A Printable Photopolymerizable Thermosensitive p(HPMAm‐lactate)‐PEG Hydrogel for Tissue Engineering

Bioprinting is a new technology in regenerative medicine that allows the engineering of tissues by specific placement of cells in biomaterials. Importantly, the porosity and the relatively small dimensions of the fibers allow rapid diffusion of nutrients and metabolites. This technology requires the availability of hydrogels that ensure viability of encapsulated cells and have adequate mechanical properties for the preparation of structurally stable and well-defined three-dimensional constructs. The aim of this study is to evaluate the suitability of a biodegradable, photopolymerizable and thermosensitive A–B–A triblock copolymer hydrogel as a synthetic extracellular matrix for engineering tissues by means of three dimensional fiber deposition. The polymer is composed of poly(N-(2-hydroxypropyl)methacrylamide lactate) A-blocks, partly derivatized with methacrylate groups, and hydrophilic poly(ethylene glycol) B-blocks of a molecular weight of 10 kDa. Gels are obtained by thermal gelation and stabilized with additional chemical cross-links by photopolymerization of the methacrylate groups coupled to the polymer. A power law dependence of the storage plateau modulus of the studied hydrogels on polymer concentration is observed for both thermally and chemically cross-linked hydrogels. The hydrogels demonstrated mechanical characteristics similar to natural semi-flexible polymers, including collagen. Moreover, the hydrogel shows suitable mechanical properties for bioprinting, allowing subsequent layer-by-layer deposition of gel fibers to form stable constructs up to at least 0.6 cm (height) with different patterns and strand spacing. The resulting constructs have reproducible vertical porosity and the ability to maintain separate localization of encapsulated fluorescent microspheres. Moreover, the constructs show an elastic modulus of 119 kPa (25 wt% polymer content) and a degradation time of approximately 190 days. Furthermore, high viability is observed for encapsulated chondrocytes after 1 and 3 days of culture. In summary, we conclude that the evaluated hydrogel is an interesting candidate for bioprinting applications.

[1]  M E Levenston,et al.  Articular chondrocytes derived from distinct tissue zones differentially respond to in vitro oscillatory tensile loading. , 2008, Osteoarthritis and cartilage.

[2]  Danièle Noël,et al.  Cartilage engineering: a crucial combination of cells, biomaterials and biofactors. , 2009, Trends in biotechnology.

[3]  P. Butler,et al.  Injectable Cartilage Using Polyethylene Oxide Polymer Substrates , 1996, Plastic and reconstructive surgery.

[4]  K. Anseth,et al.  Decorin moieties tethered into PEG networks induce chondrogenesis of human mesenchymal stem cells. , 2009, Journal of biomedical materials research. Part A.

[5]  Wim E Hennink,et al.  Poly(N-(2-hydroxypropyl) methacrylamide mono/di lactate): a new class of biodegradable polymers with tuneable thermosensitivity. , 2004, Biomacromolecules.

[6]  Michael Rubinstein,et al.  Elasticity of Polymer Networks , 2002 .

[7]  Mitsuo Ochi,et al.  Articular cartilage repair using tissue engineering technique--novel approach with minimally invasive procedure. , 2004, Artificial organs.

[8]  Dietmar W Hutmacher,et al.  Scaffold-based tissue engineering: rationale for computer-aided design and solid free-form fabrication systems. , 2004, Trends in biotechnology.

[9]  W. Hennink,et al.  Degradation Mechanism and Kinetics of Thermosensitive Polyacrylamides Containing Lactic Acid Side Chains , 2003 .

[10]  D. Maniglio,et al.  Effects on interfacial properties and cell adhesion of surface modification by pectic hairy regions. , 2004, Biomacromolecules.

[11]  Y. Bae,et al.  Thermosensitive sol-gel reversible hydrogels. , 2002, Advanced drug delivery reviews.

[12]  Alexandre Kabla,et al.  Strain-Induced Alignment in Collagen Gels , 2009, PloS one.

