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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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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 Monferrer, Ezequiel and Martín-Vañó, Susana and Carretero, Aitor and García-Lizarribar, Andrea and Burgos-Panadero, Rebeca and Navarro, Samuel and Samitier, Josep and Noguera, Rosa
Title A three-dimensional bioprinted model to evaluate the effect of stiffness on neuroblastoma cell cluster dynamics and behavior [Abstract]
Year 2020
Journal/Proceedings Scientific Reports
Reftype Monferrer2020
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Three-dimensional (3D) bioprinted culture systems allow to accurately control microenvironment components and analyze their effects at cellular and tissue levels. The main objective of this study was to identify, quantify and localize the effects of physical-chemical communication signals between tumor cells and the surrounding biomaterial stiffness over time, defining how aggressiveness increases in SK-N-BE(2) neuroblastoma (NB) cell line. Biomimetic hydrogels with SK-N-BE(2) cells, methacrylated gelatin and increasing concentrations of methacrylated alginate (AlgMA 0%, 1% and 2%) were used. Young’s modulus was used to define the stiffness of bioprinted hydrogels and NB tumors. Stained sections of paraffin-embedded hydrogels were digitally quantified. Human NB and 1% AlgMA hydrogels presented similar Young´s modulus mean, and orthotopic NB mice tumors were equally similar to 0% and 1% AlgMA hydrogels. Porosity increased over time; cell cluster density decreased over time and with stiffness, and cell cluster occupancy generally increased with time and decreased with stiffness. In addition, cell proliferation, mRNA metabolism and antiapoptotic activity advanced over time and with stiffness. Together, this rheological, optical and digital data show the potential of the 3D in vitro cell model described herein to infer how intercellular space stiffness patterns drive the clinical behavior associated with NB patients.
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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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 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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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 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 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 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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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 Aliyazdi, Samy and Frisch, Sarah and Hidalgo, Alberto and Frank, Nicolas and Krug, Daniel and Müller, Rolf and Schaefer, Ulrich F. and Vogt, Thomas and Loretz, Brigitta and Lehr, Claus-Michael
Title 3D bioprinting of E. coli MG1655 biofilms on human lung epithelial cells for building complex in vitro infection models [Abstract]
Year 2023
Journal/Proceedings Biofabrication
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Biofilm-associated infections are causing over half a million deaths each year, raising the requirement for innovative therapeutic approaches. For developing novel therapeutics against bacterial biofilm infections, complex in vitro models that allow to study drug effects on both pathogens and host cells as well as their interaction under controlled, physiologically relevant conditions appear as highly desirable. Nonetheless, building such models is quite challenging because (1) rapid bacterial growth and release of virulence factors may lead to premature host cell death and (2) maintaining the biofilm status under suitable co-culture requires a highly controlled environment. To approach that problem, we chose 3D bioprinting. However, printing living bacterial biofilms in defined shapes on human cell models, requires bioinks with very specific properties. Hence, this work aims to develop a 3D bioprinting biofilm method to build robust in vitro infection models. Based on rheology, printability and bacterial growth, a bioink containing 3% gelatin and 1% alginate in Luria-Bertani-medium was found optimal for Escherichia coli MG1655 biofilms. Biofilm properties were maintained after printing, as shown visually via microscopy techniques as well as in antibiotic susceptibility assays. Metabolic profile analysis of bioprinted biofilms showed high similarity to native biofilms. After printing on human bronchial epithelial cells (Calu-3), the shape of printed biofilms was maintained even after dissolution of non-crosslinked bioink, while no cytotoxicity was observed over 24 h. Therefore, the approach presented here may provide a platform for building complex in vitro infection models comprising bacterial biofilms and human host cells.
AUTHOR Kajtez, Janko and Wesseler, Milan Finn and Birtele, Marcella and Khorasgani, Farinaz Riyahi and Rylander Ottosson, Daniella and Heiskanen, Arto and Kamperman, Tom and Leijten, Jeroen and Martínez-Serrano, Alberto and Larsen, Niels B. and Angelini, Thomas E. and Parmar, Malin and Lind, Johan U. and Emnéus, Jenny
Title Embedded 3D Printing in Self-Healing Annealable Composites for Precise Patterning of Functionally Mature Human Neural Constructs [Abstract]
Year 2022
Journal/Proceedings Advanced Science
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Abstract Human in vitro models of neural tissue with tunable microenvironment and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing inside granular gels holds great promise as it allows precise patterning of extremely soft tissue constructs. However, granular printing support formulations are restricted to only a handful of materials. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high-fidelity support for embedded printing akin to granular gels. To address this need, Authors present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular-matrix solution to enable precise and programmable patterning of human stem cells and consequent generation mature subtype-specific neurons that extend projections into the volume of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular-like channels. Due to its modularity and versatility, SHAPE biomanufacturing toolbox has potential to be used in applications beyond functional modeling of mechanically sensitive neural constructs.
