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AUTHOR Benmeridja, Lara and De Moor, Lise and De Maere, Elisabeth and Vanlauwe, Florian and Ryx, Michelle and Tytgat, Liesbeth and Vercruysse, Chris and Dubruel, Peter and Van Vlierberghe, Sandra and Blondeel, Phillip and Declercq, Heidi
Title High-throughput fabrication of vascularized adipose microtissues for 3D bioprinting [Abstract]
Year 2020
Journal/Proceedings Journal of Tissue Engineering and Regenerative Medicine
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Abstract For patients with soft tissue defects, repair with autologous in vitro engineered adipose tissue could be a promising alternative to current surgical therapies. A volume-persistent engineered adipose tissue construct under in vivo conditions can only be achieved by early vascularization after transplantation. The combination of 3D bioprinting technology with self-assembling microvascularized units as building blocks can potentially answer the need for a microvascular network. In the present study, co-culture spheroids combining adipose-derived stem cells (ASC) and human umbilical vein endothelial cells (HUVEC) were created with an ideal geometry for bioprinting. When applying the favourable seeding technique and condition, compact viable spheroids were obtained, demonstrating high adipogenic differentiation and capillary-like network formation after 7 and 14 days of culture, as shown by live/dead analysis, immunohistochemistry and RT-qPCR. Moreover, we were able to successfully 3D bioprint the encapsulated spheroids, resulting in compact viable spheroids presenting capillary-like structures, lipid droplets and spheroid outgrowth after 14 days of culture. This is the first study that generates viable high-throughput (pre-)vascularized adipose microtissues as building blocks for bioprinting applications using a novel ASC/HUVEC co-culture spheroid model, which enables both adipogenic differentiation while simultaneously supporting the formation of prevascular-like structures within engineered tissues in vitro.
AUTHOR Peiffer, Quentin C. and de Ruijter, Mylène and van Duijn, Joost and Crottet, Denis and Dominic, Ernst and Malda, Jos and Castilho, Miguel
Title Melt electrowriting onto anatomically relevant biodegradable substrates: Resurfacing a diarthrodial joint [Abstract]
Year 2020
Journal/Proceedings Materials & Design
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Three-dimensional printed hydrogel constructs with well-organized melt electrowritten (MEW) fibre-reinforcing scaffolds have been demonstrated as a promising regenerative approach to treat small cartilage defects. Here, we investige how to translate the fabrication of small fibre-reinforced structures on flat surfaces to anatomically relevant structures. In particular, the accurate deposition of MEW-fibres onto curved surfaces of conductive and non-conductive regenerative biomaterials is studied. This study reveals that clinically relevant materials with low conductivities are compatible with resurfacing with organized MEW fibres. Importantly, accurate patterning on non-flat surfaces was successfully shown, provided that a constant electrical field strength and an electrical force normal to the substrate material is maintained. Furthermore, the application of resurfacing the geometry of the medial human femoral condyle is confirmed by the fabrication of a personalised osteochondral implant. The implant composed of an articular cartilage-resident chondroprogenitor cells (ACPCs)-laden hydrogel reinforced with a well-organized MEW scaffold retained its personalised shape, improved its compressive properties and supported neocartilage formation after 28 days in vitro culture. Overall, this study establishes the groundwork for translating MEW from planar and non-resorbable material substrates to anatomically relevant geometries and regenerative materials that the regenerative medicine field aims to create.
AUTHOR Daly, Andrew C. and Kelly, Daniel J.
Title Biofabrication of spatially organised tissues by directing the growth of cellular spheroids within 3D printed polymeric microchambers [Abstract]
Year 2019
Journal/Proceedings Biomaterials
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Successful tissue engineering requires the generation of human scale implants that mimic the structure, composition and mechanical properties of native tissues. Here, we report a novel biofabrication strategy that enables the engineering of structurally organised tissues by guiding the growth of cellular spheroids within arrays of 3D printed polymeric microchambers. With the goal of engineering stratified articular cartilage, inkjet bioprinting was used to deposit defined numbers of mesenchymal stromal cells (MSCs) and chondrocytes into pre-printed microchambers. These jetted cell suspensions rapidly underwent condensation within the hydrophobic microchambers, leading to the formation of organised arrays of cellular spheroids. The microchambers were also designed to provide boundary conditions to these spheroids, guiding their growth and eventual fusion, leading to the development of stratified cartilage tissue with a depth-dependant collagen fiber architecture that mimicked the structure of native articular cartilage. Furthermore, the composition and biomechanical properties of the bioprinted cartilage was also comparable to the native tissue. Using multi-tool biofabrication, we were also able to engineer anatomically accurate, human scale, osteochondral templates by printing this microchamber system on top of a hypertrophic cartilage region designed to support endochondral bone formation and then maintaining the entire construct in long-term bioreactor culture to enhance tissue development. This bioprinting strategy provides a versatile and scalable approach to engineer structurally organised cartilage tissues for joint resurfacing applications.
AUTHOR Laternser, Sandra and Keller, Hansjoerg and Leupin, Olivier and Rausch, Martin and Graf-Hausner, Ursula and Rimann, Markus
Title A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues [Abstract]
Year 2018
Journal/Proceedings SLAS TECHNOLOGY: Translating Life Sciences Innovation
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Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.
AUTHOR de Ruijter, Mylène and Ribeiro, Alexandre and Dokter, Inge and Castilho, Miguel and Malda, Jos
Title Simultaneous Micropatterning of Fibrous Meshes and Bioinks for the Fabrication of Living Tissue Constructs [Abstract]
Year 2018
Journal/Proceedings Advanced Healthcare Materials
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Abstract Fabrication of biomimetic tissues holds much promise for the regeneration of cells or organs that are lost or damaged due to injury or disease. To enable the generation of complex, multicellular tissues on demand, the ability to design and incorporate different materials and cell types needs to be improved. Two techniques are combined: extrusion-based bioprinting, which enables printing of cell-encapsulated hydrogels; and melt electrowriting (MEW), which enables fabrication of aligned (sub)-micrometer fibers into a single-step biofabrication process. Composite structures generated by infusion of MEW fiber structures with hydrogels have resulted in mechanically and biologically competent constructs; however, their preparation involves a two-step fabrication procedure that limits freedom of design of microfiber architectures and the use of multiple materials and cell types. How convergence of MEW and extrusion-based bioprinting allows fabrication of mechanically stable constructs with the spatial distributions of different cell types without compromising cell viability and chondrogenic differentiation of mesenchymal stromal cells is demonstrated for the first time. Moreover, this converged printing approach improves freedom of design of the MEW fibers, enabling 3D fiber deposition. This is an important step toward biofabrication of voluminous and complex hierarchical structures that can better resemble the characteristics of functional biological tissues.
AUTHOR Lee, Ji Seung and Park, Hae Sang and Jung, Harry and Lee, Hanna and Hong, Heesun and Lee, Young Jin and Suh, Ye Ji and Lee, Ok Joo and Kim, Soon Hee and Park, Chan Hum
Title 3D-printable photocurable bioink for cartilage regeneration of tonsil-derived mesenchymal stem cells [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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Cartilage regeneration is challenging because of the poor intrinsic self-repair capacity of avascular tissue. Three-dimensional (3D) bioprinting has gained significant attention in the field of tissue engineering and is a promising technology to overcome current difficulties in cartilage regeneration. Although bioink is an essential component of bioprinting technology, several challenges remain in satisfying different requirements for ideal bioink, including biocompatibility and printability based on specific biological requirements. Gelatin and hyaluronic acid (HA) have been shown to be ideal biomimetic hydrogel sources for cartilage regeneration. However, controlling their structure, mechanical properties, biocompatibility, and degradation rate for cartilage repair remains a challenge. Here, we show a photocurable bioink created by hybridization of gelatin methacryloyl (GelMA) and glycidyl-methacrylated HA (GMHA) for material extrusion 3D bioprinting in cartilage regeneration. GelMA and GMHA were mixed in various ratios, and the mixture of 7% GelMA and 5% GMHA bioink (G7H5) demonstrated the most reliable mechanical properties, rheological properties, and printability. This G7H5 bioink allowed us to build a highly complex larynx structure, including the hyoid bone, thyroid cartilage, cricoid cartilage, arytenoid cartilage, and cervical trachea. This bioink also provided an excellent microenvironment for chondrogenesis of tonsil-derived mesenchymal stem cells (TMSCs) in vitro and in vivo. In summary, this study presents the ideal formulation of GelMA/GMHA hybrid bioink to generate a well-suited photocurable bioink for cartilage regeneration of TMSCs using a material extrusion bioprinter, and could be applied to cartilage tissue engineering.
AUTHOR Colle, Julien and Blondeel, Phillip and De Bruyne, Axelle and Bochar, Silke and Tytgat, Liesbeth and Vercruysse, Chris and Van Vlierberghe, Sandra and Dubruel, Peter and Declercq, Heidi
Title Bioprinting predifferentiated adipose-derived mesenchymal stem cell spheroids with methacrylated gelatin ink for adipose tissue engineering [Abstract]
Year 2020
Journal/Proceedings Journal of Materials Science: Materials in Medicine
Reftype Colle2020
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The increasing number of mastectomies results in a greater demand for breast reconstruction characterized by simplicity and a low complication profile. Reconstructive surgeons are investigating tissue engineering (TE) strategies to overcome the current surgical drawbacks. 3D bioprinting is the rising technique for the fabrication of large tissue constructs which provides a potential solution for unmet clinical needs in breast reconstruction building on decades of experience in autologous fat grafting, adipose-derived mesenchymal stem cell (ASC) biology and TE. A scaffold was bioprinted using encapsulated ASC spheroids in methacrylated gelatin ink (GelMA). Uniform ASC spheroids with an ideal geometry and diameter for bioprinting were formed, using a high-throughput non-adhesive agarose microwell system. ASC spheroids in adipogenic differentiation medium (ADM) were evaluated through live/dead staining, histology (HE, Oil Red O), TEM and RT-qPCR. Viable spheroids were obtained for up to 14 days post-printing and showed multilocular microvacuoles and successful differentiation toward mature adipocytes shown by gene expression analysis. Moreover, spheroids were able to assemble at random in GelMA, creating a macrotissue. Combining the advantage of microtissues to self-assemble and the controlled organization by bioprinting technologies, these ASC spheroids can be useful as building blocks for the engineering of soft tissue implants.
AUTHOR Daly, Andrew C. and Pitacco, Pierluca and Nulty, Jessica and Cunniffe, Gráinne M. and Kelly, Daniel J.
