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AUTHOR Terpstra, Margo L. and Li, Jinyu and Mensinga, Anneloes and de Ruijter, Myl{`{e}}ne and van Rijen, Mattie H. P. and Androulidakis, Charalampos and Galiotis, Costas and Papantoniou, Ioannis and Matsusaki, Michiya and Malda, Jos and Levato, Riccardo
Title Bioink with cartilage-derived extracellular matrix microfibers enables spatial control of vascular capillary formation in bioprinted constructs [Abstract]
Year 2022
Journal/Proceedings Biofabrication
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Microvasculature is essential for the exchange of gas and nutrient for most tissues in our body. Some tissue structures such as the meniscus presents spatially confined blood vessels adjacent to non-vascularized regions. In biofabrication, mimicking the spatial distribution of such vascular components is paramount, as capillary ingrowth into non-vascularized tissues can lead to tissue matrix alterations and subsequent pathology. Multi-material three-dimensional (3D) bioprinting strategies have the potential to resolve anisotropic tissue features, although building complex constructs comprising stable vascularized and non-vascularized regions remains a major challenge to date. In this study, we developed endothelial cell-laden pro- and anti-angiogenic bioinks, supplemented with bioactive matrix-derived microfibers (MFs) that were created from type I collagen sponges (col-1) and cartilage decellularized extracellular matrix (CdECM), respectively. Human umbilical vein endothelial cell (HUVEC)-driven capillary networks started to form 2 d after bioprinting. Supplementing cartilage-derived MFs to endothelial-cell laden bioinks reduced the total length of neo-microvessels by 29%, and the number of microvessel junctions by 37% after 14 d, compared to bioinks with pro-angiogenic col-1 MFs. As a proof of concept, the bioinks were bioprinted into an anatomical meniscus shape with a biomimetic vascularized outer and non-vascularized inner region, using a gellan gum microgel suspension bath. These 3D meniscus-like constructs were cultured up to 14 d, with in the outer zone the HUVEC-, mural cell-, and col-1 MF-laden pro-angiogenic bioink, and in the inner zone a meniscus progenitor cell (MPC)- and CdECM MF-laden anti-angiogenic bioink, revealing successful spatial confinement of the nascent vascular network only in the outer zone. Further, to co-facilitate both microvessel formation and MPC-derived matrix formation, we formulated cell culture medium conditions with a temporal switch. Overall, this study provides a new strategy that could be applied to develop zonal biomimetic meniscal constructs. Moreover, the use of ECM-derived MFs to promote or inhibit capillary networks opens new possibilities for the biofabrication of tissues with anisotropic microvascular distribution. These have potential for many applications including in vitro models of vascular-to-avascular tissue interfaces, cancer progression, and for testing anti-angiogenic therapies.
AUTHOR Browning, James R. and Derr, Paige and Derr, Kristy and Doudican, Nicole and Michael, Sam and Lish, Samantha R. and Taylor, Nicholas A. and Krueger, James G. and Ferrer, Marc and Carucci, John A. and Gareau, Daniel S.
Title A 3D biofabricated cutaneous squamous cell carcinoma tissue model with multi-channel confocal microscopy imaging biomarkers to quantify antitumor effects of chemotherapeutics in tissue [Abstract]
Year 2020
Journal/Proceedings Oncotarget; Vol 11, No 27
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// James R. Browning 1 , Paige Derr 2 , Kristy Derr 2 , Nicole Doudican 3 , Sam Michael 2 , Samantha R. Lish 1 , Nicholas A. Taylor 3 , James G. Krueger 1 , Marc Ferrer 2 , John A. Carucci 3 and Daniel S. Gareau 1 1 Laboratory for Investigative Dermatology, The Rockefeller University, New York, New York, USA 2 National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA 3 The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA Correspondence to: Daniel S. Gareau, email: dgareau@rockefeller.edu Keywords: squamous cell carcinoma; screening; 3D printing; in vitro model; confocal microscopy Received: January 05, 2020     Accepted: April 03, 2020     Published: July 07, 2020 ABSTRACT Cutaneous squamous cell carcinoma (cSCC) causes approximately 10,000 deaths annually in the U. S. Current therapies are largely ineffective against metastatic and locally advanced cSCC. There is a need to identify novel, effective, and less toxic small molecule cSCC therapeutics. We developed a 3-dimensional bioprinted skin (3DBPS) model of cSCC tumors together with a microscopy assay to test chemotherapeutic effects in tissue. The full thickness SCC tissue model was validated using hematoxylin and eosin (H&E) and immunohistochemical histological staining, confocal microscopy, and cDNA microarray analysis. A nondestructive, 3D fluorescence confocal imaging assay with tdTomato-labeled A431 SCC and ZsGreen-labeled keratinocytes was developed to test efficacy and general toxicity of chemotherapeutics. Fluorescence-derived imaging biomarkers indicated that 50% of cancer cells were killed in the tissue after 1?M 5-Fluorouracil 48-hour treatment, compared to a baseline of 12% for untreated controls. The imaging biomarkers also showed that normal keratinocytes were less affected by treatment (11% killed) than the untreated tissue, which had no significant killing effect. Data showed that 5-Fluorouracil selectively killed cSCC cells more than keratinocytes. Our 3DBPS assay platform provides cellular-level measurement of cell viability and can be adapted to achieve nondestructive high-throughput screening (HTS) in bio-fabricated tissues.
AUTHOR Noor, Nadav and Shapira, Assaf and Edri, Reuven and Gal, Idan and Wertheim, Lior and Dvir, Tal
Title 3D Printing of Personalized Thick and Perfusable Cardiac Patches and Hearts [Abstract]
Year 2019
Journal/Proceedings Advanced Science
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Abstract Generation of thick vascularized tissues that fully match the patient still remains an unmet challenge in cardiac tissue engineering. Here, a simple approach to 3D-print thick, vascularized, and perfusable cardiac patches that completely match the immunological, cellular, biochemical, and anatomical properties of the patient is reported. To this end, a biopsy of an omental tissue is taken from patients. While the cells are reprogrammed to become pluripotent stem cells, and differentiated to cardiomyocytes and endothelial cells, the extracellular matrix is processed into a personalized hydrogel. Following, the two cell types are separately combined with hydrogels to form bioinks for the parenchymal cardiac tissue and blood vessels. The ability to print functional vascularized patches according to the patient's anatomy is demonstrated. Blood vessel architecture is further improved by mathematical modeling of oxygen transfer. The structure and function of the patches are studied in vitro, and cardiac cell morphology is assessed after transplantation, revealing elongated cardiomyocytes with massive actinin striation. Finally, as a proof of concept, cellularized human hearts with a natural architecture are printed. These results demonstrate the potential of the approach for engineering personalized tissues and organs, or for drug screening in an appropriate anatomical structure and patient-specific biochemical microenvironment.