AUTHOR García-Lizarribar, Andrea and Villasante, Aranzazu and Lopez-Martin, Jose Antonio and Flandez, Marta and Soler-Vázquez, M. Carmen and Serra, Dolors and Herrero, Laura and Sagrera, Ana and Efeyan, Alejo and Samitier, Josep
Title 3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia [Abstract]
Year 2023
Journal/Proceedings Biomaterials Advances
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Acquired muscle diseases such as cancer cachexia are responsible for the poor prognosis of many patients suffering from cancer. In vitro models are needed to study the underlying mechanisms of those pathologies. Extrusion bioprinting is an emerging tool to emulate the aligned architecture of fibers while implementing additive manufacturing techniques in tissue engineering. However, designing bioinks that reconcile the rheological needs of bioprinting and the biological requirements of muscle tissue is a challenging matter. Here we formulate a biomaterial with dual crosslinking to modulate the physical properties of bioprinted models. We design 3D bioprinted muscle models that resemble the mechanical properties of native tissue and show improved proliferation and high maturation of differentiated myotubes suggesting that the GelMA-AlgMA-Fibrin biomaterial possesses myogenic properties. The electrical stimulation of the 3D model confirmed the contractile capability of the tissue and enhanced the formation of sarcomeres. Regarding the functionality of the models, they served as platforms to recapitulate skeletal muscle diseases such as muscle wasting produced by cancer cachexia. The genetic expression of 3D models demonstrated a better resemblance to the muscular biopsies of cachectic mouse models. Altogether, this biomaterial is aimed to fabricate manipulable skeletal muscle in vitro models in a non-costly, fast and feasible manner.
AUTHOR Krstić, Nenad and Jüttner, Jens and Giegerich, Lars and Mayer, Margot and Knuth, Monika and Müller, Achim and Thielemann, Christiane
Title 3D printed biosensor for continuous glucose measurement in cell cultures [Abstract]
Year 2023
Journal/Proceedings Annals of 3D Printed Medicine
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A novel 3D-printed glucose sensor is presented for cell culture application. Glucose sensing was performed using a fluorescence resonance energy transfer (FRET)-based assay principle based on ConA and dextran. Both molecules are encapsulated in alginate microspheres and embedded in the UV-curable, stable hydrogel polyvinyl alcohol (PVA). The rheology of the formulation was adapted to obtain good properties for an extrusion-based printing process. The printed sensor structures were tested for their ability to detect glucose in vitro. A proportional increase in fluorescence intensity was observed in a concentration range of 0 - 2 g/L glucose. Tests with HEK cell cultures also showed good cell compatibility and excellent adhesion properties on plasma-treated Petri dishes. The printed sensors were able to detect the glucose decay associated with the metabolic activities of the fast-growing HEK cells in the cell culture medium over ten days. The proof-of-principle study shows that metabolic processes in cell cultures can be monitored with the new printed sensor using a standard fluorescence wide-field microscope.
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 Estermann, Manuela and Coelho, Ricardo and Jacob, Francis and Huang, Yen-Lin and Liang, Ching-Yeu and Faia-Torres, Ana Bela and Septiadi, Dedy and Drasler, Barbara and Karakocak, Bedia Begum and Dijkhoff, Irini Magdelina and Petri-Fink, Alke and Heinzelmann-Schwarz, Viola and Rothen-Rutishauser, Barbara
Title A 3D multi-cellular tissue model of the human omentum to study the formation of ovarian cancer metastasis [Abstract]
Year 2023
Journal/Proceedings Biomaterials
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Reliable and predictive experimental models are urgently needed to study metastatic mechanisms of ovarian cancer cells in the omentum. Although models for ovarian cancer cell adhesion and invasion were previously investigated, the lack of certain omental cell types, which influence the metastatic behavior of cancer cells, limits the application of these tissue models. Here, we describe a 3D multi-cellular human omentum tissue model, which considers the spatial arrangement of five omental cell types. Reproducible tissue models were fabricated combining permeable cell culture inserts and bioprinting technology to mimic metastatic processes of immortalized and patient-derived ovarian cancer cells. The implementation of an endothelial barrier further allowed studying the interaction between cancer and endothelial cells during hematogenous dissemination and the impact of chemotherapeutic drugs. This proof-of-concept study may serve as a platform for patient-specific investigations in personalized oncology in the future.