Title 3D printed microchannel networks to direct vascularisation during endochondral bone repair [Abstract]
Year 2018
Journal/Proceedings Biomaterials
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Bone tissue engineering strategies that recapitulate the developmental process of endochondral ossification offer a promising route to bone repair. Clinical translation of such endochondral tissue engineering strategies will require overcoming a number of challenges, including the engineering of large and often anatomically complex cartilage grafts, as well as the persistence of core regions of avascular cartilage following their implantation into large bone defects. Here 3D printing technology is utilized to develop a versatile and scalable approach to guide vascularisation during endochondral bone repair. First, a sacrificial pluronic ink was used to 3D print interconnected microchannel networks in a mesenchymal stem cell (MSC) laden gelatin-methacryloyl (GelMA) hydrogel. These constructs (with and without microchannels) were next chondrogenically primed in vitro and then implanted into critically sized femoral bone defects in rats. The solid and microchanneled cartilage templates enhanced bone repair compared to untreated controls, with the solid cartilage templates (without microchannels) supporting the highest levels of total bone formation. However, the inclusion of 3D printed microchannels was found to promote osteoclast/immune cell invasion, hydrogel degradation, and vascularisation following implantation. In addition, the endochondral bone tissue engineering strategy was found to support comparable levels of bone healing to BMP-2 delivery, whilst promoting lower levels of heterotopic bone formation, with the microchanneled templates supporting the lowest levels of heterotopic bone formation. Taken together, these results demonstrate that 3D printed hypertrophic cartilage grafts represent a promising approach for the repair of complex bone fractures, particularly for larger defects where vascularisation will be a key challenge.
AUTHOR Kessel, Benjamin and Lee, Mihyun and Bonato, Angela and Tinguely, Yann and Tosoratti, Enrico and Zenobi-Wong, Marcy
Title 3D Bioprinting of Macroporous Materials Based on Entangled Hydrogel Microstrands [Abstract]
Year 2020
Journal/Proceedings Advanced Science
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Abstract Hydrogels are excellent mimetics of mammalian extracellular matrices and have found widespread use in tissue engineering. Nanoporosity of monolithic bulk hydrogels, however, limits mass transport of key biomolecules. Microgels used in 3D bioprinting achieve both custom shape and vastly improved permissivity to an array of cell functions, however spherical-microbead-based bioinks are challenging to upscale, are inherently isotropic, and require secondary crosslinking. Here, bioinks based on high-aspect-ratio hydrogel microstrands are introduced to overcome these limitations. Pre-crosslinked, bulk hydrogels are deconstructed into microstrands by sizing through a grid with apertures of 40–100 µm. The microstrands are moldable and form a porous, entangled structure, stable in aqueous medium without further crosslinking. Entangled microstrands have rheological properties characteristic of excellent bioinks for extrusion bioprinting. Furthermore, individual microstrands align during extrusion and facilitate the alignment of myotubes. Cells can be placed either inside or outside the hydrogel phase with >90% viability. Chondrocytes co-printed with the microstrands deposit abundant extracellular matrix, resulting in a modulus increase from 2.7 to 780.2 kPa after 6 weeks of culture. This powerful approach to deconstruct bulk hydrogels into advanced bioinks is both scalable and versatile, representing an important toolbox for 3D bioprinting of architected hydrogels.
AUTHOR Zhang, Danwei and Peng, Erwin and Borayek, Ramadan and Ding, Jun
Title Controllable Ceramic Green-Body Configuration for Complex Ceramic Architectures with Fine Features [Abstract]
Year 2019
Journal/Proceedings Advanced Functional Materials
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Abstract Fabrication of dense ceramic articles with intricate fine features and geometrically complex morphology by using a relatively simple and the cost-effective process still remains a challenge. Ceramics, either in its green- or sintered-form, are known for being hard yet brittle which limits further shape reconfiguration. In this work, a combinatorial process of ceramic robocasting and photopolymerization is demonstrated to produce either flexible and/or stretchable ceramic green-body (Flex-Body or Stretch-Body) that can undergo a postprinting reconfiguration process. Secondary shaping may proceed through: i) self-assembly-assisted shaping and ii) mold-assisted shaping process, which allows a well-controlled ceramic structure morphology. With a proposed well-controlled thermal heating process, the ceramic Sintered-Body can achieve >99.0% theoretical density with good mechanical rigidity. Complex and dense ceramic articles with fine features down to 65 μm can be fabricated. When combined with a multi-nozzle deposition process, i) self-shaping ceramic structures can be realized through anisotropic shrinkage induced by suspensions' composition variation and ii) technical and functional multiceramic structures can be fabricated. The simplicity of the proposed technique and its inexpensive processing cost make it an attractive approach for fabricating geometrically complex ceramic articles with unique macrostructures, which complements the existing state of-the-art ceramic additive manufacturing techniques.
AUTHOR Schaffner, Manuel and Faber, Jakob A. and Pianegonda, Lucas and Rühs, Patrick A. and Coulter, Fergal and Studart, André R.
Title 3D printing of robotic soft actuators with programmable bioinspired architectures [Abstract]
Year 2018
Journal/Proceedings Nature Communications
Reftype Schaffner2018
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Soft actuation allows robots to interact safely with humans, other machines, and their surroundings. Full exploitation of the potential of soft actuators has, however, been hindered by the lack of simple manufacturing routes to generate multimaterial parts with intricate shapes and architectures. Here, we report a 3D printing platform for the seamless digital fabrication of pneumatic silicone actuators exhibiting programmable bioinspired architectures and motions. The actuators comprise an elastomeric body whose surface is decorated with reinforcing stripes at a well-defined lead angle. Similar to the fibrous architectures found in muscular hydrostats, the lead angle can be altered to achieve elongation, contraction, or twisting motions. Using a quantitative model based on lamination theory, we establish design principles for the digital fabrication of silicone-based soft actuators whose functional response is programmed within the material's properties and architecture. Exploring such programmability enables 3D printing of a broad range of soft morphing structures.
AUTHOR Nulty, Jessica and Freeman, Fiona E. and Browe, David C. and Burdis, Ross and Ahern, Daniel P. and Pitacco, Pierluca and Lee, Yu Bin and Alsberg, Eben and Kelly, Daniel J.
Title 3D Bioprinting of prevascularised implants for the repair of critically-sized bone defects [Abstract]
Year 2021
Journal/Proceedings Acta Biomaterialia
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For 3D bioprinted tissues to be scaled-up to clinically relevant sizes, effective prevascularisation strategies are required to provide the necessary nutrients for normal metabolism and to remove associated waste by-products. The aim of this study was to develop a bioprinting strategy to engineer prevascularised tissues in vitro and to investigate the capacity of such constructs to enhance the vascularisation and regeneration of large bone defects in vivo. From a screen of different bioinks, a fibrin-based hydrogel was found to best support human umbilical vein endothelial cell (HUVEC) sprouting and the establishment of a microvessel network. When this bioink was combined with HUVECs and supporting human bone marrow stem/stromal cells (hBMSCs), these microvessel networks persisted in vitro. Furthermore, only bioprinted tissues containing both HUVECs and hBMSCs, that were first allowed to mature in vitro, supported robust blood vessel development in vivo. To assess the therapeutic utility of this bioprinting strategy, these bioinks were used to prevascularise 3D printed polycaprolactone (PCL) scaffolds, which were subsequently implanted into critically-sized femoral bone defects in rats. Microcomputed tomography (µCT) angiography revealed increased levels of vascularisation in vivo, which correlated with higher levels of new bone formation. Such prevascularised constructs could be used to enhance the vascularisation of a range of large tissue defects, forming the basis of multiple new bioprinted therapeutics. Statement of Significance This paper demonstrates a versatile 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into a bone tissue engineering strategy to improve vascularisation in a rat femoral defect model.
AUTHOR Leu Alexa, Rebeca and Iovu, Horia and Ghitman, Jana and Serafim, Andrada and Stavarache, Cristina and Marin, Maria-Minodora and Ianchis, Raluca
Title 3D-Printed Gelatin Methacryloyl-Based Scaffolds with Potential Application in Tissue Engineering [Abstract]
Year 2021
Journal/Proceedings Polymers
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The development of materials for 3D printing adapted for tissue engineering represents one of the main concerns nowadays. Our aim was to obtain suitable 3D-printed scaffolds based on methacrylated gelatin (GelMA). In this respect, three degrees of GelMA methacrylation, three different concentrations of GelMA (10%, 20%, and 30%), and also two concentrations of photoinitiator (I-2959) (0.5% and 1%) were explored to develop proper GelMA hydrogel ink formulations to be used in the 3D printing process. Afterward, all these GelMA hydrogel-based inks/3D-printed scaffolds were characterized structurally, mechanically, and morphologically. The presence of methacryloyl groups bounded to the surface of GelMA was confirmed by FTIR and 1H-NMR analyses. The methacrylation degree influenced the value of the isoelectric point that decreased with the GelMA methacrylation degree. A greater concentration of photoinitiator influenced the hydrophilicity of the polymer as proved using contact angle and swelling studies because of the new bonds resulting after the photocrosslinking stage. According to the mechanical tests, better mechanical properties were obtained in the presence of the 1% initiator. Circular dichroism analyses demonstrated that the secondary structure of gelatin remained unaffected during the methacrylation process, thus being suitable for biological applications.
AUTHOR Bin Wang and Pedro J. Díaz-Payno and David C. Browe and Fiona E. Freeman and Jessica Nulty and Ross Burdis and Daniel J. Kelly
Title Affinity-bound growth factor within sulfated interpenetrate network bioinks for bioprinting cartilaginous tissues [Abstract]
Year 2021
Journal/Proceedings Acta Biomaterialia
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3D bioprinting has emerged as a promising technology in the field of tissue engineering and regenerative medicine due to its ability to create anatomically complex tissue substitutes. However, it still remains challenging to develop bioactive bioinks that provide appropriate and permissive environments to instruct and guide the regenerative process in vitro and in vivo. In this study alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize an alginate-gelatin methacryloyl (GelMA) interpenetrating network (IPN) bioink to enable the bioprinting of cartilaginous tissues. The inclusion of alginate sulfate had a limited influence on the viscosity, shear-thinning and thixotropic properties of the IPN bioink, enabling high-fidelity bioprinting and supporting mesenchymal stem cell (MSC) viability post-printing. The stiffness of printed IPN constructs greatly exceeded that achieved by printing alginate or GelMA alone, while maintaining resilience and toughness. Furthermore, given the high affinity of alginate sulfate to heparin-binding growth factors, the sulfated IPN bioink supported the sustained release of transforming growth factor-β3 (TGF-β3), providing an environment that supported robust chondrogenesis in vitro, with little evidence of hypertrophy or mineralization over extended culture periods. Such bioprinted constructs also supported chondrogenesis in vivo, with the controlled release of TGF-β3 promoting significantly higher levels of cartilage-specific extracellular matrix deposition. Altogether, these results demonstrate the potential of bioprinting sulfated bioinks as part of a ‘single-stage’ or ‘point-of-care’ strategy for regenerating cartilaginous tissues. Statement of Significance: This study highlights the potential of using sulfated interpenetrating network (IPN) bioink to support the regeneration of phenotypically stable articular cartilage. Construction of interpenetrate networks in the bioink enables unique high-fidelity bioprinting and unique synergistic mechanical properties. The presence of alginate sulfate provided the capacity of high affinity-binding of TGF-β3, which promoted robust chondrogenesis.