AUTHOR Derr, Kristy and Zou, Jinyun and Luo, Keren and Song, Min Jae and Sittampalam, G. Sitta and Zhou, Chao and Michael, Samuel and Ferrer, Marc and Derr, Paige
Title Fully 3D Bioprinted Skin Equivalent Constructs with Validated Morphology and Barrier Function [Abstract]
Year 2019
Journal/Proceedings Tissue Engineering Part C: Methods
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Development of high throughput, reproducible, three-dimensional bioprinted skin equivalents that are morphologically and functionally comparable to native skin tissue is advancing research in skin diseases, and providing a physiologically relevant platform for the development of therapeutics, transplants for regenerative medicine, and testing of skin products like cosmetics. Current protocols for the production of engineered skin rafts are limited in their ability to control three dimensional geometry of the structure and contraction leading to variability of skin function between constructs. Here we describe a method for the biofabrication of skin equivalents that are fully bioprinted using an open market bioprinter, made with commercially available primary cells and natural hydrogels. The unique hydrogel formulation allows for the production of a human-like skin equivalent with minimal lateral tissue contraction in a multiwell plate format, thus making them suitable for high throughput bioprinting in a single print with fast print and relatively short incubation times. The morphology and barrier function of the fully three-dimensional bioprinted skin equivalents are validated by immunohistochemistry staining, optical coherence tomography, and permeation assays.
AUTHOR 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 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 Cianciosi, Alessandro and Simon, Jonas and Bartolf-Kopp, Michael and Grausgruber, Heinrich and Dargaville, Tim R. and Forget, Aurélien and Groll, Jürgen and Jungst, Tomasz and Beaumont, Marco
Title Direct ink writing of multifunctional nanocellulose and allyl-modified gelatin biomaterial inks for the fabrication of mechanically and functionally graded constructs [Abstract]
Year 2023
Journal/Proceedings Carbohydrate Polymers
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Recreating the intricate mechanical and functional gradients found in natural tissues through additive manufacturing poses significant challenges, including the need for precise control over time and space and the availability of versatile biomaterial inks. In this proof-of-concept study, we developed a new biomaterial ink for direct ink writing, allowing the creation of 3D structures with tailorable functional and mechanical gradients. Our ink formulation combined multifunctional cellulose nanofibrils (CNFs), allyl-functionalized gelatin (0.8–2.0 wt%), and polyethylene glycol dithiol (3.0–7.5 wt%). The CNF served as a rheology modifier, whereas a concentration of 1.8 w/v % in the inks was chosen for optimal printability and shape fidelity. In addition, CNFs were functionalized with azido groups, enabling the spatial distribution of functional moieties within a 3D structure. These functional groups were further modified using a spontaneous click chemistry reaction. Through additive manufacturing and a readily available static mixer, we successfully demonstrated the fabrication of mechanical gradients – ranging from 3 to 6 kPa in indentation strength – and functional gradients. Additionally, we introduced dual gradients by combining gradient printing with an anisotropic photocrosslinking step. The developed biomaterial ink opens up possibilities for printing intricate multigradient structures, resembling the complex hierarchical organization seen in living tissues.
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 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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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 Züger, Fabian and Berner, Natascha and Gullo, Maurizio R.
Title Towards a Novel Cost-Effective and Versatile Bioink for 3D-Bioprinting in Tissue Engineering [Abstract]
Year 2023
Journal/Proceedings Biomimetics
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3D-bioprinting for tissue regeneration relies on, among other things, hydrogels with favorable rheological properties. These include shear thinning for cell-friendly extrusion, post-printing structural stability as well as physiologically relevant elastic moduli needed for optimal cell attachment, proliferation, differentiation and tissue maturation. This work introduces a cost-efficient gelatin-methylcellulose based hydrogel whose rheological properties can be independently optimized for optimal printability and tissue engineering. Hydrogel viscosities were designed to present three different temperature regimes: low viscosity for eased cell suspension and printing with minimal shear stress, form fidelity directly after printing and long term structural stability during incubation. Enzymatically crosslinked hydrogel scaffolds with stiffnesses ranging from 5 to 50 kPa were produced, enabling the hydrogel to biomimic cell environments for different types of tissues. The bioink showed high intrinsic cytocompatibility and tissues fabricated by embedding and bioprinting NIH 3T3 fibroblasts showed satisfactory viability. This novel hydrogel uses robust and inexpensive technology, which can be adjusted for implementation in tissue regeneration, e.g., in myocardial or neural tissue engineering.
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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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 Pitacco, Pierluca and Sadowska, Joanna M. and O'Brien, Fergal J. and Kelly, Daniel J.
Title 3D bioprinting of cartilaginous templates for large bone defect healing [Abstract]
Year 2022
Journal/Proceedings Acta Biomaterialia
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Damaged or diseased bone can be treated using autografts or a range of different bone grafting biomaterials, however limitations with such approaches has motivated increased interest in developmentally inspired bone tissue engineering (BTE) strategies that seek to recapitulate the process of endochondral ossification (EO) as a means of regenerating critically sized defects. The clinical translation of such strategies will require the engineering of scaled-up, geometrically defined hypertrophic cartilage grafts that can be rapidly vascularised and remodelled into bone in mechanically challenging defect environments. The goal of this study was to 3D bioprint mechanically reinforced cartilaginous templates and to assess their capacity to regenerate critically sized femoral bone defects. Human mesenchymal stem/stromal cells (hMSCs) were incorporated into fibrin based bioinks and bioprinted into polycaprolactone (PCL) frameworks to produce mechanically reinforced constructs. Chondrogenic priming of such hMSC laden constructs was required to support robust vascularisation and graft mineralisation in vivo following their subcutaneous implantation into nude mice. With a view towards maximising their potential to support endochondral bone regeneration, we next explored different in vitro culture regimes to produce chondrogenic and early hypertrophic engineered grafts. Following their implantation into femoral bone defects within transiently immunosuppressed rats, such bioprinted constructs were rapidly remodelled into bone in vivo, with early hypertrophic constructs supporting higher levels of vascularisation and bone formation compared to the chondrogenic constructs. Such early hypertrophic bioprinted constructs also supported higher levels of vascularisation and spatially distinct patterns of new formation compared to BMP-2 loaded collagen scaffolds (here used as a positive control). In conclusion, this study demonstrates that fibrin based bioinks support chondrogenesis of hMSCs in vitro, which enables the bioprinting of mechanically reinforced hypertrophic cartilaginous templates capable of supporting large bone defect regeneration. These results support the use of 3D bioprinting as a strategy to scale-up the engineering of developmentally inspired templates for BTE. Statement of significance Despite the promise of developmentally inspired tissue engineering strategies for bone regeneration, there are still challenges that need to be addressed to enable clinical translation. This work reports the development and assessment (in vitro and in vivo) of a 3D bioprinting strategy to engineer mechanically-reinforced cartilaginous templates for large bone defect regeneration using human MSCs. Using distinct in vitro priming protocols, it was possible to generate cartilage grafts with altered phenotypes. More hypertrophic grafts, engineered in vitro using TGF-β3 and BMP-2, supported higher levels of blood vessel infiltration and accelerated bone regeneration in vivo. This study also identifies some of the advantages and disadvantages of such endochondral bone TE strategies over the direct delivery of BMP-2 from collagen-based scaffolds.
AUTHOR Kuthe, Sudhanshu and Schlothauer, Arthur and Bodkhe, Sampada and Hulme, Christopher and Ermanni, Paolo
Title 3D printed mechanically representative aortic model made of gelatin fiber reinforced silicone composite [Abstract]
Year 2022
Journal/Proceedings Materials Letters
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Additive manufacturing (AM) is a useful technology to produce artificial aortic models for the training of transcatheter aortic valve replacement (TAVR) surgery. With AM, the models can be tailored towards the individualized aortic anatomy of patients. Most of these reported models so far are manufactured using single rubber-like materials. However, such materials do not replicate the mechanical properties of natural aortic tissue, especially the stress–strain response in higher strain (>0.1) regions. This could be problematic for surgeons training for surgeries using a model which does not exhibit properties of the real aorta. To overcome this limitation, we developed a 3D-printed, mechanically representative aortic model comprising gelatin fibers and silicone. The model is promising as a realistic analog of aortic sinus for mock TAVR surgery. Computerized tomography data was analyzed beforehand using medical imaging to identify the anatomy of a specific patient’s aortic sinus and the surrounding blood vessels. A novel silicone matrix composite reinforced with gelatin fibers designed in this work was tested and compared with the stress–strain response of aortic tissue. Such a model comprising both patient-specific geometries as well as realistic material properties of aortic tissue can be helpful for the development of next-generation medical phantoms.