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 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 Cojocaru, Elena and Ghitman, Jana and Pircalabioru, Gratiela Gradisteanu and Zaharia, Anamaria and Iovu, Horia and Sarbu, Andrei
Title Electrospun/3D-Printed Bicomponent Scaffold Co-Loaded with a Prodrug and a Drug with Antibacterial and Immunomodulatory Properties [Abstract]
Year 2023
Journal/Proceedings Polymers
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This work reports the construction of a bicomponent scaffold co-loaded with both a prodrug and a drug (BiFp@Ht) as an efficient platform for wound dressing, by combining the electrospinning and 3D-printing technologies. The outer component consisted of a chitosan/polyethylene oxide-electrospun membrane loaded with the indomethacin–polyethylene glycol–indomethacin prodrug (Fp) and served as a support for printing the inner component, a gelatin methacryloyl/sodium alginate hydrogel loaded with tetracycline hydrochloride (Ht). The different architectural characteristics of the electrospun and 3D-printed layers were very well highlighted in a morphological analysis performed by Scanning Electron Microscopy (SEM). In vitro release profile studies demonstrated that both Fp and Ht layers were capable to release the loaded therapeutics in a controlled and sustained manner. According to a quantitative in vitro biological assessment, the bicomponent BiFp@Ht scaffold showed a good biocompatibility and no cytotoxic effect on HeLa cell cultures, while the highest proliferation level was noted in the case of HeLa cells seeded onto an Fp nanofibrous membrane. Furthermore, the BiFp@Ht scaffold presented an excellent antimicrobial activity against the E. coli and S. aureus bacterial strains, along with promising anti-inflammatory and proangiogenic activities, proving its potential to be used for wound dressing.
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 Gruhn, Thomas and Monsalve, Camilo Ortiz and Müller, Claudia and Heid, Susanne and Boccaccini, Aldo R. and Salehi, Sahar
Title Fabrication of Hydrogel-Based Composite Fibers and Computer Simulation of the Filler Dynamics in the Composite Flow [Abstract]
Year 2023
Journal/Proceedings Bioengineering
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Fibrous structures with anisotropic fillers as composites have found increasing interest in the field of biofabrication since they can mimic the extracellular matrix of anisotropic tissues such as skeletal muscle or nerve tissue. In the present work, the inclusion of anisotropic fillers in hydrogel-based filaments with an interpenetrating polymeric network (IPN) was evaluated and the dynamics of such fillers in the composite flow were analyzed using computational simulations. In the experimental part, microfabricated rods (200 and 400 μm length, 50 μm width) were used as anisotropic fillers in extrusion of composite filaments using two techniques of wet spinning and 3D printing. Hydrogels such as oxidized alginate (ADA) and methacrylated gelatin (GelMA) were used as matrices. In the computational simulation, a combination of computational fluid dynamics and coarse-grained molecular dynamics was used to study the dynamics of rod-like fillers in the flow field of a syringe. It showed that, during the extrusion process, microrods are far from being well aligned. Instead, many of them tumble on their way through the needle leading to a random orientation in the fiber which was confirmed experimentally.
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 Moo, Eng Kuan and Ebrahimi, Mohammadhossein and Hrynevich, Andrei and de Ruijter, Mylène and Castilho, Miguel and Malda, Jos and Korhonen, Rami K.
Title Load-induced fluid pressurisation in hydrogel systems before and after reinforcement by melt-electrowritten fibrous meshes [Abstract]
Year 2023
Journal/Proceedings Journal of the Mechanical Behavior of Biomedical Materials
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Fluid pressure develops transiently within mechanically-loaded, cell-embedding hydrogels, but its magnitude depends on the intrinsic material properties of the hydrogel and cannot be easily altered. The recently developed melt-electrowriting (MEW) technique enables three-dimensional printing of structured fibrous mesh with small fibre diameter (20 μm). The MEW mesh with 20 μm fibre diameter can synergistically increase the instantaneous mechanical stiffness of soft hydrogels. However, the reinforcing mechanism of the MEW meshes is not well understood, and may involve load-induced fluid pressurisation. Here, we examined the reinforcing effect of MEW meshes in three hydrogels: gelatin methcryloyl (GelMA), agarose and alginate, and the role of load-induced fluid pressurisation in the MEW reinforcement. We tested the hydrogels with and without MEW mesh (i.e., hydrogel alone, and MEW-hydrogel composite) using micro-indentation and unconfined compression, and analysed the mechanical data using biphasic Hertz and mixture models. We found that the MEW mesh altered the tension-to-compression modulus ratio differently for hydrogels that are cross-linked differently, which led to a variable change to their load-induced fluid pressurisation. MEW meshes only enhanced the fluid pressurisation for GelMA, but not for agarose or alginate. We speculate that only covalently cross-linked hydrogels (GelMA) can effectively tense the MEW meshes, thereby enhancing the fluid pressure developed during compressive loading. In conclusion, load-induced fluid pressurisation in selected hydrogels was enhanced by MEW fibrous mesh, and may be controlled by MEW mesh of different designs in the future, thereby making fluid pressure a tunable cell growth stimulus for tissue engineering involving mechanical stimulation.