AUTHOR Leu Alexa, Rebeca and Iovu, Horia and Trica, Bogdan and Zaharia, Catalin and Serafim, Andrada and Alexandrescu, Elvira and Radu, Ionut-Cristian and Vlasceanu, George and Preda, Silviu and Ninciuleanu, Claudia Mihaela and Ianchis, Raluca
Title Assessment of Naturally Sourced Mineral Clays for the 3D Printing of Biopolymer-Based Nanocomposite Inks [Abstract]
Year 2021
Journal/Proceedings Nanomaterials
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The present study investigated the possibility of obtaining 3D printed composite constructs using biomaterial-based nanocomposite inks. The biopolymeric matrix consisted of methacrylated gelatin (GelMA). Several types of nanoclay were added as the inorganic component. Our aim was to investigate the influence of clay type on the rheological behavior of ink formulations and to determine the morphological and structural properties of the resulting crosslinked hydrogel-based nanomaterials. Moreover, through the inclusion of nanoclays, our goal was to improve the printability and shape fidelity of nanocomposite scaffolds. The viscosity of all ink formulations was greater in the presence of inorganic nanoparticles as shear thinning occurred with increased shear rate. Hydrogel nanocomposites presented predominantly elastic rather than viscous behavior as the materials were crosslinked which led to improved mechanical properties. The inclusion of nanoclays in the biopolymeric matrix limited hydrogel swelling due the physical barrier effect but also because of the supplementary crosslinks induced by the clay layers. The distribution of inorganic filler within the GelMA-based hydrogels led to higher porosities as a consequence of their interaction with the biopolymeric ink. The present study could be useful for the development of soft nanomaterials foreseen for the additive manufacturing of customized implants for tissue engineering.
AUTHOR Oliveira, Hugo and Médina, Chantal and Stachowicz, Marie-Laure and Paiva dos Santos, Bruno and Chagot, Lise and Dusserre, Nathalie and Fricain, Jean-Christophe
Title Extracellular matrix (ECM)-derived bioinks designed to foster vasculogenesis and neurite outgrowth: Characterization and bioprinting [Abstract]
Year 2021
Journal/Proceedings Bioprinting
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The field of bioprinting has shown a tremendous development in recent years, focusing on the development of advanced in vitro models and on regeneration approaches. In this scope, the lack of suitable biomaterials that can be efficiently formulated as printable bioinks, while supporting specific cellular events, is currently considered as one of the main limitations in the field. Indeed, extracellular matrix (ECM)-derived biomaterials formulated to enable printability and support cellular response, for instance via integrin binding, are eagerly awaited in the field of bioprinting. Several bioactive laminin sequences, including peptides such as YIGSR and IKVAV, have been identified to promote endothelial cell attachment and/or neurite outgrowth and guidance, respectively. Here, we show the development of two distinct bioinks, designed to foster vasculogenesis or neurogenesis, based on methacrylated collagen and hyaluronic acid (CollMA and HAMA, respectively), both relevant ECM-derived polymers, and on their combination with cysteine-flanked laminin-derived peptides. Using this strategy, it was possible to optimize the bioink printability, by tuning CollMA and HAMA concentration and ratio, and modulate their bioactivity, through adjustments in the cell-active peptide sequence spatial density, without compromising cell viability. We demonstrated that cell-specific bioinks could be customized for the bioprinting of both human umbilical vein cord endothelial cells (HUVECs) or adult rat sensory neurons from the dorsal root ganglia, and could stimulate both vasculogenesis and neurite outgrowth, respectively. This approach holds great potential as it can be tailored to other cellular models, due to its inherent capacity to accommodate different peptide compositions and to generate complex peptide mixtures and/or gradients.
AUTHOR Tan, Edgar Y. S. and Suntornnond, Ratima and Yeong, Wai Yee
Title High-Resolution Novel Indirect Bioprinting of Low-Viscosity Cell-Laden Hydrogels via Model-Support Bioink Interaction [Abstract]
Year 2021
Journal/Proceedings 3D Printing and Additive Manufacturing
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Abstract Bioprinting of unmodified soft extracellular matrix into complex 3D structures has remained challenging to fabricate. Herein, we established a novel process for the printing of low-viscosity hydrogel by using a unique support technique to retain the structural integrity of the support structure. We demonstrated that this process of printing could be used for different types of hydrogel, ranging from fast crosslinking gelatin methacrylate to slow crosslinking collagen type I. In addition, we evaluated the biocompatibility of the process by observing the effects of the cytotoxicity of L929 and the functionality of the human umbilical vein endothelium primary cells after printing. The results show that the bioprinted construct provided excellent biocompatibility as well as supported cell growth and differentiation. Thus, this is a novel technique that can be potentially used to enhance the resolution of the extrusion-based bioprinter.
AUTHOR De Moor, Lise and Minne, Mendy and Tytgat, Liesbeth and Vercruysse, Chris and Dubruel, Peter and Van Vlierberghe, Sandra and Declercq, Heidi
Title Tuning the Phenotype of Cartilage Tissue Mimics by Varying Spheroid Maturation and Methacrylamide-Modified Gelatin Hydrogel Characteristics [Abstract]
Year 2021
Journal/Proceedings Macromolecular Bioscience
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Abstract In hybrid bioprinting of cartilage tissue constructs, spheroids are used as cellular building blocks and combined with biomaterials for dispensing. However, biomaterial intrinsic cues can deeply affect cell fate and to date, the influence of hydrogel encapsulation on spheroid viability and phenotype has received limited attention. This study assesses this need and unravels 1) how the phenotype of spheroid-laden constructs can be tuned through adjusting the hydrogel physico–chemical properties and 2) if the spheroid maturation stage prior to encapsulation is a determining factor for the construct phenotype. Articular chondrocyte spheroids with a cartilage specific extracellular matrix (ECM) are generated and different maturation stages, early-, mid-, and late-stage (3, 7, and 14 days, respectively), are harvested and encapsulated in 10, 15, or 20 w/v% methacrylamide-modified gelatin (gelMA) for 14 days. The encapsulation of immature spheroids do not lead to a cartilage-like ECM production but when more mature mid- or late-stage spheroids are combined with a certain concentration of gelMA, a fibrocartilage-like as well as a hyaline cartilage-like phenotype can be induced. As a proof of concept, late-stage spheroids are bioprinted using a 10 w/v% gelMA–Irgacure 2959 solution with the aim to test the processing potential of the spheroid-laden bioink.
AUTHOR Hamid, Omar A. and Eltaher, Hoda M. and Sottile, Virginie and Yang, Jing
Title 3D bioprinting of a stem cell-laden, multi-material tubular composite: An approach for spinal cord repair [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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Development of a biomimetic tubular scaffold capable of recreating developmental neurogenesis using pluripotent stem cells offers a novel strategy for the repair of spinal cord tissues. Recent advances in 3D printing technology have facilitated biofabrication of complex biomimetic environments by precisely controlling the 3D arrangement of various acellular and cellular components (biomaterials, cells and growth factors). Here, we present a 3D printing method to fabricate a complex, patterned and embryoid body (EB)-laden tubular scaffold composed of polycaprolactone (PCL) and hydrogel (alginate or gelatine methacrylate (GelMA)). Our results revealed 3D printing of a strong, macro-porous PCL/hydrogel tubular scaffold with a high capacity to control the porosity of the PCL scaffold, wherein the maximum porosity in the PCL wall was 15%. The method was equally employed to create spatiotemporal protein concentration within the scaffold, demonstrating its ability to generate linear and opposite gradients of model molecules (fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) and rhodamine). 3D bioprinting of EBs-laden GelMA was introduced as a novel 3D printing strategy to incorporate EBs in a hydrogel matrix. Cell viability and proliferation were measured post-printing. Following the bioprinting of EBs-laden 5% GelMA hydrogel, neural differentiation of EBs was induced using 1 μM retinoic acid (RA). The differentiated EBs contained βIII-tubulin positive neurons displaying axonal extensions and cells migration. Finally, 3D bioprinting of EBs-laden PCL/GelMA tubular scaffold successfully supported EBs neural differentiation and patterning in response to co-printing with 1 μM RA. 3D printing of a complex heterogeneous tubular scaffold that can encapsulate EBs, spatially controlled protein concentration and promote neuronal patterning will help in developing more biomimetic scaffolds capable of replicating the neural patterning which occurs during neural tube development.
AUTHOR Chen, Shengyang and Jang, Tae-Sik and Pan, Houwen Matthew and Jung, Hyun-Do and Sia, Ming Wei and Xie, Shuying and Hang, Yao and Chong, Seow and Wong, Dongan
Title 3D Freeform Printing of Nanocomposite Hydrogels through in situ Precipitation in Reactive Viscous Fluid
Year 2020
Journal/Proceedings International Journal of Bioprinting
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AUTHOR Lee, J. M. and Sing, S. L. and Yeong, W. Y.
Title Bioprinting of Multimaterials with Computer-aided Design/Computer -aided Manufacturing [Abstract]
Year 2020
Journal/Proceedings International Journal of Bioprinting; Vol 6, No 1 (2020)
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Abstract
Multimaterials deposition, a distinct advantage in bioprinting, overcomes material’s limitation in hydrogel-based bioprinting. Multimaterials are deposited in a build/support configuration to improve the structural integrity of three-dimensional bioprinted construct. A combination of rapid cross-linking hydrogel has been chosen for the build/support setup. The bioprinted construct was further chemically cross-linked to ensure a stable construct after print. This paper also proposes a file segmentation and preparation technique to be used in bioprinting for printing freeform structures.