AUTHOR Kitana, Waseem and Apsite, Indra and Hazur, Jonas and Boccaccini, Aldo R. and Ionov, Leonid
Title 4D Biofabrication of T-Shaped Vascular Bifurcation [Abstract]
Year 2022
Journal/Proceedings Advanced Materials Technologies
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Abstract 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 Dorjsuren, Dorjbal and Eastman, Richard T. and Song, Min Jae and Yasgar, Adam and Chen, Yuchi and Bharti, Kapil and Zakharov, Alexey V. and Jadhav, Ajit and Ferrer, Marc and Shi, Pei-Yong and Simeonov, Anton
Title A platform of assays for the discovery of anti-Zika small-molecules with activity in a 3D-bioprinted outer-blood-retina model [Abstract]
Year 2022
Journal/Proceedings PLOS ONE
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The global health emergency posed by the outbreak of Zika virus (ZIKV), an arthropod-borne flavivirus causing severe neonatal neurological conditions, has subsided, but there continues to be transmission of ZIKV in endemic regions. As such, there is still a medical need for discovering and developing therapeutical interventions against ZIKV. To identify small-molecule compounds that inhibit ZIKV disease and transmission, we screened multiple small-molecule collections, mostly derived from natural products, for their ability to inhibit wild-type ZIKV. As a primary high-throughput screen, we used a viral cytopathic effect (CPE) inhibition assay conducted in Vero cells that was optimized and miniaturized to a 1536-well format. Suitably active compounds identified from the primary screen were tested in a panel of orthogonal assays using recombinant Zika viruses, including a ZIKV Renilla luciferase reporter assay and a ZIKV mCherry reporter system. Compounds that were active in the wild-type ZIKV inhibition and ZIKV reporter assays were further evaluated for their inhibitory effects against other flaviviruses. Lastly, we demonstrated that wild-type ZIKV is able to infect a 3D-bioprinted outer-blood-retina barrier tissue model and disrupt its barrier function, as measured by electrical resistance. One of the identified compounds (3-Acetyl-13-deoxyphomenone, NCGC00380955) was able to prevent the pathological effects of the viral infection on this clinically relevant ZIKV infection model.
AUTHOR Cakal, Selgin D. and Radeke, Carmen and Alcala, Juan F. and Ellman, Ditte G. and Butdayev, Sarkhan and Andersen, Ditte C. and Calloe, Kirstine and Lind, Johan U.
Title A simple and scalable 3D printing methodology for generating aligned and extended human and murine skeletal muscle tissues [Abstract]
Year 2022
Journal/Proceedings Biomedical Materials
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Preclinical biomedical and pharmaceutical research on disease causes, drug targets, and side effects increasingly relies on in vitro models of human tissue. 3D printing offers unique opportunities for generating models of superior physiological accuracy, as well as for automating their fabrication. Towards these goals, we here describe a simple and scalable methodology for generating physiologically relevant models of skeletal muscle. Our approach relies on dual-material micro-extrusion of two types of gelatin hydrogel into patterned soft substrates with locally alternating stiffness. We identify minimally complex patterns capable of guiding the large-scale self-assembly of aligned, extended, and contractile human and murine skeletal myotubes. Interestingly, we find high-resolution patterning is not required, as even patterns with feature sizes of several hundred micrometers is sufficient. Consequently, the procedure is rapid and compatible with any low-cost extrusion-based 3D printer. The generated myotubes easily span several millimeters, and various myotube patterns can be generated in a predictable and reproducible manner. The compliant nature and adjustable thickness of the hydrogel substrates, serves to enable extended culture of contractile myotubes. The method is further readily compatible with standard cell-culturing platforms as well as commercially available electrodes for electrically induced exercise and monitoring of the myotubes.
AUTHOR Anand, Resmi and Amoli, Mehdi Salar and Huysecom, An-Sofie and Amorim, Paulo Alexandre and Agten, Hannah and Geris, Liesbet and Bloemen, Veerle
Title A tunable gelatin-hyaluronan dialdehyde/methacryloyl gelatin interpenetrating polymer network hydrogel for additive tissue manufacturing [Abstract]
Year 2022
Journal/Proceedings Biomedical Materials
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Abstract
Methacryloyl gelatin (GelMA) is a versatile material for bioprinting because of its tunable physical properties and inherent bioactivity. Bioprinting of GelMA is often met with challenges such as lower viscosity of GelMA inks due to higher methacryloyl substitution and longer physical gelation time at room temperature. In this study, a tunable interpenetrating polymer network (IPN) hydrogel was prepared from gelatin-hyaluronan dialdehyde (Gel-HDA) Schiff’s polymer, and 100% methacrylamide substituted GelMA for biofabrication through extrusion based bioprinting. Temperature sweep rheology measurements show a higher sol-gel transition temperature for IPN (30 °C) compared to gold standard GelMA (27 °C). Furthermore, to determine the tunability of the IPN hydrogel, several IPN samples were prepared by combining different ratios of Gel-HDA and GelMA achieving a compressive modulus ranging from 20.6 ± 2.48 KPa to 116.7 ± 14.80 KPa. Our results showed that the mechanical properties and printability at room temperature could be tuned by adjusting the ratios of GelMA and Gel-HDA. To evaluate cell response to the material, MC3T3-E1 mouse pre-osteoblast cells were embedded in hydrogels and 3D-printed, demonstrating excellent cell viability and proliferation after 10 d of 3D in vitro culture, making the IPN an interesting bioink for the fabrication of 3D constructs for tissue engineering applications.
AUTHOR Song, Min Jae and Quinn, Russ and Nguyen, Eric and Hampton, Christopher and Sharma, Ruchi and Park, Tea Soon and Koster, Céline and Voss, Ty and Tristan, Carlos and Weber, Claire and Singh, Anju and Dejene, Roba and Bose, Devika and Chen, Yu-Chi and Derr, Paige and Derr, Kristy and Michael, Sam and Barone, Francesca and Chen, Guibin and Boehm, Manfred and Maminishkis, Arvydas and Singec, Ilyas and Ferrer, Marc and Bharti, Kapil
Title Bioprinted 3D outer retina barrier uncovers RPE-dependent choroidal phenotype in advanced macular degeneration [Abstract]
Year 2022
Journal/Proceedings Nature Methods
Reftype Song2022
DOI/URL DOI
Abstract
Age-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retina-barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch’s membrane, and choriocapillaris. The mechanisms of AMD initiation and progression remain poorly understood owing to the lack of physiologically relevant human oBRB models. To this end, we engineered a native-like three-dimensional (3D) oBRB tissue (3D-oBRB) by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB model, a fully-polarized RPE monolayer provides barrier resistance, induces choriocapillaris fenestration, and supports the formation of Bruch’s-membrane-like structure by inducing changes in gene expression in cells of the choroid. Complement activation in the 3D-oBRB triggers dry AMD phenotypes (including subRPE lipid-rich deposits called drusen and choriocapillaris degeneration), and HIF-α stabilization or STAT3 overactivation induce choriocapillaris neovascularization and type-I wet AMD phenotype. The 3D-oBRB provides a physiologically relevant model to studying RPE-choriocapillaris interactions under healthy and diseased conditions.