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 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 Kopecká, Kateřina and Vítková, Lenka and Kroneková, Zuzana and Musilová, Lenka and Smolka, Petr and Mikulka, Filip and Melánová, Klára and Knotek, Petr and Humeník, Martin and Minařík, Antonín and Mráček, Aleš
Title Synthesis and Exfoliation of Calcium Organophosphonates for Tailoring Rheological Properties of Sodium Alginate Solutions: A Path toward Polysaccharide-Based Bioink [Abstract]
Year 2023
Journal/Proceedings Biomacromolecules
Reftype
DOI/URL DOI
Abstract
Layered nanoparticles with surface charge are explored as rheological modifiers for extrudable materials, utilizing their ability to induce electrostatic repulsion and create a house-of-cards structure. These nanoparticles provide mechanical support to the polymer matrix, resulting in increased viscosity and storage modulus. Moreover, their advantageous aspect ratio allows for shear-induced orientation and decreased viscosity during flow. In this work, we present a synthesis and liquid-based exfoliation procedure of phenylphosphonate-phosphate particles with enhanced ability to be intercalated by hydrophilic polymers. These layered nanoparticles are then tested as rheological modifiers of sodium alginate. The effective rheological modification is proved as the viscosity increases from 101 up to 103 Pa·s in steady state. Also, shear-thinning behavior is observed. The resulting nanocomposite hydrogels show potential as an extrudable bioink for 3D printing in tissue engineering and other biomedical applications, with good shape fidelity, nontoxicity, and satisfactory cell viability confirmed through encapsulation and printing of mouse fibroblasts.
AUTHOR Radeke, Carmen and Pons, Raphaël and Mihajlovic, Marko and Knudsen, Jonas R. and Butdayev, Sarkhan and Kempen, Paul J. and Segeritz, Charis-Patricia and Andresen, Thomas L. and Pehmøller, Christian K. and Jensen, Thomas E. and Lind, Johan U.
Title Transparent and Cell-Guiding Cellulose Nanofiber 3D Printing Bioinks [Abstract]
Year 2023
Journal/Proceedings ACS Appl. Mater. Interfaces
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DOI/URL DOI
Abstract
For three-dimensional (3D) bioprinting to fulfill its promise and enable the automated fabrication of complex tissue-mimicking constructs, there is a need for developing bioinks that are not only printable and biocompatible but also have integrated cell-instructive properties. Toward this goal, we here present a scalable technique for generating nanofiber 3D printing inks with unique tissue-guiding capabilities. Our core methodology relies on tailoring the size and dispersibility of cellulose fibrils through a solvent-controlled partial carboxymethylation. This way, we generate partially negatively charged cellulose nanofibers with diameters of ∼250 nm and lengths spanning tens to hundreds of microns. In this range, the fibers structurally match the size and dimensions of natural collagen fibers making them sufficiently large to orient cells. Yet, they are simultaneously sufficiently thin to be optically transparent. By adjusting fiber concentration, 3D printing inks with excellent shear-thinning properties can be established. In addition, as the fibers are readily dispersible, composite inks with both carbohydrates and extracellular matrix (ECM)-derived proteins can easily be generated. We apply such composite inks for 3D printing cell-laden and cross-linkable structures, as well as tissue-guiding gel substrates. Interestingly, we find that the spatial organization of engineered tissues can be defined by the shear-induced alignment of fibers during the printing procedure. Specifically, we show how myotubes derived from human and murine skeletal myoblasts can be programmed into linear and complex nonlinear architectures on soft printed substrates with intermediate fiber contents. Our nanofibrillated cellulose inks can thus serve as a simple and scalable tool for engineering anisotropic human muscle tissues that mimic native structure and function.
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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DOI/URL URL DOI
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 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
Reftype
DOI/URL URL DOI
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 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 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 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 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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DOI/URL DOI
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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DOI/URL DOI
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 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 Govindharaj, Mano and Al Hashemi, Noura Sayed and Soman, Soja Saghar and Vijayavenkataraman, Sanjairaj
Title Bioprinting of bioactive tissue scaffolds from ecologically-destructive fouling tunicates [Abstract]
Year 2022
Journal/Proceedings Journal of Cleaner Production
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DOI/URL URL DOI
Abstract
Urochordates are the closest invertebrate relative to humans and commonly referred to as tunicates, a name ascribed to their leathery outer “tunic”. The tunic is the outer covering of the organism which functions as the exoskeleton and is rich in carbohydrates and proteins. Invasive or fouling tunicates pose a great threat to the indigenous marine ecosystem and governments spend several hundred thousand dollars for tunicate management, considering the huge adverse economic impact it has on the shipping and fishing industries. In this work, the environmentally destructive colonizing tunicate species of Polyclinum constellatum was successfully identified in the coast of Abu Dhabi and methods of sustainably using it as wound-dressing materials, decellularized extra-cellular matrix (dECM) scaffolds for tissue engineering applications and bioinks for bioprinting of tissue constructs for regenerative medicine are proposed. The intricate three-dimensional nanofibrous cellulosic networks in the tunic remain intact even after the multi-step process of decellularization and lyophilization. The lyophilized dECM tunics possess excellent biocompatibility and remarkable tensile modulus of 3.85 ± 0.93 MPa compared to ∼0.1–1 MPa of other hydrogel systems. This work demonstrates the use of lyophilized tunics as wound-dressing materials, having outperformed the commercial dressing materials with a capacity of absorbing 20 times its weight in the dry state. This work also demonstrates the biocompatibility of dECM scaffold and dECM-derived bioink (3D bioprinting with Mouse Embryonic Fibroblasts (MEFs)). Both dECM scaffolds and bioprinted dECM-based tissue constructs show enhanced metabolic activity and cell proliferation over time. Sustainable utilization of dECM-based biomaterials from ecologically-destructive fouling tunicates proposed in this work helps preserve the marine ecosystem, shipping and fishing industries worldwide, and mitigate the huge cost spent for tunicate management.