AUTHOR Müller, Michael and Fisch, Philipp and Molnar, Marc and Eggert, Sebastian and Binelli, Marco and Maniura-Weber, Katharina and Zenobi-Wong, Marcy
Title Development and thorough characterization of the processing steps of an ink for 3D printing for bone tissue engineering [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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Abstract
Achieving reproducibility in the 3D printing of biomaterials requires a robust polymer synthesis method to reduce batch-to-batch variation as well as methods to assure a thorough characterization throughout the manufacturing process. Particularly biomaterial inks containing large solid fractions such as ceramic particles, often required for bone tissue engineering applications, are prone to inhomogeneity originating from inadequate mixing or particle aggregation which can lead to inconsistent printing results. The production of such an ink for bone tissue engineering consisting of gellan gum methacrylate (GG-MA), hyaluronic acid methacrylate and hydroxyapatite (HAp) particles was therefore optimized in terms of GG-MA synthesis and ink preparation process, and the ink's printability was thoroughly characterized to assure homogeneous and reproducible printing results. A new buffer mediated synthesis method for GG-MA resulted in consistent degrees of substitution which allowed the creation of large 5 g batches. We found that both the new synthesis as well as cryomilling of the polymer components of the ink resulted in a decrease in viscosity from 113 kPa·s to 11.3 kPa·s at a shear rate of 0.1 s−1 but increased ink homogeneity. The ink homogeneity was assessed through thermogravimetric analysis and a newly developed extrusion force measurement setup. The ink displayed strong inter-layer adhesion between two printed ink layers as well as between a layer of ink with and a layer without HAp. The large polymer batch production along with the characterization of the ink during the manufacturing process allows ink production in the gram scale and could be used in applications such as the printing of osteochondral grafts.
AUTHOR Lee, Jia Min and Yeong, Wai Yee
Title Engineering macroscale cell alignment through coordinated toolpath design using support-assisted 3D bioprinting [Abstract]
Year 2020
Journal/Proceedings Journal of The Royal Society Interface
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Abstract
Aligned cells provide direction-dependent mechanical properties that influence biological and mechanical function in native tissues. Alignment techniques such as casting and uniaxial stretching cannot fully replicate the complex fibre orientation of native tissue such as the heart. In this study, bioprinting is used to direct the orientation of cell alignment. A 0°–90° grid structure was printed to assess the robustness of the support-assisted bioprinting technique. The variation in the angles of the grid pattern is designed to mimic the differences in fibril orientation of native tissues, where angles of cell alignment vary across the different layers. Through bioprinting of a cell–hydrogel mixture, C2C12 cells displayed directed alignment along the longitudinal axis of printed struts. Cell alignment is induced through firstly establishing structurally stable constructs (i.e. distinct 0°–90° structures) and secondly, allowing cells to dynamically remodel the bioprinted construct. Herein reports a method of inducing a macroscale level of controlled cell alignment with angle variation. This was not achievable both in terms of methods (i.e. conventional alignment techniques such as stretching and electrical stimulation) and magnitude (i.e. hydrogel features with less than 100 µm features).
AUTHOR Hauptstein, Julia and Böck, Thomas and Bartolf-Kopp, Michael and Forster, Leonard and Stahlhut, Philipp and Nadernezhad, Ali and Blahetek, Gina and Zernecke-Madsen, Alma and Detsch, Rainer and Jüngst, Tomasz and Groll, Jürgen and Teßmar, Jörg and Blunk, Torsten
Title Hyaluronic Acid-Based Bioink Composition Enabling 3D Bioprinting and Improving Quality of Deposited Cartilaginous Extracellular Matrix [Abstract]
Year 2020
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract In 3D bioprinting, bioinks with high concentrations of polymeric materials are frequently used to enable fabrication of 3D cell-hydrogel constructs with sufficient stability. However, this is often associated with restricted cell bioactivity and an inhomogeneous distribution of newly produced extracellular matrix (ECM). Therefore, this study investigates bioink compositions based on hyaluronic acid (HA), an attractive material for cartilage regeneration, which allow for reduction of polymer content. Thiolated HA and allyl-modified poly(glycidol) in varying concentrations are UV-crosslinked. To adapt bioinks to poly(ε-caprolactone) (PCL)-supported 3D bioprinting, the gels are further supplemented with 1 wt% unmodified high molecular weight HA (hmHA) and chondrogenic differentiation of incorporated human mesenchymal stromal cells is assessed. Strikingly, addition of hmHA to gels with a low polymer content (3 wt%) results in distinct increase of construct quality with a homogeneous ECM distribution throughout the constructs, independent of the printing process. Improved ECM distribution in those constructs is associated with increased construct stiffness after chondrogenic differentiation, as compared to higher concentrated constructs (10 wt%), which only show pericellular matrix deposition. The study contributes to effective bioink development, demonstrating dual function of a supplement enabling PCL-supported bioprinting and at the same time improving biological properties of the resulting constructs.
AUTHOR De Moor, Lise and Fernandez, Sélina and Vercruysse, Chris and Tytgat, Liesbeth and Asadian, Mahtab and De Geyter, Nathalie and Van Vlierberghe, Sandra and Dubruel, Peter and Declercq, Heidi
Title Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids [Abstract]
Year 2020
Journal/Proceedings Frontiers in Bioengineering and Biotechnology
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DOI/URL DOI
Abstract
To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 μm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting.
AUTHOR López-Carrasco, Amparo and Martín-Vañó, Susana and Burgos-Panadero, Rebeca and Monferrer, Ezequiel and Berbegall, Ana P. and Fernández-Blanco, Beatriz and Navarro, Samuel and Noguera, Rosa
Title Impact of extracellular matrix stiffness on genomic heterogeneity in MYCN-amplified neuroblastoma cell line [Abstract]
Year 2020
Journal/Proceedings Journal of Experimental & Clinical Cancer Research
Reftype López-Carrasco2020
DOI/URL DOI
Abstract
Increased tissue stiffness is a common feature of malignant solid tumors, often associated with metastasis and poor patient outcomes. Vitronectin, as an extracellular matrix anchorage glycoprotein related to a stiff matrix, is present in a particularly increased quantity and specific distribution in high-risk neuroblastoma. Furthermore, as cells can sense and transform the proprieties of the extracellular matrix into chemical signals through mechanotransduction, genotypic changes related to stiffness are possible.
AUTHOR Ruiz-Cantu, Laura and Gleadall, Andrew and Faris, Callum and Segal, Joel and Shakesheff, Kevin and Yang, Jing
Title Multi-material 3D bioprinting of porous constructs for cartilage regeneration [Abstract]
Year 2020
Journal/Proceedings Materials Science and Engineering: C
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DOI/URL URL DOI
Abstract
The current gold standard for nasal reconstruction after rhinectomy or severe trauma includes transposition of autologous cartilage grafts in conjunction with coverage using an autologous skin flap. Harvesting autologous cartilage requires a major additional procedure that may create donor site morbidity. Major nasal reconstruction also requires sculpting autologous cartilages to form a cartilage framework, which is complex, highly skill-demanding and very time consuming. These limitations have prompted facial reconstructive surgeons to explore different techniques such as tissue engineered cartilage. This work explores the use of multi-material 3D bioprinting with chondrocyte-laden gelatin methacrylate (GelMA) and polycaprolactone (PCL) to fabricate constructs that can potentially be used for nasal reconstruction. In this study, we have investigated the effect of 3D manufacturing parameters including temperature, needle gauge, UV exposure time, and cell carrier formulation (GelMA) on the viability and functionality of chondrocytes in bioprinted constructs. Furthermore, we printed chondrocyte-laden GelMA and PCL into composite constructs to combine biological and mechanical properties. It was found that 20% w/v GelMA was the best concentration for the 3D bioprinting of the chondrocytes without comprising the scaffold's porous structure and cell functionality. In addition, the 3D bioprinted constructs showed neocartilage formation and similar mechanical properties to nasal alar cartilage after a 50-day culture period. Neocartilage formation was also observed in the composite constructs evidenced by the presence of glycosaminoglycans and collagen type II. This study shows the feasibility of manufacturing neocartilage using chondrocytes/GelMA/PCL 3D bioprinted porous constructs which could be applied as a method for fabricating implants for nose reconstruction.
AUTHOR Lim, Khoon S. and Abinzano, Florencia and Bernal, Paulina Nuñez and Albillos Sanchez, Ane and Atienza-Roca, Pau and Otto, Iris A. and Peiffer, Quentin C. and Matsusaki, Michiya and Woodfield, Tim B. F. and Malda, Jos and Levato, Riccardo
Title One-Step Photoactivation of a Dual-Functionalized Bioink as Cell Carrier and Cartilage-Binding Glue for Chondral Regeneration [Abstract]
Year 2020
Journal/Proceedings Advanced Healthcare Materials
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Abstract
Abstract Cartilage defects can result in pain, disability, and osteoarthritis. Hydrogels providing a chondroregeneration-permissive environment are often mechanically weak and display poor lateral integration into the surrounding cartilage. This study develops a visible-light responsive gelatin ink with enhanced interactions with the native tissue, and potential for intraoperative bioprinting. A dual-functionalized tyramine and methacryloyl gelatin (GelMA-Tyr) is synthesized. Photo-crosslinking of both groups is triggered in a single photoexposure by cell-compatible visible light in presence of tris(2,2′-bipyridyl)dichlororuthenium(II) and sodium persulfate as initiators. Neo-cartilage formation from embedded chondroprogenitor cells is demonstrated in vitro, and the hydrogel is successfully applied as bioink for extrusion-printing. Visible light in situ crosslinking in cartilage defects results in no damage to the surrounding tissue, in contrast to the native chondrocyte death caused by UV light (365–400 nm range), commonly used in biofabrication. Tyramine-binding to proteins in native cartilage leads to a 15-fold increment in the adhesive strength of the bioglue compared to pristine GelMA. Enhanced adhesion is observed also when the ink is extruded as printable filaments into the defect. Visible-light reactive GelMA-Tyr bioinks can act as orthobiologic carriers for in situ cartilage repair, providing a permissive environment for chondrogenesis, and establishing safe lateral integration into chondral defects.
