrobial properties and ability to trigger hemostasis. In this paper, we report the modification of chitosan-based biomaterials for functional 3D bioprinting. Modification to the chitosan was carried out via the oppositely charged functional groups from chitosan and gelatin at a specific pH of ~pH 6.5 to form polyelectrolyte complexes. The polyelectrolyte hydrogels were evaluated in terms of chemical interactions within polymer blend, rheological properties (viscosities, storage and loss modulus), printing resolution at varying pressures and feed rates and biocompatibility. The chitosan-based hydrogels formulated in this work exhibited good printability at room temperature, high shape fidelity of the printed 3D constructs and good biocompatibility with fibroblast skin cells.
AUTHOR Markstedt, Kajsa and Mantas, Athanasios and Tournier, Ivan and Mart{'{i}}nez {{'{A}}}vila, H{'{e}}ctor and H{"{a}}gg, Daniel and Gatenholm, Paul
Title 3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications [Abstract]
Year 2015
Journal/Proceedings Biomacromolecules
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Abstract
The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs. The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs.
AUTHOR Ho, Chee Meng Benjamin and Ng, Sum Huan and Yoon, Yong-Jin
Title A review on 3D printed bioimplants [Abstract]
Year 2015
Journal/Proceedings International Journal of Precision Engineering and Manufacturing
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Additive manufacturing (AM) also known as 3D printing have been making inroads into medical applications such as surgical models and tools, tooling equipment, medical devices. One key area researchers are looking into is bioimplants. With the improvement and development of AM technologies, many different bioimplants can be made using 3D printing. Different biomaterials and various AM technologies can be used to create customized bioimplants to suit the individual needs. With the aid of 3D printing this could lead to new foam and design of bioimplants in the near further. Therefore, the purpose of this review articles is to (1) Describe the various AM technologies and process used to make bioimplants, (2) Different types of bioimplants printed with AM and (3) Discuss some of the challenges and future developments for 3D printed bioimplants.
AUTHOR Kesti, Matti and Eberhardt, Christian and Pagliccia, Guglielmo and Kenkel, David and Grande, Daniel and Boss, Andreas and Zenobi-Wong, Marcy
Title Bioprinting Complex Cartilaginous Structures with Clinically Compliant Biomaterials [Abstract]
Year 2015
Journal/Proceedings Advanced Functional Materials
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Abstract
Bioprinting is an emerging technology for the fabrication of patient-specific, anatomically complex tissues and organs. A novel bioink for printing cartilage grafts is developed based on two unmodified FDA-compliant polysaccharides, gellan and alginate, combined with the clinical product BioCartilage (cartilage extracellular matrix particles). Cell-friendly physical gelation of the bioink occurs in the presence of cations, which are delivered by co-extrusion of a cation-loaded transient support polymer to stabilize overhanging structures. Rheological properties of the bioink reveal optimal shear thinning and shear recovery properties for high-fidelity bioprinting. Tensile testing of the bioprinted grafts reveals a strong, ductile material. As proof of concept, 3D auricular, nasal, meniscal, and vertebral disk grafts are printed based on computer tomography data or generic 3D models. Grafts after 8 weeks in vitro are scanned using magnetic resonance imaging and histological evaluation is performed. The bioink containing BioCartilage supports proliferation of chondrocytes and, in the presence of transforming growth factor beta-3, supports strong deposition of cartilage matrix proteins. A clinically compliant bioprinting method is presented which yields patient-specific cartilage grafts with good mechanical and biological properties. The versatile method can be used with any type of tissue particles to create tissue-specific and bioactive scaffolds.
AUTHOR Moussa, Mira and Carrel, Jean-Pierre and Scherrer, Susanne and Cattani-Lorente, Maria and Wiskott, Anselm and Durual, Stéphane
Title Medium-Term Function of a 3D Printed TCP/HA Structure as a New Osteoconductive Scaffold for Vertical Bone Augmentation: A Simulation by BMP-2 Activation [Abstract]
Year 2015
Journal/Proceedings Materials
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Abstract
Introduction: A 3D-printed construct made of orthogonally layered strands of tricalcium phosphate (TCP) and hydroxyapatite has recently become available. The material provides excellent osteoconductivity. We simulated a medium-term experiment in a sheep calvarial model by priming the blocks with BMP-2. Vertical bone growth/maturation and material resorption were evaluated. Materials and methods: Titanium hemispherical caps were filled with either bare- or BMP-2 primed constructs and placed onto the calvaria of adult sheep (n = 8). Histomorphometry was performed after 8 and 16 weeks. Results: After 8 weeks, relative to bare constructs, BMP-2 stimulation led to a two-fold increase in bone volume (Bare: 22% ± 2.1%; BMP-2 primed: 50% ± 3%) and a 3-fold decrease in substitute volume (Bare: 47% ± 5%; BMP-2 primed: 18% ± 2%). These rates were still observed at 16 weeks. The new bone grew and matured to a haversian-like structure while the substitute material resorbed via cell- and chemical-mediation. Conclusion: By priming the 3D construct with BMP-2, bone metabolism was physiologically accelerated, that is, enhancing vertical bone growth and maturation as well as material bioresorption. The scaffolding function of the block was maintained, leaving time for the bone to grow and mature to a haversian-like structure. In parallel, the material resorbed via cell-mediated and chemical processes. These promising results must be confirmed in clinical tests.
AUTHOR Rimann, Markus and Bono, Epifania and Annaheim, Helene and Bleisch, Matthias and Graf-Hausner, Ursula
Title Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells. [Abstract]
Year 2015
Journal/Proceedings Journal of laboratory automation
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Abstract
Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application.