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 Geevarghese, Rency and Somasekharan, Lakshmi T. and Bhatt, Anugya and Kasoju, Naresh and Nair, Renjith P.
Title Development and evaluation of a multicomponent bioink consisting of alginate, gelatin, diethylaminoethyl cellulose and collagen peptide for 3D bioprinting of tissue construct for drug screening application [Abstract]
Year 2022
Journal/Proceedings International Journal of Biological Macromolecules
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DOI/URL URL DOI
Abstract
Three dimensional (3D) bioprinting technology has been making a progressive advancement in the field of tissue engineering to produce tissue constructs that mimic the shape, framework, and microenvironment of an organ. The technology has not only paved the way to organ development but has been widely studied for its application in drug and cosmetic testing using 3D bioprinted constructs. However, not much has been explored on the utilization of bioprinting technology for the development of tumor models to test anti-cancer drug efficacy. The conventional methodology involves a two dimensional (2D) monolayer model to test cellular drug response which has multiple limitations owing to its inability to mimic the natural tissue environment. The choice of bioink for 3D bioprinting is critical as cell morphology and proliferation depend greatly on the property of bioink. In this study, we developed a multicomponent bioink composed of alginate, diethylaminoethyl cellulose, gelatin, and collagen peptide to generate a 3D bioprinted construct. The bioink has been characterised and validated for its printability, shape fidelity and biocompatibility to be used for generating tumor models. Further, a bioprinted tumor model was developed using lung cancer cell line and the efficacy of 3D printed construct for drug screening application was established.
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 Da Silva, Aruã Clayton and Wang, Junzhi and Minev, Ivan Rusev
Title Electro-assisted printing of soft hydrogels via controlled electrochemical reactions [Abstract]
Year 2022
Journal/Proceedings Nature Communications
Reftype Da Silva2022
DOI/URL DOI
Abstract
Hydrogels underpin many applications in tissue engineering, cell encapsulation, drug delivery and bioelectronics. Methods improving control over gelation mechanisms and patterning are still needed. Here we explore a less-known gelation approach relying on sequential electrochemical-chemical-chemical (ECC) reactions. An ionic species and/or molecule in solution is oxidised over a conductive surface at a specific electric potential. The oxidation generates an intermediate species that reacts with a macromolecule, forming a hydrogel at the electrode-electrolyte interface. We introduce potentiostatic control over this process, allowing the selection of gelation reactions and control of hydrogel growth rate. In chitosan and alginate systems, we demonstrate precipitation, covalent and ionic gelation mechanisms. The method can be applied in the polymerisation of hybrid systems consisting of more than one polymer. We demonstrate concomitant deposition of the conductive polymer Poly(3,4-ethylenedioxythiophene) (PEDOT) and alginate. Deposition of the hydrogels occurs in small droplets held between a conductive plate (working electrode, WE), a printing nozzle (counter electrode, CE) and a pseudoreference electrode (reference electrode, RE). We install this setup on a commercial 3D printer to demonstrate patterning of adherent hydrogels on gold and flexible ITO foils. Electro-assisted printing may contribute to the integration of well-defined hydrogels on hybrid electronic-hydrogel devices for bioelectronics applications.
AUTHOR Strauß, Svenja and Schroth, Bianca and Hubbuch, Jürgen
Title Evaluation of the Reproducibility and Robustness of Extrusion-Based Bioprinting Processes Applying a Flow Sensor [Abstract]
Year 2022
Journal/Proceedings Frontiers in bioengineering and biotechnology
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DOI/URL URL DOI
Abstract
Bioprinting is increasingly regarded as a suitable additive manufacturing method in biopharmaceutical process development and formulation. In order to manage the leap from research to industrial application, higher levels of reproducibility and a standardized bioprinting process are prerequisites. This said, the concept of process analytical technologies, standard in the biopharmaceutical industry, is still at its very early steps. To date most extrusion-based printing processes are controlled over penumatic pressure and thus not adaptive to environmental or system related changes over several experimental runs. A constant set pressure applied over a number of runs, might lead to variations in flow rate and thus to unreliable printed constructs. With this in mind, the simple question arises whether a printing process based on a set flow rate could improve reproduciblity and transfer to different printing systems. The control and monitoring of flow rate aim to introduce the concept of PAT in the field of bioprinting. This study investigates the effect of different processing modes (set pressure vs. set flow rate) on printing reproducibility occurring during an extrusion-based printing process consisting of 6 experimental runs consisting of 3 printed samples each. Additionally, the influence of different filling levels of the ink containing cartridge during a printing process was determined. Different solutions based on a varying amount of alginate polymer and Kolliphor hydrogels in varying concentrations showed the need for individual setting of printing parameter. To investigate parameter transferability among different devices two different printers were used and the flow was monitored using a flow sensor attached to the printing unit. It could be demonstrated that a set flow rate controlled printing process improved accuracy and the filling level also affects the accuracy of printing, the magnitude of this effects varies as the cartridge level declined. The transferability between printed devices was eased by setting the printing parameters according to a set flow rate of each bioink disregarding the value of the set pressure. Finally, by a bioprinting porcess control based on a set flow rate, the coefficient of variance for printed objects could be reduced from 0.2 to 0.02 for 10% (w/v) alginate polymer solutions.