AUTHOR Schipani, Rossana and Scheurer, Stefan and Florentin, Romain and Critchley, Susan E. and Kelly, Daniel John
Title Reinforcing interpenetrating network hydrogels with 3D printed polymer networks to engineer cartilage mimetic composites [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Abstract
Engineering constructs that mimic the complex structure, composition and biomechanics of the articular cartilage represents a promising route to joint regeneration. Such tissue engineering strategies require the development of biomaterials that mimic the mechanical properties of articular cartilage whilst simultaneously providing an environment supportive of chondrogenesis. Here three-dimensional (3D) bioprinting is used to develop polycaprolactone (PCL) fibre networks to mechanically reinforce interpenetrating network (IPN) hydrogels consisting of alginate and gelatin methacryloyl (GelMA). Inspired by the significant tension-compression nonlinearity of the collagen network in articular cartilage, we printed reinforcing PCL networks with different ratios of tensile to compressive modulus. Synergistic increases in compressive modulus were observed when IPN hydrogels were reinforced with PCL networks that were relatively soft in compression and stiff in tension. The resulting composites possessed equilibrium and dynamic mechanical properties that matched or approached that of native articular cartilage. Finite Element (FE) modelling revealed that the reinforcement of IPN hydrogels with specific PCL networks limited radial expansion and increased the hydrostatic pressure generated within the IPN upon the application of compressive loading. Next, multiple-tool biofabrication techniques were used to 3D bioprint PCL reinforced IPN hydrogels laden with a co-culture of bone marrow-derived stromal cells (BMSCs) and chondrocytes (CCs). The bioprinted biomimetic composites were found to support robust chondrogenesis, with encapsulated cells producing hyaline-like cartilage that stained strongly for sGAG and type II collagen deposition, and negatively for type X collagen and calcium deposition. Taken together, these results demonstrate how 3D bioprinting can be used to engineer constructs that are both pro-chondrogenic and biomimetic of the mechanical properties of articular cartilage.
AUTHOR Li, Huijun and Tan, Yu Jun and Kiran, Raj and Tor, Shu Beng and Zhou, Kun
Title Submerged and non-submerged 3D bioprinting approaches for the fabrication of complex structures with the hydrogel pair GelMA and alginate/methylcellulose [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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Abstract
The extrusion-based bioprinting of hydrogels such as gelatin methacrylate (GelMA) into structures with complex shape suffers from poor printability due to their low viscosity. The present study deals with hydrogel materials by using the mixture of cell-laden photopolymerizable GelMA as a main printing material and the mixture of alginate and methylcellulose (Alg/MC) as a support material because of its high viscosity and good thixotropic property. One extrusion-based approach is developed by printing the two mixtures into structures in an alternating layer-by-layer manner, with the electrostatic interactions between polycationic GelMA and polyanionic Alg/MC contributing to the integrity of the structures. The final printed structures are exposed to ultraviolet (UV) light to form crosslinks in GelMA through photopolymerization for further structural strengthening. The one-time UV exposure minimizes cell damage in cell-GelMA, demonstrating an advantage over those in previously reported studies that required repeated UV exposures upon the printing of each layer of a structure. The other approach is developed by submerging the extrusion nozzle into a bath of Alg/MC to print cell-laden GelMA structures, which, upon printing completion, are also subject to one-time UV exposure before the removal of the support material Alg/MC. A flower with living cells is printed to demonstrate the capability of the second approach of fabricating structures with geometric complexity. The structures printed using both approaches demonstrate a well-maintained shape fidelity, structural integrity and cell viability of over 93% up to five culturing days. The proposed two printing approaches based on the cell-GelMA and Alg/MC pair will be beneficial for exploring new opportunities in bioprinting.
AUTHOR Augurio, Adriana and Cortelletti, Paolo and Tognato, Riccardo and Rios, Anne and Levato, Riccardo and Malda, Jos and Alini, Mauro and Eglin, David and Giancane, Gabriele and Speghini, Adolfo and Serra, Tiziano
Title A Multifunctional Nanocomposite Hydrogel for Endoscopic Tracking and Manipulation [Abstract]
Year 2019
Journal/Proceedings Advanced Intelligent Systems
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Abstract
In this work, the fabrication of multi-responsive and hierarchically organized nanomaterial by using core-shell SrF2 upconverting nanoparticles, doped with Yb3+, Tm3+, Nd3+ incorporated into gelatin methacryloyl matrix, is reported. Upon 800 nm excitation, deep monitoring of 3D printed constructs is demonstrated. Addition of magnetic self-assembly of iron oxide nanoparticles within the hydrogel provides anisotropic structuration from the nano- to the macro-scale and magnetic responsiveness permitting remote manipulation. The present study provides a new strategy for the fabrication of a novel highly organized multi-responsive material using additive manufacturing, which could have important implications in biomedicine. This article is protected by copyright. All rights reserved.
AUTHOR Creusen, Guido and Roshanasan, Ardeshir and Garcia Lopez, Javier and Peneva, Kalina and Walther, Andreas
Title Bottom-up design of model network elastomers and hydrogels from precise star polymers [Abstract]
Year 2019
Journal/Proceedings Polymer Chemistry
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Abstract
We introduce a platform for the simultaneous design of model network hydrogels and bulk elastomers based on well-defined water-soluble star polymers with a low glass transition temperature (Tg). This platform is enabled via the development of a synthetic route to a new family of 4-arm star polymers based on water-soluble poly(triethylene glycol methyl ether acrylate) (p(mTEGA)){,} which after quantitative introduction of functional end-groups can serve as suitable building blocks for model network formation. We first describe in detail the synthesis of highly defined star polymers using light and Cu-wire mediated Cu-based reversible deactivation radical polymerization. The resulting polymers exhibit narrow dispersities and controlled arm length at very high molecular weights{,} and feature a desirable low Tg of −55 °C. Subsequently{,} we elucidate the rational design of the stiffness and elasticity in covalent model network elastomers and hydrogels formed by fast photo-crosslinking for different arm lengths{,} and construct thermally reversible model network hydrogels based on dynamic supramolecular bonds. In addition{,} we describe preliminary 3D-printing applications. This work provides a key alternative to commonly used star-poly(ethylene glycol) (PEG) for model hydrogel networks{,} and demonstrates access to new main and side chain chemistries{,} thus chain stiffnesses and entanglement molecular weight{,} and{,} critically{,} enables the simultaneous study of the mechanical behavior of bulk network elastomers and swollen hydrogels with the same network topology. In a wider perspective{,} this work also highlights the need for advancing precision polymer chemistry to allow for an understanding of architectural control for the rational design of functional mechanical network materials.
AUTHOR Zhuang, Pei and Ng, Wei Long and An, Jia and Chua, Chee Kai and Tan, Lay Poh
Title Layer-by-layer ultraviolet assisted extrusion-based (UAE) bioprinting of hydrogel constructs with high aspect ratio for soft tissue engineering applications [Abstract]
Year 2019
Journal/Proceedings PLOS ONE
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Abstract
One of the major challenges in the field of soft tissue engineering using bioprinting is fabricating complex tissue constructs with desired structure integrity and mechanical property. To accomplish such requirements, most of the reported works incorporated reinforcement materials such as poly(ϵ-caprolactone) (PCL) polymer within the 3D bioprinted constructs. Although this approach has made some progress in constructing soft tissue-engineered scaffolds, the mechanical compliance mismatch and long degradation period are not ideal for soft tissue engineering. Herein, we present a facile bioprinting strategy that combines the rapid extrusion-based bioprinting technique with an in-built ultraviolet (UV) curing system to facilitate the layer-by-layer UV curing of bioprinted photo-curable GelMA-based hydrogels to achieve soft yet stable cell-laden constructs with high aspect ratio for soft tissue engineering. GelMA is supplemented with a viscosity enhancer (gellan gum) to improve the bio-ink printability and shape fidelity while maintaining the biocompatibility before crosslinking via a layer-by-layer UV curing process. This approach could eventually fabricate soft tissue constructs with high aspect ratio (length to diameter) of ≥ 5. The effects of UV source on printing resolution and cell viability were also studied. As a proof-of-concept, small building units (3D lattice and tubular constructs) with high aspect ratio are fabricated. Furthermore, we have also demonstrated the ability to perform multi-material printing of tissue constructs with high aspect ratio along both the longitudinal and transverse directions for potential applications in tissue engineering of soft tissues. This layer-by-layer ultraviolet assisted extrusion-based (UAE) Bioprinting may provide a novel strategy to develop soft tissue constructs with desirable structure integrity.
AUTHOR Zhou, Miaomiao and Lee, Bae Hoon and Tan, Yu Jun and Tan, Lay Poh
Title Microbial transglutaminase induced controlled crosslinking of gelatin methacryloyl to tailor rheological properties for 3D printing [Abstract]
Year 2019
Journal/Proceedings Biofabrication
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Abstract
Gelatin methacryloyl (GelMA) is a versatile biomaterial that has been shown to possess many advantages such as good biocompatibility, support for cell growth, tunable mechanical properties, photocurable capability, and low material cost. Due to these superior properties, much research has been carried out to develop GelMA as a bioink for bioprinting. However, there are still many challenges, and one major challenge is the control of its rheological properties to yield good printability. Herein, this study presents a strategy to control the rheology of GelMA through partial enzymatic crosslinking. Unlike other enzymatic crosslinking strategies where the rheological properties could not be controlled once reaction takes place, we could, to a large extent, keep the rheological properties stable by introducing a deactivation step after obtaining the optimized rheological properties. Ca2+-independent microbial transglutaminase (MTGase) was introduced to partially catalyze covalent bond formation between chains of GelMA. The enzyme was then deactivated to prevent further uncontrolled crosslinking that would render the hydrogel not printable. After printing, a secondary post-printing crosslinking step (photo crosslinking) was then introduced to ensure long-term stability of the printed structure for subsequent cell studies. Biocompatibility studies carried out using cells encapsulated in the printed structure showed excellent cell viability for at least 7 d. This strategy for better control of rheological properties of GelMA could more significantly enhance the usability of this material as bioink for bioprinting of cell-laden structures for soft tissue engineering.
AUTHOR Pan, Houwen Matthew and Chen, Shengyang and Jang, Tae-Sik and Han, Win Tun and Jung, Hyun-do and Li, Yaning and Song, Juha
Title Plant seed-inspired cell protection, dormancy, and growth for large-scale biofabrication [Abstract]
Year 2019
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
Biofabrication technologies have endowed us with the capability to fabricate complex biological constructs. However, cytotoxic biofabrication conditions have been a major challenge for their clinical application, leading to a trade-off between cell viability and scalability of biofabricated constructs. Taking inspiration from nature, we proposed a cell protection strategy which mimicks the protected and dormant state of plant seeds in adverse external conditions and their germination in response to appropriate environmental cues. Applying this bioinspired strategy to biofabrication, we successfully preserved cell viability and enhanced the seeding of cell-laden biofabricated constructs via a cytoprotective pyrogallol (PG)-alginate encapsulation system. Our cytoprotective encapsulation technology utilizes PG-triggered sporulation and germination processes to preserve cells, is mechanically robust, chemically resistant, and highly customizable to adequately match cell protectability with cytotoxicity of biofabrication conditions. More importantly, the facile and tunable decapsulation of our PG-alginate system allows for effective germination of dormant cells, under typical culture conditions. With this approach, we have successfully achieved a biofabrication process which is reproducible, scalable, and provided a practical solution for off-the-shelf availability, shipping and temporary storage of fabricated bio-constructs.