AUTHOR Carrel, Jean-Pierre and Wiskott, Anselm and Moussa, Mira and Rieder, Philippe and Scherrer, Susanne and Durual, St{'{e}}phane
Title A 3D printed TCP/HA structure as a new osteoconductive scaffold for vertical bone augmentation [Abstract]
Year 2014
Journal/Proceedings Clinical Oral Implants Research
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Abstract
Introduction OsteoFlux® (OF) is a 3D printed porous block of layered strands of tricalcium phosphate (TCP) and hydroxyapatite. Its porosity and interconnectivity are defined, and it can be readily shaped to conform the bone bed's morphology. We investigated the performance of OF as a scaffold to promote the vertical growth of cortical bone in a sheep calvarial model. Materials and methods Six titanium hemispheres were filled with OF, Bio-Oss (particulate bovine bone, BO), or Ceros (particulate TCP, CO) and placed onto the calvaria of 12 adult sheep (6 hemispheres/sheep). Histomorphometric analyses were performed after 8 and 16 weeks. Results OF led to substantial vertical bone growth by 8 weeks and outperformed BO and CO by a factor 2 yielding OF 22% ± 2.1; BO 11.5% ± 1.9; and CO 12.9% ± 2.1 total new bone. 3 mm away from the bony bed, OF led to a fourfold increase in new bone relative to BO and CO (n = 8, P < 0.002). At 16 weeks, OF, BO, and CO behaved similarly and showed marked new bone synthesis. A moderate degradation was observed at 16 weeks for all bone substitutes. Conclusion When compared to existing bone substitutes, OF enhances vertical bone growth during the first 2 months after implantation in a sheep calvarial model. The controlled porous structure translated in a high osteoconductivity and resulted in a bone mass 3 mm above the bony bed that was four times greater than that obtained with standard substitutes. These results are promising but must be confirmed in clinical tests.
AUTHOR Kesti, Matti and M{"{u}}ller, Michael and Becher, Jana and Schnabelrauch, Matthias and D{textquoteright}Este, Matteo and Eglin, David and Zenobi-Wong, Marcy
Title A versatile bioink for three-dimensional printing of cellular scaffolds based on thermally and photo-triggered tandem gelation [Abstract]
Year 2014
Journal/Proceedings Acta Biomaterialia
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Abstract
Abstract Layer-by-layer bioprinting is a logical choice for the fabrication of stratified tissues like articular cartilage. Printing of viable organ replacements, however, is dependent on bioinks with appropriate rheological and cytocompatible properties. In cartilage engineering, photocrosslinkable glycosaminoglycan-based hydrogels are chondrogenic, but alone have generally poor printing properties. By blending the thermoresponsive polymer poly(N-isopropylacrylamide) grafted hyaluronan (HA-pNIPAAM) with methacrylated hyaluronan (HAMA), high-resolution scaffolds with good viability were printed. HA-pNIPAAM provided fast gelation and immediate post-printing structural fidelity, while {HAMA} ensured long-term mechanical stability upon photocrosslinking. The bioink was evaluated for rheological properties, swelling behavior, printability and biocompatibility of encapsulated bovine chondrocytes. Elution of HA-pNIPAAM from the scaffold was necessary to obtain good viability. HA-pNIPAAM can therefore be used to support extrusion of a range of biopolymers which undergo tandem gelation, thereby facilitating the printing of cell-laden, stratified cartilage constructs with zonally varying composition and stiffness.
AUTHOR Neubauer, Vanessa J. and Trossmann, Vanessa T. and Jacobi, Sofia and Döbl, Annika and Scheibel, Thomas
Title Aqueous-Organic Solvent Derived Recombinant Spider Silk Gels as Depots for Drugs [Abstract]
Year 2
Journal/Proceedings Angewandte Chemie International Edition
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Abstract
Hydrogels are widely used in various biomedical applications, as they cannot only serve as materials for biofabrication but also as depots for the administration of drugs. However, the possibilities of formulation of water-insoluble drugs in hydrogels are rather limited. In this study, we assembled recombinant spider silk gels using a new processing route with aqueous-organic co-solvents, and the properties of these gels could be controlled by the choice of the co-solvent. The presence of the organic co-solvent further enabled the incorporation of hydrophobic drugs as exemplary shown for 6-mercaptopurine. The developed gels showed shear-thinning behaviour and could be easily injected to serve e.g. as drug depots and could even be 3D printed to serve as scaffolds for biofabrication. With this new processing route, the formulation of water-insoluble drugs in spider silk-based depots is possible, circumventing common pharmaceutical solubility issues.
AUTHOR Quadri, Faisal and Soman, Soja Saghar and Vijayavenkataraman, Sanjairaj
Title Progress in cardiovascular bioprinting [Abstract]
Year 2
Journal/Proceedings Artificial Organs
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Abstract Cardiovascular disease has been the leading cause of death globally for the past 15 years. Following a major cardiac disease episode, the ideal treatment would be the replacement of the damaged tissue, due to the limited regenerative capacity of cardiac tissues. However, we suffer from a chronic organ donor shortage which causes approximately 20 people to die each day waiting to receive an organ. Bioprinting of tissues and organs can potentially alleviate this burden by fabricating low cost tissue and organ replacements for cardiac patients. Clinical adoption of bioprinting in cardiovascular medicine is currently limited by the lack of systematic demonstration of its effectiveness, high costs, and the complexity of the workflow. Here, we give a concise review of progress in cardiovascular bioprinting and its components. We further discuss the challenges and future prospects of cardiovascular bioprinting in clinical applications.