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
Reftype
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 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
Reftype
DOI/URL URL DOI
Abstract
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 Schmieg, Barbara and Gretzinger, Sarah and Schuhmann, Sebastian and Guthausen, Gisela and Hubbuch, Jürgen
Title Magnetic resonance imaging as a tool for quality control in extrusion-based bioprinting [Abstract]
Year 2022
Journal/Proceedings Biotechnology Journal
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Abstract
Abstract Bioprinting is gaining importance for the manufacturing of tailor-made hydrogel scaffolds in tissue engineering, pharmaceutical research and cell therapy. However, structure fidelity and geometric deviations of printed objects heavily influence mass transport and process reproducibility. Fast, three-dimensional and nondestructive quality control methods will be decisive for the approval in larger studies or industry. Magnetic resonance imaging (MRI) meets these requirements for characterizing heterogeneous soft materials with different properties. Complementary to the idea of decentralized 3D printing, magnetic resonance tomography is common in medicine, and image data processing tools can be transferred system-independently. In this study, a MRI measurement and image analysis protocol was evaluated to jointly assess the reproducibility of three different hydrogels and a reference material. Critical parameters for object quality, namely porosity, hole areas and deviations along the height of the scaffolds are discussed. Geometric deviations could be correlated to specific process parameters, anomalies of the ink or changes of ambient conditions. This strategy allows the systematic investigation of complex 3D objects as well as an implementation as a process control tool. Combined with the monitoring of metadata this approach might pave the way for future industrial applications of 3D printing in the field of biopharmaceutics.
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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Abstract
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 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 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 Gretzinger, Sarah and Schmieg, Barbara and Guthausen, Gisela and Hubbuch, Jürgen
Title Virtual Reality as Tool for Bioprinting Quality Inspection: A Proof of Principle [Abstract]
Year 2022
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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Abstract
As virtual reality (VR) has drastically evolved over the past few years, the field of applications of VR flourished way beyond the gaming industry. While commercial VR solutions might be available, there is a need to develop a workflow for specific applications. Bioprinting represents such an example. Here, complex 3D data is generated and needs to be visualized in the context of quality control. We demonstrate that the transfer to a commercially available VR software is possible by introducing an optimized workflow. In the present work, we developed a workflow for the visualization of the critical quality attribute (cQA) cell distribution in bioprinted (extrusion-based) samples in VR. The cQA cell distribution is directly influenced by the pre-processing step mixing of cell material in the bioink. Magnetic Resonance Imaging (MRI) was used as an analytical tool to generate spatially resolved 2.5 and 3D data of the bioprinted objects. A sample with poor quality in respect of the cQA cell distribution was identified as its inhomogeneous cell distribution could be displayed spatially resolved in VR. The described workflow facilitates the usage of VR as a tool for quality inspection in the field of bioprinting and represents a powerful tool for visualization of complex 3D MRI data.
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 Rößler, Sina and Brückner, Andreas and Kruppke, Iris and Wiesmann, Hans-Peter and Hanke, Thomas and Kruppke, Benjamin
Title 3D Plotting of Silica/Collagen Xerogel Granules in an Alginate Matrix for Tissue-Engineered Bone Implants [Abstract]
Year 2021
Journal/Proceedings Materials
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Abstract
Today, materials designed for bone regeneration are requested to be degradable and resorbable, bioactive, porous, and osteoconductive, as well as to be an active player in the bone-remodeling process. Multiphasic silica/collagen Xerogels were shown, earlier, to meet these requirements. The aim of the present study was to use these excellent material properties of silica/collagen Xerogels and to process them by additive manufacturing, in this case 3D plotting, to generate implants matching patient specific shapes of fractures or lesions. The concept is to have Xerogel granules as active major components embedded, to a large proportion, in a matrix that binds the granules in the scaffold. By using viscoelastic alginate as matrix, pastes of Xerogel granules were processed via 3D plotting. Moreover, alginate concentration was shown to be the key to a high content of irregularly shaped Xerogel granules embedded in a minimum of matrix phase. Both the alginate matrix and Xerogel granules were also shown to influence viscoelastic behavior of the paste, as well as the dimensionally stability of the scaffolds. In conclusion, 3D plotting of Xerogel granules was successfully established by using viscoelastic properties of alginate as matrix phase.