AUTHOR Petta, D. and Armiento, A. R. and Grijpma, D. and Alini, M. and Eglin, D. and D'Este, M.
Title 3D bioprinting of a hyaluronan bioink through enzymatic-and visible light-crosslinking [Abstract]
Year 2018
Journal/Proceedings Biofabrication
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DOI/URL DOI
Abstract
Extrusion-based three-dimensional bioprinting relies on bioinks engineered to combine viscoelastic properties for extrusion and shape retention, and biological properties for cytocompatibility and tissue regeneration. To satisfy these conflicting requirements, bioinks often utilize either complex mixtures or complex modifications of biopolymers. In this paper we introduce and characterize a bioink exploiting a dual crosslinking mechanism, where an enzymatic reaction forms a soft gel suitable for cell encapsulation and extrusion, while a visible light photo-crosslinking allows shape retention of the printed construct. The influence of cell density and cell type on the rheological and printability properties was assessed correlating the printing outcomes with the damping factor, a rheological characteristic independent of the printing system. Stem cells, chondrocytes and fibroblasts were encapsulated, and their viability was assessed up to 14 days with live/dead, alamar blue and trypan blue assays. Additionally, the impact of the printing parameters on cell viability was investigated. Owing to its straightforward preparation, low modification, presence of two independent crosslinking mechanisms for tuning shear-thinning independently of the final shape fixation, the use of visible green instead of UV light, the possibility of encapsulating and sustaining the viability of different cell types, the hyaluronan bioink here presented is a valid biofabrication tool for producing 3D printed tissue-engineered constructs.
AUTHOR Agarwala, Shweta and Lee, Jia Min and Ng, Wei Long and Layani, Michael and Yeong, Wai Yee and Magdassi, Shlomo
Title A novel 3D bioprinted flexible and biocompatible hydrogel bioelectronic platform [Abstract]
Year 2018
Journal/Proceedings Biosensors and Bioelectronics
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DOI/URL URL DOI
Abstract
Abstract Bioelectronics platforms are gaining widespread attention as they provide a template to study the interactions between biological species and electronics. Decoding the effect of the electrical signals on the cells and tissues holds the promise for treating the malignant tissue growth, regenerating organs and engineering new-age medical devices. This work is a step forward in this direction, where bio- and electronic materials co-exist on one platform without any need for post processing. We fabricate a freestanding and flexible hydrogel based platform using 3D bioprinting. The fabrication process is simple, easy and provides a flexible route to print materials with preferred shapes, size and spatial orientation. Through the design of interdigitated electrodes and heating coil, the platform can be tailored to print various circuits for different functionalities. The biocompatibility of the printed platform is tested using C2C12 murine myoblasts cell line. Furthermore, normal human dermal fibroblasts (primary cells) are also seeded on the platform to ascertain the compatibility.
AUTHOR Tognato, Riccardo and Armiento, Angela R. and Bonfrate, Valentina and Levato, Riccardo and Malda, Jos and Alini, Mauro and Eglin, David and Giancane, Gabriele and Serra, Tiziano
Title A Stimuli-Responsive Nanocomposite for 3D Anisotropic Cell-Guidance and Magnetic Soft Robotics [Abstract]
Year 2018
Journal/Proceedings Advanced Functional Materials
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DOI/URL DOI
Abstract
Abstract Stimuli-responsive materials have the potential to enable the generation of new bioinspired devices with unique physicochemical properties and cell-instructive ability. Enhancing biocompatibility while simplifying the production methodologies, as well as enabling the creation of complex constructs, i.e., via 3D (bio)printing technologies, remains key challenge in the field. Here, a novel method is presented to biofabricate cellularized anisotropic hybrid hydrogel through a mild and biocompatible process driven by multiple external stimuli: magnetic field, temperature, and light. A low-intensity magnetic field is used to align mosaic iron oxide nanoparticles (IOPs) into filaments with tunable size within a gelatin methacryloyl matrix. Cells seeded on top or embedded within the hydrogel align to the same axes of the IOPs filaments. Furthermore, in 3D, C2C12 skeletal myoblasts differentiate toward myotubes even in the absence of differentiation media. 3D printing of the nanocomposite hydrogel is achieved and creation of complex heterogeneous structures that respond to magnetic field is demonstrated. By combining the advanced, stimuli-responsive hydrogel with the architectural control provided by bioprinting technologies, 3D constructs can also be created that, although inspired by nature, express functionalities beyond those of native tissue, which have important application in soft robotics, bioactuators, and bionic devices.
AUTHOR Schmieg, Barbara and Schimek, Adrian and Franzreb, Matthias
Title Development and performance of a 3D‐printable Polyethylenglycol‐Diacrylate hydrogel suitable for enzyme entrapment and long‐term biocatalytic applications [Abstract]
Year 2018
Journal/Proceedings Engineering in Life Sciences
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Abstract
Physical entrapment of enzymes within a porous matrix is a fast and gentle process to immobilize biocatalysts to enable their recycling and long‐term use. This study introduces the development of a biocompatible 3D‐printing material suitable for enzyme entrapment, while having good rheological and UV‐hardening properties. Three different viscosity‐enhancing additives have been tested in combination with a polyethylenglycol‐diacrylate‐based hydrogel system. The addition of polyxanthan or hectorite clay particles results in hydrogels that degrade over hours or days, releasing entrapped compounds. In contrast, the addition of nanometer‐sized silicate particles ensures processability while preventing disintegration of the hydrogel. Lattice structures with a total height of 6 mm consisting of 40 layers were 3D‐printed with all materials and characterized by image analysis. Rheological measurements identified a shear stress window of 200 < τ < 500 Pa at shear rates of 25 s−1 and 25°C for well‐defined geometries with an extrusion‐based printhead. Enzymes immobilized in these long‐term stable hydrogel structures retained an effective activity of approximately 10% compared to the free enzyme in solution. It could be shown that the reduction of effective activity isn't caused by a significant reduction of the intrinsic enzyme activity but by mass transfer limitations within the printed hydrogel structures. This article is protected by copyright. All rights reserved
AUTHOR Bastola, A. K. and Paudel, M. and Li, L.
Title Development of hybrid magnetorheological elastomers by 3D printing [Abstract]
Year 2018
Journal/Proceedings Polymer
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DOI/URL URL DOI
Abstract
Intelligent or smart materials have one or more properties that can be significantly changed in a controlled fashion by external stimuli, such as temperature, pH, electric or magnetic fields, etc. Magnetorheological (MR) materials are a class of smart materials whose properties can be varied by applying an external magnetic field. In this work, the possibility of employing a suitable 3D printing technology for the development of one of the smart MR materials, the magnetorheological elastomer (MRE) has been explored. In order to achieve such 3D printing, a multi-material printing is implemented, where a controlled volume of MR fluid is encapsulated within an elastomer matrix in the layer-by-layer fashion. The choice of printing materials determines the final structure of the 3D printed hybrid MR elastomer. Printing with a vulcanizing MR suspension produces the solid MR structure inside the elastomer matrix while printing with a non-vulcanizing MR suspension (MR fluid) results in the structures that the MR fluid is encapsulated inside the elastomer matrix. The 3D printability of different materials has been studied by measuring their rheological properties and we found that the highly shear thinning and thixotropic properties are important for 3D printability. The quality of the printed filaments strongly depends on the key printing parameters such as extrusion pressure, initial height and feed rate. The experimental results from the forced vibration testing show that the 3D printed MR elastomers could change their elastic and damping properties when exposed to the external magnetic field. Furthermore, the 3D printed MR elastomer also exhibits the anisotropic behavior when the direction of the magnetic field is changed with respect to the orientation of the printed filaments. This study has demonstrated that the 3D printing is viable for fabrication of hybrid MR elastomers with controlled structures of magnetic particles or MR fluids.
AUTHOR Li, Huijun and Tan, Yu Jun and Liu, Sijun and Li, Lin
Title Three-Dimensional Bioprinting of Oppositely Charged Hydrogels with Super Strong Interface Bonding [Abstract]
Year 2018
Journal/Proceedings ACS Applied Materials and Interfaces
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DOI/URL DOI
Abstract
A novel strategy to improve the adhesion between printed layers of three-dimensional (3D) printed constructs is developed by exploiting the interaction between two oppositely charged hydrogels. Three anionic hydrogels [alginate, xanthan, and κ-carrageenan (Kca)] and three cationic hydrogels [chitosan, gelatin, and gelatin methacrylate (GelMA)] are chosen to find the optimal combination of two oppositely charged hydrogels for the best 3D printability with strong interface bonding. Rheological properties and printability of the hydrogels, as well as structural integrity of printed constructs in cell culture medium, are studied as functions of polymer concentration and the combination of hydrogels. Kca2 (2 wt % Kca hydrogel) and GelMA10 (10 wt % GelMA hydrogel) are found to be the best combination of oppositely charged hydrogels for 3D printing. The interfacial bonding between a Kca layer and a GelMA layer is proven to be significantly higher than that of the bilayered Kca or bilayered GelMA because of the formation of polyelectrolyte complexes between the oppositely charged hydrogels. A good cell viability of >96% is obtained for the 3D-bioprinted Kca–GelMA construct. This novel strategy has a great potential for 3D bioprinting of layered constructs with a strong interface bonding.
AUTHOR Suntornnond, R. and Tan, E. Y. S. and An, J. and Chua, C. K.
Title A highly printable and biocompatible hydrogel composite for direct printing of soft and perfusable vasculature-like structures [Abstract]
Year 2017
Journal/Proceedings Scientific Reports
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Abstract
Vascularization is one major obstacle in bioprinting and tissue engineering. In order to create thick tissues or organs that can function like original body parts, the presence of a perfusable vascular system is essential. However, it is challenging to bioprint a hydrogel-based three-dimensional vasculature-like structure in a single step. In this paper, we report a new hydrogel-based composite that offers impressive printability, shape integrity, and biocompatibility for 3D bioprinting of a perfusable complex vasculature-like structure. The hydrogel composite can be used on a non-liquid platform and is printable at human body temperature. Moreover, the hydrogel composite supports both cell proliferation and cell differentiation. Our results represent a potentially new vascularization strategy for 3D bioprinting and tissue engineering.
AUTHOR Bastola, A. K. and Hoang, V. T. and Li, L.