AUTHOR Blanco-Fernandez, Barbara and Rey-Vinolas, Sergi and Bağcı, Gülsün and Rubi-Sans, Gerard and Otero, Jorge and Navajas, Daniel and Perez-Amodio, Soledad and Engel, Elisabeth
Title Bioprinting Decellularized Breast Tissue for the Development of Three-Dimensional Breast Cancer Models [Abstract]
Year 2022
Journal/Proceedings ACS Appl. Mater. Interfaces
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DOI/URL DOI
Abstract
The tumor extracellular matrix (ECM) plays a vital role in tumor progression and drug resistance. Previous studies have shown that breast tissue-derived matrices could be an important biomaterial to recreate the complexity of the tumor ECM. We have developed a method for decellularizing and delipidating a porcine breast tissue (TDM) compatible with hydrogel formation. The addition of gelatin methacrylamide and alginate allows this TDM to be bioprinted by itself with good printability, shape fidelity, and cytocompatibility. Furthermore, this bioink has been tuned to more closely recreate the breast tumor by incorporating collagen type I (Col1). Breast cancer cells (BCCs) proliferate in both TDM bioinks forming cell clusters and spheroids. The addition of Col1 improves the printability of the bioink as well as increases BCC proliferation and reduces doxorubicin sensitivity due to a downregulation of HSP90. TDM bioinks also allow a precise three-dimensional printing of scaffolds containing BCCs and stromal cells and could be used to fabricate artificial tumors. Taken together, we have proven that these novel bioinks are good candidates for biofabricating breast cancer models.
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 Ramakrishnan, Rashmi and Kasoju, Naresh and Raju, Riya and Geevarghese, Rency and Gauthaman, Ashna and Bhatt, Anugya
Title Exploring the Potential of Alginate-Gelatin-Diethylaminoethyl Cellulose-Fibrinogen based Bioink for 3D Bioprinting of Skin Tissue Constructs [Abstract]
Year 2022
Journal/Proceedings Carbohydrate Polymer Technologies and Applications
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DOI/URL URL DOI
Abstract
Designing printable bioinks for 3D bioprinting capable of supporting cellular viability with post-printing functionality remains challenging. Native ECM offers several physical, chemical, and biological cues that are difficult to restore using only a single component. Herein, we have optimized a multicomponent-based bioink formulation comprising alginate (ALG), gelatin (GEL), diethylaminoethyl cellulose (DCEL) and fibrinogen (FIB), termed as ALG-GEL-DCEL-FIB bioink for potential application in bioprinting and biofabrication of skin tissue equivalents. The designed formulation was extensively studied for its printability, physico-chemical, rheological, and biocompatibility properties. Excellent printability, shape fidelity and cell-laden tissue equivalent printing were established using the RegenHu 3D Discovery Bioprinter. The human primary fibroblast and keratinocyte-laden bioprinted constructs exhibited good cell viability. Long term culture of 4 weeks comprising 5 days of air-liquid-interphase followed by 21 days of submerged culture produced biomimetic tissue histology in the ALG-GEL-DCEL-FIB bioink printed constructs. Specific epidermal-dermal marker expressions proving functionality were evident in immunohistochemical, biochemical and gene expression analysis. The ALG-GEL-DCEL-FIB bioink may be explored further for potential biofabrication and therapeutic applications.
AUTHOR Rahimnejad, Maedeh and Adoungotchodo, Atma and Demarquette, Nicole R. and Lerouge, Sophie
Title FRESH bioprinting of biodegradable chitosan thermosensitive hydrogels [Abstract]
Year 2022
Journal/Proceedings Bioprinting
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DOI/URL URL DOI
Abstract
Thermosensitive chitosan (CH)-based hydrogels prepared with a mix of sodium bicarbonate and β-glycerophosphate as gelling agents rapidly pass from a liquid at room temperature to a mechanically strong solid at body temperature without any crosslinker. They show excellent potential for tissue engineering applications and could be interesting candidates for bioprinting. Unfortunately, since gelation is not instantaneous, formulations compatible with cell encapsulation (chitosan concentrations around 2% or lower) lead to very poor resolution and fidelity due to filament spreading. Here, we investigate the FRESH bioprinting approach with a warm sacrificial support bath, to overcome these limitations and enhance their bioprintability. First, a support bath, made of Pluronic including sodium chloride salt as a rheology modifier agent, was designed to meet the specific physical state requirements (solid at 37 °C and liquid at room temperature) and rheological properties appropriate for bioprinting. This support bath presented yield stress of over 100 Pa, a shear thinning behavior, and fast self-healing during cyclic recovery tests. Three different chitosan hydrogels (CH2%w/v, CH3%w/v, and a mixture of CH and gelatin) were tested for their ability to form filament and 3D structures, with and without a support bath. Both the resolution and mechanical properties of the printed structure were drastically enhanced using the FRESH method, with an approximate four fold decrease of the filament diameter which is close to the needle diameter. The printed structures were easily harvested without altering their shape by cooling down the support bath, and do not swell when immersed in PBS. Live/dead assays confirmed that the viability of encapsulated mesenchymal stem cells was highest in CH2% and that the support bath-assisted bioprinting process did not adversely impact cell viability. This study demonstrates that using a warm FRESH-like approach drastically enhances the potential for bioprinting of the thermosensitive biodegradable chitosan hydrogels and opens up a wide range of applications for 3D models and tissue engineering.
AUTHOR Lai, Jiahui and Wang, Chong and Liu, Jia and Chen, Shangsi and Liu, Chaoyu and Huang, Xiangxuan and Wu, Jing and Pan, Yue and Xie, Yuancai and Wang, Min
Title Low temperature hybrid 3D printing of hierarchically porous bone tissue engineering scaffolds with in situ delivery of osteogenic peptide and mesenchymal stem cells [Abstract]
Year 2022
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
Compared to other conventional scaffold fabrication techniques, three-dimensional (3D) printing is advantageous in producing bone tissue engineering scaffolds with customized shape, tailored pore size/porosity, required mechanical properties and even desirable biomolecule delivery capability. However, for scaffolds with a large volume, it is highly difficult to get seeded cells to migrate to the central region of the scaffolds, resulting in an inhomogeneous cell distribution and therefore lowering the bone forming ability. To overcome this major obstacle, in this study, cell-laden bone tissue engineering scaffolds consisting of osteogenic peptide (OP) loaded β-tricalcium phosphate (TCP)/poly(lactic-co-glycolic acid) (PLGA) (OP/TCP/PLGA, designated as OTP) nanocomposite struts and rat bone marrow derived mesenchymal stem cell (rBMSC)-laden gelatin/GelMA hydrogel rods were produced through ‘dual-nozzle’ low temperature hybrid 3D printing. The cell-laden scaffolds exhibited a bi-phasic structure and had a mechanical modulus of about 19.6 MPa, which was similar to that of human cancellous bone. OP can be released from the hybrid scaffolds in a sustained manner and achieved a cumulative release level of about 78% after 24 d. rBMSCs encapsulated in the hydrogel rods exhibited a cell viability of about 87.4% right after low temperature hybrid 3D printing and could be released from the hydrogel rods to achieve cell anchorage on the surface of adjacent OTP struts. The OP released from OTP struts enhanced rBMSCs proliferation. Compared to rBMSC-laden hybrid scaffolds without OP incorporation, the rBMSC-laden hybrid scaffolds incorporated with OP significantly up-regulated osteogenic differentiation of rBMSCs by showing a higher level of alkaline phosphatase expression and calcium deposition. This ‘proof-of-concept’ study has provided a facile method to form cell-laden bone tissue engineering scaffolds with not only required mechanical strength, biomimetic structure and sustained biomolecule release profile but also excellent cell delivery capability with uniform cell distribution, which can improve the bone forming ability in the body.