AUTHOR Leu Alexa, Rebeca and Ianchis, Raluca and Savu, Diana and Temelie, Mihaela and Trica, Bogdan and Serafim, Andrada and Vlasceanu, George Mihail and Alexandrescu, Elvira and Preda, Silviu and Iovu, Horia
Title 3D Printing of Alginate-Natural Clay Hydrogel-Based Nanocomposites [Abstract]
Year 2021
Journal/Proceedings Gels
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Abstract
Biocompatibility, biodegradability, shear tinning behavior, quick gelation and an easy crosslinking process makes alginate one of the most studied polysaccharides in the field of regenerative medicine. The main purpose of this study was to obtain tissue-like materials suitable for use in bone regeneration. In this respect, alginate and several types of clay were investigated as components of 3D-printing, nanocomposite inks. Using the extrusion-based nozzle, the nanocomposites inks were printed to obtain 3D multilayered scaffolds. To observe the behavior induced by each type of clay on alginate-based inks, rheology studies were performed on composite inks. The structure of the nanocomposites samples was examined using Fourier Transform Infrared Spectrometry and X-ray Diffraction (XRD), while the morphology of the 3D-printed scaffolds was evaluated using Electron Microscopy (SEM, TEM) and Micro-Computed Tomography (Micro-CT). The swelling and dissolvability of each composite scaffold in phosfate buffer solution were followed as function of time. Biological studies indicated that the cells grew in the presence of the alginate sample containing unmodified clay, and were able to proliferate and generate calcium deposits in MG-63 cells in the absence of specific signaling molecules. This study provides novel information on potential manufacturing methods for obtaining nanocomposite hydrogels suitable for 3D printing processes, as well as valuable information on the clay type selection for enabling accurate 3D-printed constructs. Moreover, this study constitutes the first comprehensive report related to the screening of several natural clays for the additive manufacturing of 3D constructs designed for bone reconstruction therapy.
AUTHOR Jiahui Lai and Xinliang Ye and Jia Liu and Chong Wang and Junzhi Li and Xiang Wang and Mingze Ma and Min Wang
Title 4D printing of highly printable and shape morphing hydrogels composed of alginate and methylcellulose [Abstract]
Year 2021
Journal/Proceedings Materials & Design
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Abstract
4D printing of swellable/shrinkable hydrogels has been viewed as an appealing approach for fabricating dynamic structures for various biomedical applications. However, 4D printing of precise hydrogel structures is still highly challenging due to the relatively poor printability of hydrogels and high surface roughness of printed patterns, when micro extrusion-based 3D printers are used. In this study, a highly printable and shape morphing hydrogel was investigated for 4D printing by blending alginate (Alg) and methylcellulose (MC). The optimized Alg/MC hydrogel exhibited excellent rheological properties, extrudability and shape fidelity of printed structures. The printable Alg/MC hydrogel was 4D printed into a series of patterned 2D architectures which were encoded with anisotropic stiffness and swelling behaviors by strategically controlling the network density gradients vertical to the orientation of the patterned strips. By controlling the strip interspacing and angle, these 2D architectures could transform into various prescribed simple 3D morphologies (e.g., tube-curling and helix) and complex 3D morphologies (e.g., double helix and flowers) after immersion in a calcium chloride solution. This shape morphing Alg/MC hydrogel with excellent printability has high potential for 4D printing of delicate hydrogel patterns, which are increasingly needed in the tissue engineering, biomedical device and soft robotics fields.
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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Abstract
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 Curti, Filis and Drăgușin, Diana-Maria and Serafim, Andrada and Iovu, Horia and Stancu, Izabela-Cristina
Title Development of thick paste-like inks based on superconcentrated gelatin/alginate for 3D printing of scaffolds with shape fidelity and stability [Abstract]
Year 2021
Journal/Proceedings Materials Science and Engineering: C
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Abstract
Shape fidelity and integrity are serious challenges in the 3D printing of hydrogel precursors, as they can influence the overall performance of 3D scaffolds. This work reports the development of superconcentrated inks based on sodium alginate and fish gelatin as an appealing strategy to satisfy such challenges and dictate the quality of the printed scaffolds, without using crosslinking strategies during 3D printing. SEM micrographs and micro-CT images indicate the homogeneous distribution of the polysaccharide in the gelatin-based matrix, suggesting its potential to act as a reinforcing additive. The high concentration of gelatin aqueous solution (50 wt%) and substantial incorporation of alginate have facilitated the highly accurate printability and influence the in vitro stability and mechanical properties of the printed scaffolds. An improvement of the stiffness is dictated by the increase of alginate concentration from 20 wt% to 25 wt%, and an increase of Young modulus with about 46% is reached, confirming the reinforcing effect of polysaccharide. This study highlights the potential of paste-type inks to provide high resolution 3D printed structures with appealing structural and dimensional stability, in vitro degradability and mechanical properties for biomedical applications.
