Effect of gelatin source and photoinitiator type on chondrocyte redifferentiation in gelatin methacryloyl-based tissue-engineered cartilage constructs.

Gelatin methacryloyl (GelMA) hydrogels are a mechanically and biochemically tuneable biomaterial, facilitating chondrocyte culture for tissue engineering applications. However, a lack of characterisation and standardisation of fabrication methodologies for GelMA restricts its utilisation in surgical interventions for articular cartilage repair. The purpose of this study was to determine the effects of gelatin source and photoinitiator type on the redifferentiation capacity of monolayer-expanded human articular chondrocytes encapsulated in GelMA/hyaluronic acid methacrylate (HAMA) hydrogels. Chondrocyte-laden hydrogels reinforced with multiphasic melt-electrowritten (MEW) medical grade polycaprolactone (mPCL) microfibre scaffolds were prepared using bovine (B) or porcine-derived (P) GelMA, and photocrosslinked with either lithium acylphosphinate (LAP) and visible light (405 nm) or Irgacure 2959 (IC) and UV light (365 nm). Bulk physical properties, cell viability and biochemical features of hydrogel constructs were measured at day 1 and day 28 of chondrogenic cell culture. The compressive moduli of all groups increased after 28 days of cell culture, with B-IC displaying similar compressive strength to that of native articular cartilage (∼1.5 MPa). Compressive moduli correlated with an increase in total glycosaminoglycan (GAG) content for each group. Gene expression analysis revealed upregulation of chondrogenic marker genes in IC-crosslinked groups, whilst dedifferentiation gene markers were upregulated in LAP-crosslinked groups. mPCL reinforcement correlated with increased accumulation of collagen I and II in B-IC, B-LAP and P-IC groups compared to non-reinforced hydrogels. A reduction in cell viability was noted in all samples at day 28, potentially due to the generation of free radicals during photocrosslinking or cytotoxicity of the photoinitiators. In summary, hydrogel constructs prepared with bovine-derived GelMA and photocrosslinked with Irgacure 2959 and 365 nm light displayed properties most similar to native articular cartilage after 28 days of cell culture. The differences in biological response between investigated construct types emphasises the necessity to characterise and standardise biomaterials before translating in vitro tissue engineering research to preclinical applications for articular cartilage injuries.

[1]  Jui-Sheng Sun,et al.  Fabrication of large perfusable macroporous cell-laden hydrogel scaffolds using microbial transglutaminase. , 2014, Acta biomaterialia.

[2]  S. Roberts,et al.  Immunohistochemical study of collagen types I and II and procollagen IIA in human cartilage repair tissue following autologous chondrocyte implantation , 2009, The Knee.

[3]  Jerry C. Hu,et al.  Cell-based tissue engineering strategies used in the clinical repair of articular cartilage. , 2016, Biomaterials.

[4]  K. Draget,et al.  Mechanical properties of mammalian and fish gelatins based on their weight average molecular weight and molecular weight distribution. , 2009 .

[5]  K. Leong,et al.  Chemical modification of collagen improves glycosaminoglycan retention of their co-precipitates. , 2013, Acta biomaterialia.

[6]  Siegfried Trattnig,et al.  Clinical and Radiological Outcomes 5 Years After Matrix-Induced Autologous Chondrocyte Implantation in Patients With Symptomatic, Traumatic Chondral Defects , 2012, The American journal of sports medicine.

[7]  James D. Kang,et al.  Nucleous Pulposus Tissue Engineering Using a Novel Photopolymerizable Hydrogel and Minimally Invasive Delivery , 2014 .

[8]  C. Gielens,et al.  Gelatin degradation at elevated temperature. , 2003, International journal of biological macromolecules.

[9]  Xi Liang,et al.  BMP2 induces chondrogenic differentiation, osteogenic differentiation and endochondral ossification in stem cells , 2016, Cell and Tissue Research.

[10]  Lay Poh Tan,et al.  Synthesis and Characterization of Types A and B Gelatin Methacryloyl for Bioink Applications , 2016, Materials.

[11]  Peter Pivonka,et al.  Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair , 2017, Scientific Reports.

[12]  Brendon M. Baker,et al.  Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues , 2012, Journal of Cell Science.

[13]  P. R. van Weeren,et al.  Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs. , 2013, Macromolecular bioscience.