AUTHOR Sarmin, Atiya M. and El Moussaid, Nadia and Suntornnond, Ratima and Tyler, Eleanor J. and Kim, Yang-Hee and Di Cio, Stefania and Megone, William V. and Pearce, Oliver and Gautrot, Julien E. and Dawson, Jonathan and Connelly, John T.
Title Multi-Scale Analysis of the Composition, Structure, and Function of Decellularized Extracellular Matrix for Human Skin and Wound Healing Models [Abstract]
Year 2022
Journal/Proceedings Biomolecules
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Abstract
The extracellular matrix (ECM) is a complex mixture of structural proteins, proteoglycans, and signaling molecules that are essential for tissue integrity and homeostasis. While a number of recent studies have explored the use of decellularized ECM (dECM) as a biomaterial for tissue engineering, the complete composition, structure, and mechanics of these materials remain incompletely understood. In this study, we performed an in-depth characterization of skin-derived dECM biomaterials for human skin equivalent (HSE) models. The dECM materials were purified from porcine skin, and through mass spectrometry profiling, we quantified the presence of major ECM molecules, including types I, III, and VI collagen, fibrillin, and lumican. Rheological analysis demonstrated the sol-gel and shear-thinning properties of dECM materials, indicating their physical suitability as a tissue scaffold, while electron microscopy revealed a complex, hierarchical structure of nanofibers in dECM hydrogels. The dECM materials were compatible with advanced biofabrication techniques, including 3D printing within a gelatin microparticle support bath, printing with a sacrificial material, or blending with other ECM molecules to achieve more complex compositions and structures. As a proof of concept, we also demonstrate how dECM materials can be fabricated into a 3D skin wound healing model using 3D printing. Skin-derived dECM therefore represents a complex and versatile biomaterial with advantageous properties for the fabrication of next-generation HSEs.
AUTHOR Pai, Roopesh R. and Ajit, Shilpa and Sekar J, Anupama and Nair, Sarath S. and Anil Kumar, P. R. and Velayudhan, Shiny
Title Radical scavenging gelatin methacrylamide based bioink formulation for three dimensional bioprinting of parenchymal liver construct [Abstract]
Year 2022
Journal/Proceedings Bioprinting
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DOI/URL URL DOI
Abstract
Methacrylated gelatin (GelMA) in the form of methacryloyl, methacrylate, and methacrylamide is an established and widely accepted photocrosslinkable bioink, for three dimensional bioprinting of various tissues. One of the limitations of photocrosslinkable bioinks is the inability to control the free radicals generated by photoinitiators and ultraviolet (UV) rays. The presence of excess free radicals compromises the viability and functionality of cells during crosslinking. In this study, ascorbic acid, a known free radical scavenger (FRS) molecule, was introduced into the GelMA bioink formulation to protect the cell viability, proliferation, and tissue functions of 3D bioprinted parenchymal liver constructs. The concentration of FRS in the bioink was optimized and used for 3D bioprinting of HepG2 cells. The results confirmed that the inclusion of 3.4 mM FRS in the GelMA bioink formulation nullified the excess ROS formed inside the cells. Furthermore, the optimized GelMA formulation containing FRS preserved and improved the cell activity, albumin, and urea synthesis in the 3D construct over 7 days in culture. In the future, this concept could be implemented in the biofabrication of large liver constructs that require multiple or longer durations of UV irradiation.
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 Salar Amoli, Mehdi and Anand, Resmi and EzEldeen, Mostafa and Amorim, Paulo Alexandre and Geris, Liesbet and Jacobs, Reinhilde and Bloemen, Veerle
Title The development of a 3D printable chitosan-based copolymer with tunable properties for dentoalveolar regeneration [Abstract]
Year 2022
Journal/Proceedings Carbohydrate Polymers
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DOI/URL URL DOI
Abstract
Dentoalveolar tissue engineering is an emerging yet challenging field, considering the lack of suitable materials and difficulty to produce patient-specific hydrogel scaffolds. The present paper aims to produce a 3D printable and tuneable biomaterial by copolymerizing a synthesized water-soluble chitosan derivative called maleic anhydride grafted chitosan (MA-C) with gelatin using genipin, a natural crosslinking agent. Development and testing of this material for 3D printing, degradation, and swelling demonstrated the ability to fabricate scaffolds with controlled physical properties based on pre-determined designs. The MA-C-gelatin copolymer demonstrated excellent biocompatibility, which was verified by analyzing the viability, growth and proliferation of human dental pulp stem cells seeded on MA-C-gelatin constructs through live/dead, alamar blue and DNA quantification assays. Based on the present findings, the proposed material might be a suitable candidate for dentoalveolar tissue engineering, while further research is required to achieve this goal.
AUTHOR Yu, Haiyang and Gong, Wen and Mei, Junhao and Qin, Lihao and Piao, Zeyu and You, Deshu and Gu, Wenxian and Jia, Zhongzhi
Title The efficacy of a paeoniflorin-sodium alginate-gelatin skin scaffold for the treatment of diabetic wound: An in vivo study in a rat model [Abstract]
Year 2022
Journal/Proceedings Biomedicine & Pharmacotherapy
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Abstract
Objective To investigate the efficacy of a paeoniflorin-sodium alginate (SA)-gelatin skin scaffold for treating diabetic wound in a rat model. Methods Bioinks were prepared using various percentages of paeoniflorin in the total weight of a solution containing SA and gelatin. Skin scaffolds containing 0%, 1%, 3%, 5%, and 10% paeoniflorin were printed using 3D bioprinting technology, and scaffold microstructure was observed with scanning electron microscopy. Skin scaffolds were then used in rats with diabetic wounds. H&E staining, Masson staining, and immunohistochemical staining for IL-1β and CD31 were performed on days 7 and 14. Results All skin scaffolds had a mesh-like structure with uniform pore distribution. Wounds healed well in each group, with the 1% and 3% groups demonstrating the most complete healing. H&E staining showed that skin accessory organs had appeared in each group. On day 7, collagen deposition in the 3% group was higher than in the other groups (P<0.05), and IL-1β infiltration was lower in the 10% group than in the 3% group (P = 0.002). On day 14, IL-1β infiltration was not significantly different between the 10% and 3% groups (P = 0.078). The CD31 level was higher in the 3% group than in the other groups on days 7 and 14 (P<0.05). Conclusion A 3% paeoniflorin-SA-gelatin skin scaffold promoted the healing of diabetic wounds in rats. This scaffold promoted collagen deposition and microvascular regeneration and demonstrated anti-inflammatory properties, suggesting that this scaffold type could be used to treat diabetic wounds.
AUTHOR 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 Nulty, Jessica and Burdis, Ross and Kelly, Daniel J.