Title A novel hybrid magnetorheological elastomer developed by 3D printing [Abstract]
Year 2017
Journal/Proceedings Materials and Design
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Abstract
Abstract In this study, a novel magnetorheological (MR) hybrid elastomer has been developed using a 3D printing method. In such an MR hybrid elastomer, a controlled volume of an MR fluid was encapsulated layer by layer into an elastomer matrix by means of a 3D printer and each layer was a composite structure consisting of an MR fluid and an elastomer. Similar to current MR fluids and MR elastomers, mechanical properties of 3D printed MR hybrid elastomers could be controlled via an externally applied magnetic field. The experimental results showed that the relative change in the damping capability of the new MR elastomer was more pronounced than the change in its stiffness when exposed to an external magnetic field. The study demonstrated that the 3D printing technique is feasible for fabrication of MR elastomers with controlled microstructures including magnetic particles or MR fluids. The 3D printed MR hybrid elastomer is also a potential material as a tunable spring-damper element.
AUTHOR Siqueira, Gilberto and Kokkinis, Dimitri and Libanori, Rafael and Hausmann, Michael K. and Gladman, Amelia Sydney and Neels, Antonia and Tingaut, Philippe and Zimmermann, Tanja and Lewis, Jennifer A. and Studart, André R.
Title Cellulose Nanocrystal Inks for 3D Printing of Textured Cellular Architectures [Abstract]
Year 2017
Journal/Proceedings Advanced Functional Materials
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DOI/URL DOI
Abstract
3D printing of renewable building blocks like cellulose nanocrystals offers an attractive pathway for fabricating sustainable structures. Here, viscoelastic inks composed of anisotropic cellulose nanocrystals (CNC) that enable patterning of 3D objects by direct ink writing are designed and formulated. These concentrated inks are composed of CNC particles suspended in either water or a photopolymerizable monomer solution. The shear-induced alignment of these anisotropic building blocks during printing is quantified by atomic force microscopy, polarized light microscopy, and 2D wide-angle X-ray scattering measurements. Akin to the microreinforcing effect in plant cell walls, the alignment of CNC particles during direct writing yields textured composites with enhanced stiffness along the printing direction. The observations serve as an important step forward toward the development of sustainable materials for 3D printing of cellular architectures with tailored mechanical properties.
AUTHOR Baumann, Bernhard and Jungst, Tomasz and Stichler, Simone and Feineis, Susanne and Wiltschka, Oliver and Kuhlmann, Matthias and Lindén, Mika and Groll, Jürgen
Title Control of Nanoparticle Release Kinetics from 3D Printed Hydrogel Scaffolds [Abstract]
Year 2017
Journal/Proceedings Angewandte Chemie International Edition
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DOI/URL DOI
Abstract
The convergence of biofabrication with nanotechnology is largely unexplored but enables geometrical control of cell-biomaterial arrangement combined with controlled drug delivery and release. As a step towards integration of these two fields of research, this study demonstrates that modulation of electrostatic nanoparticle–polymer and nanoparticle–nanoparticle interactions can be used for tuning nanoparticle release kinetics from 3D printed hydrogel scaffolds. This generic strategy can be used for spatiotemporal control of the release kinetics of nanoparticulate drug vectors in biofabricated constructs.
AUTHOR Mouser, V. H. M. and Abbadessa, A. and Levato, R. and Hennink, W. E. and Vermonden, T. and Gawlitta, D. and Malda, J.
Title Development of a thermosensitive HAMA-containing bio-ink for the fabrication of composite cartilage repair constructs [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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Abstract
Fine-tuning of bio-ink composition and material processing parameters is crucial for the development of biomechanically relevant cartilage constructs. This study aims to design and develop cartilage constructs with tunable internal architectures and relevant mechanical properties. More specifically, the potential of methacrylated hyaluronic acid (HAMA) added to thermosensitive hydrogels composed of methacrylated poly[ N -(2-hydroxypropyl)methacrylamide mono/dilactate] (pHPMA-lac)/polyethylene glycol (PEG) triblock copolymers, to optimize cartilage-like tissue formation by embedded chondrocytes, and enhance printability was explored. Additionally, co-printing with polycaprolactone (PCL) was performed for mechanical reinforcement. Chondrocyte-laden hydrogels composed of pHPMA-lac-PEG and different concentrations of HAMA (0%–1% w/w) were cultured for 28 d in vitro and subsequently evaluated for the presence of cartilage-like matrix. Young’s moduli were determined for hydrogels with the different HAMA concentrations. Additionally, hydrogel/PCL constructs with different internal architectures were co-printed and analyzed for their mechanical properties. The results of this study demonstrated a dose-dependent effect of HAMA concentration on cartilage matrix synthesis by chondrocytes. Glycosaminoglycan (GAG) and collagen type II content increased with intermediate HAMA concentrations (0.25%–0.5%) compared to HAMA-free controls, while a relatively high HAMA concentration (1%) resulted in increased fibrocartilage formation. Young’s moduli of generated hydrogel constructs ranged from 14 to 31 kPa and increased with increasing HAMA concentration. The pHPMA-lac-PEG hydrogels with 0.5% HAMA were found to be optimal for cartilage-like tissue formation. Therefore, this hydrogel system was co-printed with PCL to generate porous or solid constructs with different mesh sizes. Young’s moduli of these composite constructs were in the range of native cartilage (3.5–4.6 MPa). Interestingly, the co-printing procedure influenced the mechanical properties of the final constructs. These findings are relevant for future bio-ink development, as they demonstrate the importance of selecting proper HAMA concentrations, as well as appropriate print settings and construct designs for optimal cartilage matrix deposition and final mechanical properties of constructs, respectively.
AUTHOR Stichler, Simone and Böck, Thomas and Paxton, Naomi Claire and Bertlein, Sarah and Levato, Riccardo and Schill, Verena and Smolan, Willi and Malda, Jos and Tessmar, Joerg and Blunk, Torsten and Groll, Juergen
Title Double printing of hyaluronic acid / poly(glycidol) hybrid hydrogels with poly(ε-caprolactone) for MSC chondrogenesis [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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Abstract
Abstract This study investigates the use of allyl-functionalized poly(glycidol)s (P(AGE-co-G)) as cytocompatible cross-linker for thiol-functionalized hyaluronic acid (HA-SH) and the optimization of this hybrid hydrogel as bioink for 3D bioprinting. Chemical cross-linking of gels with 10 wt.% overall polymer concentration was achieved by UV-induced radical thiol-ene coupling between the thiol and allyl groups. Addition of unmodified high molecular weight HA (1.36 MDa) allowed tuning of the rheology for extrusion based bioprinting. Incorporation of additional HA resulted in hydrogels with lower Young’s modulus and higher swelling ratio especially in the first 24 h, but a comparable equilibrium swelling for all gels after 24 h. Embedding of human and equine mesenchymal stem cells (MSCs) in the gels and subsequent in vitro culture showed promising chondrogenic differentiation after 21 d for cells from both origins. Moreover, cells could be printed with these gels, and embedded hMSCs showed good cell survival for at least 21 d in culture. To achieve mechanical stable and robust constructs for the envisioned application in articular cartilage, the formulations were adjusted for double printing with the thermoplastic poly--caprolactone (PCL).
AUTHOR Levato, Riccardo and Webb, William R. and Otto, Iris A. and Mensinga, Anneloes and Zhang, Yadan and van Rijen, Mattie and van Weeren, René and Khan, Ilyas M. and Malda, Jos
Title The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells
Year 2017
Journal/Proceedings Acta Biomaterialia
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DOI/URL URL
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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DOI/URL DOI
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 Abbadessa, Anna and Landín, Mariana and Oude Blenke, Erik and Hennink, Wim E. and Vermonden, Tina
Title Two-component thermosensitive hydrogels: Phase separation affecting rheological behavior [Abstract]
Year 2017
Journal/Proceedings European Polymer Journal
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Abstract
Abstract Extracellular matrices are mainly composed of a mixture of different biopolymers and therefore the use of two or more building blocks for the development of tissue-mimicking hydrogels is nowadays an attractive strategy in tissue-engineering. Multi-component hydrogel systems may undergo phase separation, which in turn can lead to new, unexpected material properties. The aim of this study was to understand the role of phase separation on the mechanical properties and 3D printability of hydrogels composed of triblock copolymers of polyethylene glycol (PEG) and methacrylated poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate) (pHPMAlac) blended with methacrylated hyaluronic acid (HAMA). To this end, hydrogels composed of different concentrations of PEG/pHPMAlac and HAMA, were analyzed for phase behavior and rheological properties. Subsequently, phase separation and rheological behavior as function of the two polymer concentrations were mathematically processed to generate a predictive model. Results showed that PEG/pHPMAlac/HAMA hydrogels were characterized by hydrophilic, HAMA-richer internal domains dispersed in a more hydrophobic continuous phase, composed of PEG/pHPMAlac, and that the volume fraction of the dispersed phase increased by increasing HAMA concentration. Storage modulus, yield stress and viscosity increased with increasing HAMA concentration for low/medium HAMA contents (≤0.75% w/w), while a further increase of HAMA resulted in a decrease of the mentioned properties. On the other hand, by increasing the concentration of PEG/pHPMAlac these rheological properties were enhanced. The generated models showed a good fitting with experimental data, and were used to identify an exemplary 3D printability window for PEG/pHPMAlac/HAMA hydrogels, which was verified by rheological characterization and preparation of 3D printed scaffolds. In conclusion, a clear relationship between phase separation and rheological behavior in these two-component hydrogels can be described by complex functions of the two polymer concentrations. The predictive model generated in this study can be used as a valid tool for the identification of hydrogel compositions with desired, selected characteristics.
AUTHOR Daly, Andrew C. and Critchley, Susan E. and Rencsok, Emily M. and Kelly, Daniel J.
Title A comparison of different bioinks for 3D bioprinting of fibrocartilage and hyaline cartilage [Abstract]
Year 2016
Journal/Proceedings Biofabrication
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Abstract
Cartilage is a dense connective tissue with limited self-repair capabilities. Mesenchymal stem cell (MSC) laden hydrogels are commonly used for fibrocartilage and articular cartilage tissue engineering, however they typically lack the mechanical integrity for implantation into high load bearing environments. This has led to increased interested in 3D bioprinting of cell laden hydrogel bioinks reinforced with stiffer polymer fibres. The objective of this study was to compare a range of commonly used hydrogel bioinks (agarose, alginate, GelMA and BioINK™) for their printing properties and capacity to support the development of either hyaline cartilage or fibrocartilage in vitro . Each hydrogel was seeded with MSCs, cultured for 28 days in the presence of TGF- β 3 and then analysed for markers indicative of differentiation towards either a fibrocartilaginous or hyaline cartilage-like phenotype. Alginate and agarose hydrogels best supported the development of hyaline-like cartilage, as evident by the development of a tissue staining predominantly for type II collagen. In contrast, GelMA and BioINK ™ (a PEGMA based hydrogel) supported the development of a more fibrocartilage-like tissue, as evident by the development of a tissue containing both type I and type II collagen. GelMA demonstrated superior printability, generating structures with greater fidelity, followed by the alginate and agarose bioinks. High levels of MSC viability were observed in all bioinks post-printing (∼80%). Finally we demonstrate that it is possible to engineer mechanically reinforced hydrogels with high cell viability by co-depositing a hydrogel bioink with polycaprolactone filaments, generating composites with bulk compressive moduli comparable to articular cartilage. This study demonstrates the importance of the choice of bioink when bioprinting different cartilaginous tissues for musculoskeletal applications.