[14]  R. Tuan,et al.  Cartilage tissue engineering application of injectable gelatin hydrogel with in situ visible-light-activated gelation capability in both air and aqueous solution. , 2014, Tissue engineering. Part A.

[15]  David J Mooney,et al.  Mechanical confinement regulates cartilage matrix formation by chondrocytes , 2017, Nature materials.

[16]  Pauline M. Doran,et al.  Strategies for Enhancing the Accumulation and Retention of Extracellular Matrix in Tissue-Engineered Cartilage Cultured in Bioreactors , 2011, PloS one.

[17]  Dietmar W. Hutmacher,et al.  A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin, hyaluronic acid and chondroitin sulfate. , 2014, Acta biomaterialia.

[18]  D. Hutmacher,et al.  Effect of preculture and loading on expression of matrix molecules, matrix metalloproteinases, and cytokines by expanded osteoarthritic chondrocytes. , 2013, Arthritis and rheumatism.

[19]  M. Gómez-Guillén,et al.  Structural and physical properties of gelatin extracted from different marine species: A comparative study , 2002 .

[20]  Tsai-Yu Lin,et al.  Comparative study of visible light polymerized gelatin hydrogels for 3D culture of hepatic progenitor cells , 2017 .

[21]  Paulo Jorge Da Silva Bartolo,et al.  3D Photo-Fabrication for Tissue Engineering and Drug Delivery , 2015 .

[22]  D. Speer,et al.  Biological effects of residual glutaraldehyde in glutaraldehyde-tanned collagen biomaterials. , 1980, Journal of biomedical materials research.

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

[24]  D. Buttle,et al.  Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. , 1986, Biochimica et biophysica acta.

[25]  P. Petersen,et al.  Endotoxins-the invisible companion in biomaterials research. , 2013, Tissue engineering. Part B, Reviews.

[26]  T. Aigner,et al.  Synergistic effect of IGF-1 and OP-1 on matrix formation by normal and OA chondrocytes cultured in alginate beads. , 2007, Osteoarthritis and cartilage.

[27]  B. Feeley,et al.  Management of articular cartilage defects of the knee. , 2010, The Journal of bone and joint surgery. American volume.

[28]  J. Verhaar,et al.  Multiplication of human chondrocytes with low seeding densities accelerates cell yield without losing redifferentiation capacity. , 2004, Tissue engineering.

[29]  Guangdong Zhou,et al.  Recent Progress in Cartilage Tissue Engineering—Our Experience and Future Directions , 2017 .

[30]  Dietmar W. Hutmacher,et al.  Rational design and fabrication of multiphasic soft network composites for tissue engineering articular cartilage: A numerical model-based approach , 2018 .

[31]  T. Vos,et al.  The global burden of hip and knee osteoarthritis: estimates from the Global Burden of Disease 2010 study , 2014, Annals of the rheumatic diseases.

[32]  A. Zhang,et al.  Digital microfabrication of user‐defined 3D microstructures in cell‐laden hydrogels , 2013, Biotechnology and bioengineering.

[33]  Kirsten Borchers,et al.  Methacrylated gelatin and mature adipocytes are promising components for adipose tissue engineering , 2016, Journal of biomaterials applications.

[34]  K. Chua,et al.  Insulin-transferrin-selenium prevent human chondrocyte dedifferentiation and promote the formation of high quality tissue engineered human hyaline cartilage. , 2005, European cells & materials.

[35]  Jos Malda,et al.  Gelatin-Methacryloyl Hydrogels: Towards Biofabrication-Based Tissue Repair. , 2016, Trends in biotechnology.

[36]  A. Mikos,et al.  The effect of hypoxia on the chondrogenic differentiation of co-cultured articular chondrocytes and mesenchymal stem cells in scaffolds. , 2013, Biomaterials.

[37]  Dietmar W Hutmacher,et al.  Direct Writing By Way of Melt Electrospinning , 2011, Advanced materials.

[38]  D. Mooney,et al.  Hydrogels for tissue engineering. , 2001, Chemical Reviews.

[39]  Biman B Mandal,et al.  Tissue-engineered cartilage: the crossroads of biomaterials, cells and stimulating factors. , 2015, Macromolecular bioscience.