Title Biofabrication of Prevascularised Hypertrophic Cartilage Microtissues for Bone Tissue Engineering [Abstract]
Year 2021
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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DOI/URL DOI
Abstract
Bone tissue engineering (TE) has the potential to transform the treatment of challenging musculoskeletal pathologies. To date, clinical translation of many traditional TE strategies has been impaired by poor vascularisation of the implant. Addressing such challenges has motivated research into developmentally inspired TE strategies, whereby implants mimicking earlier stages of a tissue’s development are engineered in vitro and then implanted in vivo to fully mature into the adult tissue. The goal of this study was to engineer in vitro tissues mimicking the immediate developmental precursor to long bones, specifically a vascularised hypertrophic cartilage template, and to then assess the capacity of such a construct to support endochondral bone formation in vivo. To this end, we first developed a method for the generation of large numbers of hypertrophic cartilage microtissues using a microwell system, and encapsulated these microtissues into a fibrin-based hydrogel capable of supporting vasculogenesis by human umbilical vein endothelial cells (HUVECs). The microwells supported the formation of bone marrow derived stem/stromal cell (BMSC) aggregates and their differentiation toward a hypertrophic cartilage phenotype over 5 weeks of cultivation, as evident by the development of a matrix rich in sulphated glycosaminoglycan (sGAG), collagen types I, II, and X, and calcium. Prevascularisation of these microtissues, undertaken in vitro 1 week prior to implantation, enhanced their capacity to mineralise, with significantly higher levels of mineralised tissue observed within such implants after 4 weeks in vivo within an ectopic murine model for bone formation. It is also possible to integrate such microtissues into 3D bioprinting systems, thereby enabling the bioprinting of scaled-up, patient-specific prevascularised implants. Taken together, these results demonstrate the development of an effective strategy for prevascularising a tissue engineered construct comprised of multiple individual microtissue “building blocks,” which could potentially be used in the treatment of challenging bone defects.
AUTHOR 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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DOI/URL URL DOI
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 Trucco, Diego and Sharma, Aarushi and Manferdini, Cristina and Gabusi, Elena and Petretta, Mauro and Desando, Giovanna and Ricotti, Leonardo and Chakraborty, Juhi and Ghosh, Sourabh and Lisignoli, Gina
Title Modeling and Fabrication of Silk Fibroin-Gelatin-Based Constructs Using Extrusion-Based Three-Dimensional Bioprinting [Abstract]
Year 2021
Journal/Proceedings ACS Biomater. Sci. Eng.
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DOI/URL DOI
Abstract
Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure’s fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs’ fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications. Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure’s fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs’ fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications.
AUTHOR Chawla, Shikha and Desando, Giovanna and Gabusi, Elena and Sharma, Aarushi and Trucco, Diego and Chakraborty, Juhi and Manferdini, Cristina and Petretta, Mauro and Lisignoli, Gina and Ghosh, Sourabh
Title The effect of silk-gelatin bioink and TGF-β3 on mesenchymal stromal cells in 3D bioprinted chondrogenic constructs: A proteomic study [Abstract]
Year 2021
Journal/Proceedings Journal of Materials Research
Reftype Chawla2021
DOI/URL DOI
Abstract
Major limitation of 3D bioprinting is the poor understanding of the role of bioink in modulating molecular signaling pathways. Phenotypically stable engineered articular cartilage was fabricated using silk fibroin-gelatin (SF-G) bioink and progenitor cells or mature articular chondrocytes. In the current study, role of SF-G bioink in modulating in vitro chondrogenic signaling pathways in human bone marrow-derived stromal cells (hMSCs) is elucidated. The interaction between SF-G bioink and hMSCs augmented several chondrogenic pathways, including Wnt, HIF-1, and Notch. We explored the debatable role of TGF-β signaling, by assessing the differential protein expression by hMSCs-laden bioprinted constructs in the presence and absence of TGF-β3. hMSCs-laden bioprinted constructs contained a large percentage of collagen type II and Filamin-B, typical to the native articular cartilage. Hypertrophy markers were not identified following TGF-β3 addition. This is first detailed proteomics analysis to identify articular cartilage-specific pathways in SF-G-based 3D bioprinted construct.
AUTHOR Göckler, Tobias and Haase, Sonja and Kempter, Xenia and Pfister, Rebecca and Maciel, Bruna R. and Grimm, Alisa and Molitor, Tamara and Willenbacher, Norbert and Schepers, Ute
Title Tuning Superfast Curing Thiol-Norbornene-Functionalized Gelatin Hydrogels for 3D Bioprinting [Abstract]
Year 2021
Journal/Proceedings Advanced Healthcare Materials
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DOI/URL DOI
Abstract
Abstract Photocurable gelatin-based hydrogels have established themselves as powerful bioinks in tissue engineering due to their excellent biocompatibility, biodegradability, light responsiveness, thermosensitivity and bioprinting properties. While gelatin methacryloyl (GelMA) has been the gold standard for many years, thiol-ene hydrogel systems based on norbornene-functionalized gelatin (GelNB) and a thiolated crosslinker have recently gained increasing importance. In this paper, a highly reproducible water-based synthesis of GelNB is presented, avoiding the use of dimethyl sulfoxide (DMSO) as organic solvent and covering a broad range of degrees of functionalization (DoF: 20% to 97%). Mixing with thiolated gelatin (GelS) results in the superfast curing photoclick hydrogel GelNB/GelS. Its superior properties over GelMA, such as substantially reduced amounts of photoinitiator (0.03% (w/v)), superfast curing (1–2 s), higher network homogeneity, post-polymerization functionalization ability, minimal cross-reactivity with cellular components, and improved biocompatibility of hydrogel precursors and degradation products lead to increased survival of primary cells in 3D bioprinting. Post-printing viability analysis revealed excellent survival rates of > 84% for GelNB/GelS bioinks of varying crosslinking density, while cell survival for GelMA bioinks is strongly dependent on the DoF. Hence, the semisynthetic and easily accessible GelNB/GelS hydrogel is a highly promising bioink for future medical applications and other light-based biofabrication techniques.
AUTHOR Somasekharan, Lakshmi and Kasoju, Naresh and Raju, Riya and Bhatt, Anugya
Title Formulation and Characterization of Alginate Dialdehyde, Gelatin, and Platelet-Rich Plasma-Based Bioink for Bioprinting Applications [Abstract]
Year 2020
Journal/Proceedings Bioengineering
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Abstract
Layer-by-layer additive manufacturing process has evolved into three-dimensional (3D) “bio-printing” as a means of constructing cell-laden functional tissue equivalents. The process typically involves the mixing of cells of interest with an appropriate hydrogel, termed as “bioink”, followed by printing and tissue maturation. An ideal bioink should have adequate mechanical, rheological, and biological features of the target tissues. However, native extracellular matrix (ECM) is made of an intricate milieu of soluble and non-soluble extracellular factors, and mimicking such a composition is challenging. To this end, here we report the formulation of a multi-component bioink composed of gelatin and alginate -based scaffolding material, as well as a platelet-rich plasma (PRP) suspension, which mimics the insoluble and soluble factors of native ECM respectively. Briefly, sodium alginate was subjected to controlled oxidation to yield alginate dialdehyde (ADA), and was mixed with gelatin and PRP in various volume ratios in the presence of borax. The formulation was systematically characterized for its gelation time, swelling, and water uptake, as well as its morphological, chemical, and rheological properties; furthermore, blood- and cytocompatibility were assessed as per ISO 10993 (International Organization for Standardization). Printability, shape fidelity, and cell-laden printing was evaluated using the RegenHU 3D Discovery bioprinter. The results indicated the successful development of ADA–gelatin–PRP based bioink for 3D bioprinting and biofabrication applications.