[13]  R. Kandel,et al.  The use of specific chondrocyte populations to modulate the properties of tissue‐engineered cartilage , 2003, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[14]  G. Whitesides,et al.  Soft lithography for micro- and nanoscale patterning , 2010, Nature Protocols.

[15]  W. Dhert,et al.  Three-dimensional fiber deposition of cell-laden, viable, patterned constructs for bone tissue printing. , 2008, Tissue engineering. Part A.

[16]  T. Vermonden,et al.  Rheological studies of thermosensitive triblock copolymer hydrogels. , 2006, Langmuir : the ACS journal of surfaces and colloids.

[17]  C. V. van Blitterswijk,et al.  Evaluation of photocrosslinked Lutrol hydrogel for tissue printing applications. , 2009, Biomacromolecules.

[18]  Sunny Kim.  Changes in surgical loads and economic burden of hip and knee replacements in the US: 1997-2004. , 2008, Arthritis and rheumatism.

[19]  J. Mewis,et al.  General nonlinear rheological behavior of associative polymers , 2004 .

[20]  C. V. van Blitterswijk,et al.  Design of porous scaffolds for cartilage tissue engineering using a three-dimensional fiber-deposition technique. , 2004, Biomaterials.

[21]  M. Matsusaki,et al.  Scaffold‐Mediated 2D Cellular Orientations for Construction of Three Dimensionally Engineered Tissues Composed of Oriented Cells and Extracellular Matrices , 2009 .

[22]  E. Thonar,et al.  Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. , 1994, Journal of cell science.

[23]  J. van der Gucht,et al.  Macromolecular Diffusion in Self-Assembling Biodegradable Thermosensitive Hydrogels. , 2010, Macromolecules.

[24]  Anna Gutowska,et al.  Lessons from nature: stimuli-responsive polymers and their biomedical applications. , 2002, Trends in biotechnology.

[25]  Vladimir Mironov,et al.  Organ printing: tissue spheroids as building blocks. , 2009, Biomaterials.

[26]  Kahp-Yang Suh,et al.  Capillary Force Lithography: Large‐Area Patterning, Self‐Organization, and Anisotropic Dewetting , 2002 .

[27]  K Masuda,et al.  Tissue engineering of stratified articular cartilage from chondrocyte subpopulations. , 2003, Osteoarthritis and cartilage.

[28]  Wim E Hennink,et al.  The effect of photopolymerization on stem cells embedded in hydrogels. , 2009, Biomaterials.

[29]  Stephanie J Bryant,et al.  Encapsulating chondrocytes in degrading PEG hydrogels with high modulus: Engineering gel structural changes to facilitate cartilaginous tissue production , 2004, Biotechnology and bioengineering.

[30]  S. Narine,et al.  Fractal nature of fat crystal networks , 1999 .

[31]  M. Martina,et al.  Culture and differentiation of chondrocytes entrapped in alginate gels , 2004, Calcified Tissue International.

[32]  J. Suh,et al.  Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. , 2000, Biomaterials.

[33]  Y. Ohya,et al.  Temperature‐Induced Hydrogels Through Self‐Assembly of Cholesterol‐Substituted Star PEG‐b‐PLLA Copolymers: An Injectable Scaffold for Tissue Engineering , 2008 .

[34]  A. Miller,et al.  Thermo-reversible protein fibrillar hydrogels as cell scaffolds. , 2008, Faraday discussions.

[35]  Y. Mintz,et al.  The burden of terrorism: high rate of recurrent hospital referrals. , 2008, Injury.

[36]  H. Deschout,et al.  Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery. , 2009, Journal of controlled release : official journal of the Controlled Release Society.

[37]  Hod Lipson,et al.  Direct Freeform Fabrication of Seeded Hydrogels in Arbitrary Geometries , 2022 .

[38]  Xiaofeng Cui,et al.  Application of inkjet printing to tissue engineering , 2006, Biotechnology journal.