AUTHOR Abbadessa, Anna and Mouser, Vivian H. M. and Blokzijl, Maarten M. and Gawlitta, Debby and Dhert, Wouter J. A. and Hennink, Wim E. and Malda, Jos and Vermonden, Tina
Title A Synthetic Thermosensitive Hydrogel for Cartilage Bioprinting and Its Biofunctionalization with Polysaccharides [Abstract]
Year 2016
Journal/Proceedings Biomacromolecules
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DOI/URL DOI
Abstract
Hydrogels based on triblock copolymers of polyethylene glycol and partially methacrylated poly[N-(2-hydroxypropyl) methacrylamide mono/dilactate] make up an attractive class of biomaterials because of their biodegradability, cytocompatibility, and tunable thermoresponsive and mechanical properties. If these properties are fine-tuned, the hydrogels can be three-dimensionally bioprinted, to generate, for instance, constructs for cartilage repair. This study investigated whether hydrogels based on the polymer mentioned above with a 10% degree of methacrylation (M10P10) support cartilage formation by chondrocytes and whether the incorporation of methacrylated chondroitin sulfate (CSMA) or methacrylated hyaluronic acid (HAMA) can improve the mechanical properties, long-term stability, and printability. Chondrocyte-laden M10P10 hydrogels were cultured for 42 days to evaluate chondrogenesis. M10P10 hydrogels with or without polysaccharides were evaluated for their mechanical properties (before and after UV photo-cross-linking), degradation kinetics, and printability. Extensive cartilage matrix production occurred in M10P10 hydrogels, highlighting their potential for cartilage repair strategies. The incorporation of polysaccharides increased the storage modulus of polymer mixtures and decreased the degradation kinetics in cross-linked hydrogels. Addition of HAMA to M10P10 hydrogels improved printability and resulted in three-dimensional constructs with excellent cell viability. Hence, this novel combination of M10P10 with HAMA forms an interesting class of hydrogels for cartilage bioprinting.
AUTHOR Abbadessa, A. and Blokzijl, M. M. and Mouser, V. H. M. and Marica, P. and Malda, J. and Hennink, W. E. and Vermonden, T.
Title A thermo-responsive and photo-polymerizable chondroitin sulfate-based hydrogel for 3D printing applications [Abstract]
Year 2016
Journal/Proceedings Carbohydrate Polymers
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Abstract
Abstract The aim of this study was to design a hydrogel system based on methacrylated chondroitin sulfate (CSMA) and a thermo-sensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate)-polyethylene glycol triblock copolymer (M15P10) as a suitable material for additive manufacturing of scaffolds. {CSMA} was synthesized by reaction of chondroitin sulfate with glycidyl methacrylate (GMA) in dimethylsulfoxide at 50 °C and its degree of methacrylation was tunable up to 48.5%, by changing reaction time and {GMA} feed. Unlike polymer solutions composed of {CSMA} alone (20% w/w), mixtures based on 2% w/w of {CSMA} and 18% of {M15P10} showed strain-softening, thermo-sensitive and shear-thinning properties more pronounced than those found for polymer solutions based on {M15P10} alone. Additionally, they displayed a yield stress of 19.2 ± 7.0 Pa. The 3D printing of this hydrogel resulted in the generation of constructs with tailorable porosity and good handling properties. Finally, embedded chondrogenic cells remained viable and proliferating over a culture period of 6 days. The hydrogel described herein represents a promising biomaterial for cartilage 3D printing applications.
AUTHOR Melchels, Ferry P. W. and Blokzijl, Maarten M. and Levato, Riccardo and Peiffer, Quentin C. and de Ruijter, Myl{`{e}}ne and Hennink, Wim E. and Vermonden, Tina and Malda, Jos
Title Hydrogel-based reinforcement of 3D bioprinted constructs [Abstract]
Year 2016
Journal/Proceedings Biofabrication
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Abstract
Progress within the field of biofabrication is hindered by a lack of suitable hydrogel formulations. Here, we present a novel approach based on a hybrid printing technique to create cellularized 3D printed constructs. The hybrid bioprinting strategy combines a reinforcing gel for mechanical support with a bioink to provide a cytocompatible environment. In comparison with thermoplastics such as IMG [http://ej.iop.org/images/1758-5090/8/3/035004/bfaa2f97ieqn1.gif] {$epsilon $} -polycaprolactone, the hydrogel-based reinforcing gel platform enables printing at cell-friendly temperatures, targets the bioprinting of softer tissues and allows for improved control over degradation kinetics. We prepared amphiphilic macromonomers based on poloxamer that form hydrolysable, covalently cross-linked polymer networks. Dissolved at a concentration of 28.6%w/w in water, it functions as reinforcing gel, while a 5%w/w gelatin-methacryloyl based gel is utilized as bioink. This strategy allows for the creation of complex structures, where the bioink provides a cytocompatible environment for encapsulated cells. Cell viability of equine chondrocytes encapsulated within printed constructs remained largely unaffected by the printing process. The versatility of the system is further demonstrated by the ability to tune the stiffness of printed constructs between 138 and 263 kPa, as well as to tailor the degradation kinetics of the reinforcing gel from several weeks up to more than a year.
AUTHOR Stichler, Simone and Jungst, Tomasz and Schamel, Martha and Zilkowski, Ilona and Kuhlmann, Matthias and Bock, Thomas and Blunk, Torsten and Tessmar, Jorg and Groll, Jurgen
Title Thiol-ene Clickable Poly(glycidol) Hydrogels for Biofabrication. [Abstract]
Year 2016
Journal/Proceedings Annals of biomedical engineering
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DOI/URL DOI
Abstract
In this study we introduce linear poly(glycidol) (PG), a structural analog of poly(ethylene glycol) bearing side chains at each repeating unit, as polymer basis for bioink development. We prepare allyl- and thiol-functional linear PG that can rapidly be polymerized to a three-dimensionally cross-linked hydrogel network via UV mediated thiol-ene click reaction. Influence of polymer concentration and UV irradiation on mechanical properties and swelling behavior was examined. Thiol-functional PG was synthesized in two structural variations, one containing ester groups that are susceptible to hydrolytic cleavage, and the other one ester-free and stable against hydrolysis. This allowed the preparation of degradable and non-degradable hydrogels. Cytocompatibility of the hydrogel was demonstrated by encapsulation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Rheological properties of the hydrogels were adjusted for dispense plotting by addition of high molecular weight hyaluronic acid. The optimized formulation enabled highly reproducible plotting of constructs composed of 20 layers with an overall height of 3.90 mm.
AUTHOR M{"{u}}ller, Michael and Becher, Jana and Schnabelrauch, Matthias and Zenobi-Wong, Marcy
Title Nanostructured Pluronic hydrogels as bioinks for 3D bioprinting [Abstract]
Year 2015
Journal/Proceedings Biofabrication
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Abstract
Bioprinting is an emerging technology in the field of tissue engineering as it allows the precise positioning of biologically relevant materials in 3D, which more resembles the native tissue in our body than current homogenous, bulk approaches. There is however a lack of materials to be used with this technology and materials such as the block copolymer Pluronic have good printing properties but do not allow long-term cell culture. Here we present an approach called nanostructuring to increase the biocompatibility of Pluronic gels at printable concentrations. By mixing acrylated with unmodified Pluronic F127 it was possible to maintain the excellent printing properties of Pluronic and to create stable gels via UV crosslinking. By subsequent elution of the unmodified Pluronic from the crosslinked network we were able to increase the cell viability of encapsulated chondrocytes at day 14 from 62% for a pure acrylated Pluronic hydrogel to 86% for a nanostructured hydrogel. The mixed Pluronic gels also showed good printability when cells where included in the bioink. The nanostructured gels were, with a compressive modulus of 1.42 kPa, mechanically weak, but we were able to increase the mechanical properties by the addition of methacrylated hyaluronic acid. Our nanostructuring approach enables Pluronic hydrogels to have the desired set of properties in all stages of the bioprinting process.
AUTHOR Rimann, Markus and Bono, Epifania and Annaheim, Helene and Bleisch, Matthias and Graf-Hausner, Ursula
Title Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells. [Abstract]
Year 2015
Journal/Proceedings Journal of laboratory automation
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DOI/URL DOI
Abstract
Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application.
AUTHOR Kesti, Matti and M{"{u}}ller, Michael and Becher, Jana and Schnabelrauch, Matthias and D{textquoteright}Este, Matteo and Eglin, David and Zenobi-Wong, Marcy
Title A versatile bioink for three-dimensional printing of cellular scaffolds based on thermally and photo-triggered tandem gelation [Abstract]
Year 2014
Journal/Proceedings Acta Biomaterialia
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DOI/URL URL DOI
Abstract
Abstract Layer-by-layer bioprinting is a logical choice for the fabrication of stratified tissues like articular cartilage. Printing of viable organ replacements, however, is dependent on bioinks with appropriate rheological and cytocompatible properties. In cartilage engineering, photocrosslinkable glycosaminoglycan-based hydrogels are chondrogenic, but alone have generally poor printing properties. By blending the thermoresponsive polymer poly(N-isopropylacrylamide) grafted hyaluronan (HA-pNIPAAM) with methacrylated hyaluronan (HAMA), high-resolution scaffolds with good viability were printed. HA-pNIPAAM provided fast gelation and immediate post-printing structural fidelity, while {HAMA} ensured long-term mechanical stability upon photocrosslinking. The bioink was evaluated for rheological properties, swelling behavior, printability and biocompatibility of encapsulated bovine chondrocytes. Elution of HA-pNIPAAM from the scaffold was necessary to obtain good viability. HA-pNIPAAM can therefore be used to support extrusion of a range of biopolymers which undergo tandem gelation, thereby facilitating the printing of cell-laden, stratified cartilage constructs with zonally varying composition and stiffness.