AUTHOR Figueiredo, Lara and Le Visage, Catherine and Weiss, Pierre and Yang, Jing
Title Quantifying Oxygen Levels in 3D Bioprinted Cell-Laden Thick Constructs with Perfusable Microchannel Networks [Abstract]
Year 2020
Journal/Proceedings Polymers
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Abstract
The survival and function of thick tissue engineered implanted constructs depends on pre-existing, embedded, functional, vascular-like structures that are able to integrate with the host vasculature. Bioprinting was employed to build perfusable vascular-like networks within thick constructs. However, the improvement of oxygen transportation facilitated by these vascular-like networks was directly quantified. Using an optical fiber oxygen sensor, we measured the oxygen content at different positions within 3D bioprinted constructs with and without perfusable microchannel networks. Perfusion was found to play an essential role in maintaining relatively high oxygen content in cell-laden constructs and, consequently, high cell viability. The concentration of oxygen changes following switching on and off the perfusion. Oxygen concentration depletes quickly after pausing perfusion but recovers rapidly after resuming the perfusion. The quantification of oxygen levels within cell-laden hydrogel constructs could provide insight into channel network design and cellular responses.
AUTHOR Mestre, Rafael and Patiño, Tania and Barceló, Xavier and Anand, Shivesh and Pérez-Jiménez, Ariadna and Sánchez, Samuel
Title Force Modulation and Adaptability of 3D-Bioprinted Biological Actuators Based on Skeletal Muscle Tissue [Abstract]
Year 2019
Journal/Proceedings Advanced Materials Technologies
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DOI/URL DOI
Abstract
Abstract The integration of biological systems into robotic devices might provide them with capabilities acquired from natural systems and significantly boost their performance. These abilities include real-time bio-sensing, self-organization, adaptability, or self-healing. As many muscle-based bio-hybrid robots and bio-actuators arise in the literature, the question of whether these features can live up to their expectations becomes increasingly substantial. Herein, the force generation and adaptability of skeletal-muscle-based bio-actuators undergoing long-term training protocols are analyzed. The 3D-bioprinting technique is used to fabricate bio-actuators that are functional, responsive, and have highly aligned myotubes. The bio-actuators are 3D-bioprinted together with two artificial posts, allowing to use it as a force measuring platform. In addition, the force output evolution and dynamic gene expression of the bio-actuators are studied to evaluate their degree of adaptability according to training protocols of different frequencies and mechanical stiffness, finding that their force generation could be modulated to different requirements. These results shed some light into the fundamental mechanisms behind the adaptability of muscle-based bio-actuators and highlight the potential of using 3D bioprinting as a rapid and cost-effective tool for the fabrication of custom-designed soft bio-robots.
AUTHOR Sharma, Aarushi and Desando, Giovanna and Petretta, Mauro and Chawla, Shikha and Bartolotti, Isabella and Manferdini, Cristina and Paolella, Francesca and Gabusi, Elena and Trucco, Diego and Ghosh, Sourabh and Lisignoli, Gina
Title Investigating the Role of Sustained Calcium Release in Silk-Gelatin-Based Three-Dimensional Bioprinted Constructs for Enhancing the Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stromal Cells
Year 2019
Journal/Proceedings ACS Biomaterials Science & Engineering
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AUTHOR Xu, Yichi and Peng, Jiang and Richards, Geoff and Lu, Shibi and Eglin, David
Title Optimization of electrospray fabrication of stem cell–embedded alginate–gelatin microspheres and their assembly in 3D-printed poly(ε-caprolactone) scaffold for cartilage tissue engineering [Abstract]
Year 2019
Journal/Proceedings Journal of Orthopaedic Translation
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Abstract
Objective Our study reports the optimization of electrospray human bone marrow stromal cell (hBMSCs)–embedded alginate–gelatin (Alg-Gel, same as following) microspheres for the purpose of their assembly in 3D-printed poly(ε-caprolactone) (PCL) scaffold for the fabrication of a mechanically stable and biological supportive tissue engineering cartilage construct. Methods The fabrication of the Alg-Gel microspheres using an electrospray technique was optimized in terms of polydispersity, yield of microspheres and circularity and varying fabrication conditions. PCL scaffolds were designed and printed by melt extrusion. Then, four groups were set: Alg-hBMSC microspheres cultured in the 2D well plate (Alg-hBMSCs+2D) group, Alg-Gel-hBMSC microspheres cultured in the 2D well plate (Alg-Gel-hBMSCs+2D) group, Alg-Gel-hBMSC microspheres embedded in PCL scaffold cultured in the 2D well plate (Alg-Gel-hBMSCs+2D) group and Alg-Gel-hBMSCs microspheres cultured in the 3D bioreactor (Alg-Gel-hBMSCs+3D) group. Cell viability, proliferation and chondrogenic differentiation were evaluated, and mechanical test was performed. Results Nonaggregated, low polydispersity and almost spherical microspheres of average diameter of 200–300 μm were produced with alginate 1.5 w: v%, gelatin (Type B) concentration of 0.5 w: v % and CaCl2 coagulating bath concentration of 3.0 w: v %, using 30G needle size and 8 kV and 0.6 bar voltage and air pressure, respectively. Alginate with gelatin hydrogel improved viability and promoted hBMSC proliferation better than alginate microspheres. Interestingly, hBMSCs embedded in microspheres assembled in 3D-printed PCL scaffold and cultured in a 3D bioreactor were more proliferative in comparison to the previous two groups (p < 0.05). Similarly, the GAG content, GAG/DNA ratio as well as Coll 2 and Aggr gene expression were increased in the last two groups. Conclusion Optimization of hBMSC-embedded Alg-Gel microspheres produced by electrospray has been performed. The Alg-Gel composition selected allows conservation of hBMSC viability and supports proliferation and matrix deposition. The possibility to seed and assemble microspheres in designed 3D-printed PCL scaffolds for the fabrication of a mechanically stable and biological supportive tissue engineering cartilage construct was demonstrated. Translational potential of this article We optimize and demonstrate that electrospray microsphere fabrication is a cytocompatible and facile process to produce the hBMSC-embedded microsize tissue-like particles that can easily be assembled into a stable construct. This finding could have application in the development of mechanically competent stem cell–based tissue engineering of cartilage regeneration.
AUTHOR Li, Huijun and Tan, Yu Jun and Li, Lin
Title A strategy for strong interface bonding by 3D bioprinting of oppositely charged κ-carrageenan and gelatin hydrogels [Abstract]
Year 2018
Journal/Proceedings Carbohydrate Polymers
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DOI/URL URL
Abstract
A promising approach for improving the interfacial bonding of a three-dimensionally (3D) printed multilayered structure has been investigated by taking advantage of the electrostatic interactions between two hydrogels with oppositely charges. Here, two hydrogels namely gelatin and κ-carrageenan, which are the cationic and anionic hydrogels respectively, are used. It is found that the interfacial bonding strength between these two oppositely charged hydrogels is significantly higher than that of a bilayered gelatin or a bilayered κ-carrageenan. The bioprinted multilayered κ-carrageenan-gelatin hydrogel construct demonstrates a very good biocompatibility and a good structure integrity at 37 °C. Our strategy also overcomes the limitation of using gelatin for bio-fabrication at 37 °C, without further post crosslinking.