[39]  T B F Woodfield,et al.  Scaffolds for tissue engineering of cartilage. , 2002, Critical reviews in eukaryotic gene expression.

[40]  Robert DiRaddo,et al.  Design and Dynamic Culture of 3D-Scaffolds for Cartilage Tissue Engineering , 2011, Journal of biomaterials applications.

[41]  W. Hennink,et al.  In situ gelling hydrogels for pharmaceutical and biomedical applications. , 2008, International journal of pharmaceutics.

[42]  H. Deschout,et al.  Photopolymerized thermosensitive poly(HPMAlactate)-PEG-based hydrogels: effect of network design on mechanical properties, degradation, and release behavior. , 2010, Biomacromolecules.

[43]  Robert L Sah,et al.  Tissue engineering of articular cartilage with biomimetic zones. , 2009, Tissue engineering. Part B, Reviews.

[44]  Wim E. Hennink,et al.  Thermoresponsive Polymeric Micelles with Controlled Instability Based on Hydrolytically Sensitive N-Isopropylacrylamide Copolymers , 2001 .

[45]  Antonios G Mikos,et al.  Thermoresponsive hydrogels in biomedical applications. , 2008, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[46]  K. Shakesheff,et al.  Thermoresponsive and photocrosslinkable PEGMEMA-PPGMA-EGDMA copolymers from a one-step ATRP synthesis. , 2009, Biomacromolecules.

[47]  S. Hollister.  Porous scaffold design for tissue engineering , 2005, Nature materials.

[48]  Tao Xu,et al.  Advances in tissue engineering: cell printing. , 2005, The Journal of thoracic and cardiovascular surgery.

[49]  K. Shull,et al.  Strain stiffening in synthetic and biopolymer networks. , 2010, Biomacromolecules.

[50]  P. Janmey,et al.  Elasticity of semiflexible biopolymer networks. , 1995, Physical review letters.

[51]  A. Crosby,et al.  Photo-cross-linked PLA-PEO-PLA hydrogels from self-assembled physical networks: mechanical properties and influence of assumed constitutive relationships. , 2008, Biomacromolecules.

[52]  F. Chaubet,et al.  A Novel Cross‐linked Poly(vinyl alcohol) (PVA) for Vascular Grafts , 2008 .

[53]  S. Rammensee,et al.  Rheological characterization of hydrogels formed by recombinantly produced spider silk , 2006 .

[54]  Farshid Guilak,et al.  A biomimetic three-dimensional woven composite scaffold for functional tissue engineering of cartilage. , 2007, Nature materials.

[55]  F. Guillemot,et al.  Laser assisted bioprinting of engineered tissue with high cell density and microscale organization. , 2010, Biomaterials.

[56]  G. Prestwich,et al.  Photocrosslinkable hyaluronan-gelatin hydrogels for two-step bioprinting. , 2010, Tissue engineering. Part A.

[57]  C. van Nostrum,et al.  Hydrolysable core-crosslinked thermosensitive polymeric micelles: synthesis, characterisation and in vivo studies. , 2007, Biomaterials.

[58]  T. Matsuda,et al.  System-engineered cartilage using poly(N-isopropylacrylamide)-grafted gelatin as in situ-formable scaffold: in vivo performance. , 2003, Tissue engineering.

[59]  Jos Malda,et al.  Strategies for zonal cartilage repair using hydrogels. , 2009, Macromolecular bioscience.

[60]  Johan Bellemans,et al.  Treatment of Symptomatic Cartilage Defects of the Knee , 2009, The American journal of sports medicine.

[61]  T. Vermonden,et al.  Photopolymerized thermosensitive hydrogels: synthesis, degradation, and cytocompatibility. , 2008, Biomacromolecules.

[62]  Syed H. Masood,et al.  The design and manufacturing of porous scaffolds for tissue engineering using rapid prototyping , 2005 .

[63]  W. Hennink,et al.  Hydrogels as extracellular matrices for skeletal tissue engineering: state-of-the-art and novel application in organ printing. , 2007, Tissue engineering.