[40]  Ali Khademhosseini,et al.  Photocrosslinkable Gelatin Hydrogel for Epidermal Tissue Engineering , 2016, Advanced healthcare materials.

[41]  J. Malda,et al.  Chondrocyte redifferentiation and construct mechanical property development in single-component photocrosslinkable hydrogels. , 2014, Journal of biomedical materials research. Part A.

[42]  G. Schulze-Tanzil,et al.  Osteochondral articular defect repair using auricle-derived autologous chondrocytes in a rabbit model. , 2014, Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft.

[43]  Scott A. Rodeo,et al.  The Basic Science of Articular Cartilage , 2009, Sports health.

[44]  D. Hutmacher,et al.  A novel bioreactor system for biaxial mechanical loading enhances the properties of tissue-engineered human cartilage , 2017, Scientific Reports.

[45]  Ali Khademhosseini,et al.  Functionalization, preparation and use of cell-laden gelatin methacryloyl–based hydrogels as modular tissue culture platforms , 2016, Nature Protocols.

[46]  A. Misra,et al.  Self-Strengthening Hybrid Dental Adhesive via Visible-light Irradiation Triple Polymerization. , 2016, RSC advances.

[47]  C. Rorabeck,et al.  Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. , 1997, The Journal of clinical investigation.

[48]  M. Djabourov,et al.  Structural and mechanical properties of fish gelatin as a function of extraction conditions , 2009 .

[49]  S. Reichenbach,et al.  All cause and disease specific mortality in patients with knee or hip osteoarthritis: population based cohort study , 2011, BMJ : British Medical Journal.

[50]  M. Woodruff,et al.  Protective effects of reactive functional groups on chondrocytes in photocrosslinkable hydrogel systems. , 2015, Acta biomaterialia.

[51]  Hitomi Shirahama,et al.  Precise Tuning of Facile One-Pot Gelatin Methacryloyl (GelMA) Synthesis , 2016, Scientific Reports.

[52]  Geniece L. Hallett-Tapley,et al.  Photochemical Norrish type I reaction as a tool for metal nanoparticle synthesis: importance of proton coupled electron transfer. , 2012, Chemical communications.

[53]  Paul N Manson,et al.  Variable cytocompatibility of six cell lines with photoinitiators used for polymerizing hydrogels and cell encapsulation. , 2005, Biomaterials.

[54]  R. Müller,et al.  Effect of matrix elasticity on the maintenance of the chondrogenic phenotype. , 2010, Tissue engineering. Part A.

[55]  Kristi S Anseth,et al.  Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility. , 2009, Biomaterials.

[56]  Ernst Rank,et al.  Biofabricated soft network composites for cartilage tissue engineering , 2017, Biofabrication.

[57]  Jos Malda,et al.  Reinforcement of hydrogels using three-dimensionally printed microfibres , 2015, Nature Communications.

[58]  A. Khademhosseini,et al.  Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. , 2015, Biomaterials.

[59]  Jerry C. Hu,et al.  Repair and tissue engineering techniques for articular cartilage , 2015, Nature Reviews Rheumatology.

[60]  A. Khademhosseini,et al.  Cell-laden microengineered gelatin methacrylate hydrogels. , 2010, Biomaterials.

[61]  S. Van Vlierberghe,et al.  Immunocompatibility evaluation of hydrogel-coated polyimide implants for applications in regenerative medicine. , 2014, Journal of biomedical materials research. Part A.

[62]  Michael V Sefton,et al.  Endotoxin: the uninvited guest. , 2005, Biomaterials.

[63]  V. Bampidis,et al.  Risks associated with endotoxins in feed additives produced by fermentation , 2016, Environmental Health.

[64]  R. Mason,et al.  Bovine articular chondrocyte function in vitro depends upon oxygen tension. , 2000, Osteoarthritis and cartilage.

[65]  G. Schulze-Tanzil,et al.  PGA‐associated heterotopic chondrocyte cocultures: implications of nasoseptal and auricular chondrocytes in articular cartilage repair , 2013, Journal of tissue engineering and regenerative medicine.

[66]  Sabu Thomas,et al.  Mechanism of phase separation in a weakly interacting system with strong dynamic asymmetry , 2017 .

[67]  Jiake Xu,et al.  Matrix-induced autologous chondrocyte implantation (MACI): biological and histological assessment. , 2007, Tissue engineering.