AUTHOR Fortunato, Gabriele Maria and Maria, Carmelo De and Eglin, David and Serra, Tiziano and Vozzi, Giovanni
Title An ink-jet printed electrical stimulation platform for muscle tissue regeneration [Abstract]
Year 2018
Journal/Proceedings Bioprinting
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DOI/URL URL DOI
Abstract
Conducting polymeric materials have been used to modulate response of cells seeded on their surfaces. However, there is still major improvement to be made related to their biocompatibility, conductivity, stability in biological milieu, and processability toward truly tissue engineered functional device. In this work, conductive polymer, poly(3,4-ethylene-dioxythiophene):polystyrene-sulfonate (PEDOT:PSS), and its possible applications in tissue engineering were explored. In particular PEDOT:PSS solution was inkjet printed onto a gelatin substrate for obtaining a conductive structure. Mechanical and electrical characterizations, structural stability by swelling and degradation tests were carried out on different PEDOT-based samples obtained by varying the number of printed PEDOT layers from 5 to 50 on gelatin substrate. Biocompatibility of substrates was investigated on C2C12 myoblasts, through metabolic activity assay and imaging analysis during a 7-days culture period, to assess cell morphology, differentiation and alignment. The results of this first part allowed to proceed with the second part of the study in which these substrates were used for the design of an electrical stimulation device, with the aim of providing the external stimulus (3 V amplitude square wave at 1 and 2 Hz frequency) to guide myotubes alignment and enhance differentiation, having in this way promising applications in the field of muscle tissue engineering.
AUTHOR Paxton, Naomi Claire and Smolan, Willi and Böck, Thomas and Melchels, Ferry P. W. and Groll, Juergen and Juengst, Tomasz
Title Proposal to Assess Printability of Bioinks for Extrusion-Based Bioprinting and Evaluation of Rheological Properties Governing Bioprintability [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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Abstract
Abstract The development and formulation of printable inks for extrusion-based 3D bioprinting has been a major challenge in the field of biofabrication. Inks, often polymer solutions with the addition of crosslinking to form hydrogels, must not only display adequate mechanical properties for the chosen application, but also show high biocompatibility as well as printability. Here we describe a reproducible two-step method for the assessment of the printability of inks for bioprinting, focussing firstly on screening ink formulations to assess fibre formation and the ability to form 3D constructs before presenting a method for the rheological evaluation of inks to characterise the yield point, shear thinning and recovery behaviour. In conjunction, a mathematical model was formulated to provide a theoretical understanding of the pressure-driven, shear thinning extrusion of inks through needles in a bioprinter. The assessment methods were trialled with a commercially-available crème, poloxamer 407, alginate-based inks and an alginate-gelatin composite material. Yield stress was investigated by applying a stress ramp to a number of inks, which demonstrated the necessity of high yield for printable materials. The shear thinning behaviour of the inks was then characterised by quantifying the degree of shear thinning and using the mathematical model to predict the window of printer operating parameters in which the materials could be printed. Furthermore, the model predicted high shear conditions and high residence times for cells at the walls of the needle and effects on cytocompatibility at different printing conditions. Finally, the ability of the materials to recover to their original viscosity after extrusion was examined using rotational recovery rheological measurements. Taken together, these assessment techniques revealed significant insights into the requirements for printable inks and shear conditions present during the extrusion process and allow the rapid and reproducible characterisation of a wide variety of inks for bioprinting.
AUTHOR DeSimone, Elise and Schacht, Kristin and Pellert, Alexandra and Scheibel, Thomas
Title Recombinant spider silk-based bioinks [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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Abstract
Bioinks, 3D cell culture systems which can be printed, are still in the early development stages. Currently, extensive research is going into designing printers to be more accommodating to bioinks, designing scaffolds with stiff materials as support structures for the often soft bioinks, and modifying the bioinks themselves. Recombinant spider silk proteins, a potential biomaterial component for bioinks, have high biocompatibility, can be processed into several morphologies and can be modified with cell adhesion motifs to enhance their bioactivity. In this work, thermally gelled hydrogels made from recombinant spider silk protein encapsulating mouse fibroblast cell line BALB/3T3 were prepared and characterized. The bioinks were evaluated for performance in vitro both before and after printing, and it was observed that unprinted bioinks provided a good platform for cell spreading and proliferation, while proliferation in printed scaffolds was prohibited. To improve the properties of the printed hydrogels, gelatin was given as an additive and thereby served indirectly as a plasticizer, improving the resolution of printed strands. Taken together, recombinant spider silk proteins and hydrogels made thereof show good potential as a bioink, warranting further development.
AUTHOR Bertlein, Sarah and Brown, Gabriella and Lim, Khoon and Jungst, Tomasz and Boeck, Thomas and Blunk, Torsten and Tessmar, Joerg and J. Hooper, Gary and Woodfield, Tim and Groll, Jürgen
Title Thiol-Ene Clickable Gelatin: A Platform Bioink for Multiple 3D Biofabrication Technologies [Abstract]
Year 2017
Journal/Proceedings Advanced Materials
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Abstract
Bioprinting can be defined as the art of combining materials and cells to fabricate designed, hierarchical 3D hybrid constructs. Suitable materials, so called bioinks, have to comply with challenging rheological processing demands and rapidly form a stable hydrogel postprinting in a cytocompatible manner. Gelatin is often adopted for this purpose, usually modified with (meth-)acryloyl functionalities for postfabrication curing by free radical photopolymerization, resulting in a hydrogel that is cross-linked via nondegradable polymer chains of uncontrolled length. The application of allylated gelatin (GelAGE) as a thiol-ene clickable bioink for distinct biofabrication applications is reported. Curing of this system occurs via dimerization and yields a network with flexible properties that offer a wider biofabrication window than (meth-)acryloyl chemistry, and without additional nondegradable components. An in-depth analysis of GelAGE synthesis is conducted, and standard UV-initiation is further compared with a recently described visible-light-initiator system for GelAGE hydrogel formation. It is demonstrated that GelAGE may serve as a platform bioink for several biofabrication technologies by fabricating constructs with high shape fidelity via lithography-based (digital light processing) 3D printing and extrusion-based 3D bioprinting, the latter supporting long-term viability postprinting of encapsulated chondrocytes.
AUTHOR Ng, Wei Long and Yeong, Wai Yee and Naing, May Win
Title Polyelectrolyte gelatin-chitosan hydrogel optimized for 3D bioprinting in skin tissue engineering [Abstract]
Year 2016
Journal/Proceedings International Journal of Bioprinting
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DOI/URL URL DOI
Abstract
Bioprinting is a promising automated platform that enables the simultaneous deposition of multiple types of cells and biomaterials to fabricate complex three-dimensional (3D) tissue constructs. Most of the previous bioprinting works focused on collagen-based biomaterial, which has poor printability and long crosslinking time. This posed a immerse challenge to create a 3D construct with pre-determined shape and configuration. There is a need for a functional material with good printability in order to fabricate a 3D skin construct. Recently, the use of chitosan for wound healing applications has attracted huge attention due to its attractive traits such as its antimicrobial properties and ability to trigger hemostasis. In this paper, we report the modification of chitosan-based biomaterials for functional 3D bioprinting. Modification to the chitosan was carried out via the oppositely charged functional groups from chitosan and gelatin at a specific pH of ~pH 6.5 to form polyelectrolyte complexes. The polyelectrolyte hydrogels were evaluated in terms of chemical interactions within polymer blend, rheological properties (viscosities, storage and loss modulus), printing resolution at varying pressures and feed rates and biocompatibility. The chitosan-based hydrogels formulated in this work exhibited good printability at room temperature, high shape fidelity of the printed 3D constructs and good biocompatibility with fibroblast skin cells.