AUTHOR Chen, Shengyang and Shi, Qian and Jang, Taesik and Ibrahim, Mohammed Shahrudin Bin and Deng, Jingyu and Ferracci, Gaia and Tan, Wen See and Cho, Nam-Joon and Song, Juha
Title Engineering Natural Pollen Grains as Multifunctional 3D Printing Materials [Abstract]
Year 2021
Journal/Proceedings Advanced Functional Materials
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Abstract
Abstract The development of multifunctional 3D printing materials from sustainable natural resources is a high priority in additive manufacturing. Using an eco-friendly method to transform hard pollen grains into stimulus-responsive microgel particles, we engineered a pollen-derived microgel suspension that can serve as a functional reinforcement for composite hydrogel inks and as a supporting matrix for versatile freeform 3D printing systems. The pollen microgel particles enabled the printing of composite inks and improved the mechanical and physiological stabilities of alginate and hyaluronic acid hydrogel scaffolds for 3D cell culture applications. Moreover, the particles endowed the inks with stimulus-responsive controlled release properties. The suitability of the pollen microgel suspension as a supporting matrix for freeform 3D printing of alginate and silicone rubber inks was demonstrated and optimized by tuning the rheological properties of the microgel. Compared with other classes of natural materials, pollen grains have several compelling features, including natural abundance, renewability, affordability, processing ease, monodispersity, and tunable rheological features, which make them attractive candidates to engineer advanced materials for 3D printing applications.
AUTHOR Tan, Edgar Y. S. and Suntornnond, Ratima and Yeong, Wai Yee
Title High-Resolution Novel Indirect Bioprinting of Low-Viscosity Cell-Laden Hydrogels via Model-Support Bioink Interaction [Abstract]
Year 2021
Journal/Proceedings 3D Printing and Additive Manufacturing
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DOI/URL DOI
Abstract
Abstract Bioprinting of unmodified soft extracellular matrix into complex 3D structures has remained challenging to fabricate. Herein, we established a novel process for the printing of low-viscosity hydrogel by using a unique support technique to retain the structural integrity of the support structure. We demonstrated that this process of printing could be used for different types of hydrogel, ranging from fast crosslinking gelatin methacrylate to slow crosslinking collagen type I. In addition, we evaluated the biocompatibility of the process by observing the effects of the cytotoxicity of L929 and the functionality of the human umbilical vein endothelium primary cells after printing. The results show that the bioprinted construct provided excellent biocompatibility as well as supported cell growth and differentiation. Thus, this is a novel technique that can be potentially used to enhance the resolution of the extrusion-based bioprinter.
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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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 Balaji Mahendiran and Shalini Muthusamy and Sowndarya Sampath and S.N. Jaisankar and Ketul C. Popat and R. Selvakumar and Gopal Shankar Krishnakumar
Title Recent trends in natural polysaccharide based bioinks for multiscale 3D printing in tissue regeneration: A review [Abstract]
Year 2021
Journal/Proceedings International Journal of Biological Macromolecules
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Abstract
Biofabrication by three-dimensional (3D) printing has been an attractive technology in harnessing the possibility to print anatomical shaped native tissues with controlled architecture and resolution. 3D printing offers the possibility to reproduce complex microarchitecture of native tissues by printing live cells in a layer by layer deposition to provide a biomimetic structural environment for tissue formation and host tissue integration. Plant based biomaterials derived from green and sustainable sources have represented to emulate native physicochemical and biological cues in order to direct specific cellular response and formation of new tissues through biomolecular recognition patterns. This comprehensive review aims to analyze and identify the most commonly used plant based bioinks for 3D printing applications. An overview on the role of different plant based biomaterial of terrestrial origin (Starch, Nanocellulose and Pectin) and marine origin (Ulvan, Alginate, Fucoidan, Agarose and Carrageenan) used for 3D printing applications are discussed elaborately. Furthermore, this review will also emphasis in the functional aspects of different 3D printers, appropriate printing material, merits and demerits of numerous plant based bioinks in developing 3D printed tissue-like constructs. Additionally, the underlying potential benefits, limitations and future perspectives of plant based bioinks for tissue engineering (TE) applications are also 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 Chen, Shengyang and Jang, Tae-Sik and Pan, Houwen Matthew and Jung, Hyun-Do and Sia, Ming Wei and Xie, Shuying and Hang, Yao and Chong, Seow and Wong, Dongan
Title 3D Freeform Printing of Nanocomposite Hydrogels through in situ Precipitation in Reactive Viscous Fluid
Year 2020
Journal/Proceedings International Journal of Bioprinting
Reftype
DOI/URL DOI
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