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Preparation and In Vitro Characterization of Gelatin Methacrylate for Corneal Tissue Engineering

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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

BACKGROUND:

Corneal disease is second only to cataract considered as the leading cause of blindness in the world, with high morbidity. Construction of corneal substitutes in vitro by tissue engineering technology to achieve corneal regeneration has become a research hotspot in recent years. We conducted in-depth research on the biocompatibility, physicochemical and mechanical properties of rat bone marrow mesenchymal stem cells (rBM-MSCs)-seeded gelatin methacrylate (GelMA) as a bioengineered cornea.

METHODS:

Four kinds of GelMA with different concentrations (7, 10, 15 and 30%) were prepared, and their physic-chemical, optical properties, and biocompatibility with rBM-MSCs were characterized. MTT, live/dead staining, cell morphology, immunofluorescence staining and gene expression of keratocyte markers were performed.

RESULTS:

7%GelMA hydrogel had higher equilibrium water content and porosity, better optical properties and hydrophilicity. In addition, it is more beneficial to the growth and proliferation of rBM-MSCs. However, the 30%GelMA hydrogel had the best mechanical properties, and could be more conducive to promote the differentiation of rBM-MSCs into keratocyte-like cells.

CONCLUSION:

As a natural biological scaffold, GelMA hydrogel has good biocompatibility. And it has the ability to promote the differentiation of rBM-MSCs into keratocyte-like cells, which laid a theoretical and experimental foundation for further tissue-engineered corneal stromal transplantation, and provided a new idea for the source of seeded cells in corneal tissue engineering.

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References

  1. Duan X, Sheardown H. Dendrimer crosslinked collagen as a corneal tissue engineering scaffold: mechanical properties and corneal epithelial cell interactions. Biomaterials. 2006;27:4608–17.

    Article  CAS  PubMed  Google Scholar 

  2. Vrana NE, Builles N, Kocak H, Gulay P, Justin V, Malbouyres M, et al. EDC/NHS cross-linked collagen foams as scaffolds for artificial corneal stroma. J Biomater Sci Polym Ed. 2007;18:1527–45.

    Article  CAS  PubMed  Google Scholar 

  3. Cao D, Zhang Y, Cui Z, Du Y, Shi Z. New strategy for design and fabrication of polymer hydrogel with tunable porosity as artificial corneal skirt. Mater Sci Eng C Mater Biol Appl. 2017;70:665–72.

    Article  CAS  PubMed  Google Scholar 

  4. Islam MM, Buznyk O, Reddy JC, Pasyechnikova N, Alarcon EI, Hayes S, et al. Biomaterials-enabled cornea regeneration in patients at high risk for rejection of donor tissue transplantation. NPJ Regen Med. 2018;3:2.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Chen F, Le P, Fernandes-Cunha GM, Heilshorn SC, Myung D. Bio-orthogonally crosslinked hyaluronate-collagen hydrogel for suture-free corneal defect repair. Biomaterials. 2020;255:120176.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Karamichos D. Ocular tissue engineering: current and future directions. J Funct Biomater. 2015;6:77–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Feiz V, Mannis MJ, Kandavel G, McCarthy M, Izquierdo L, Eckert M, et al. Surface keratopathy after penetrating keratoplasty. Trans Am Ophthalmol Soc. 2001;99:159–68.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Reinhart WJ, Musch DC, Jacobs DS, Lee WB, Kaufman SC, Shtein RM. Deep anterior lamellar keratoplasty as an alternative to penetrating keratoplasty a report by the american academy of ophthalmology. Ophthalmology. 2011;118:209–18.

    Article  PubMed  Google Scholar 

  9. Jirásková N, Rozsival P, Burova M, Kalfertova M. AlphaCor artificial cornea: clinical outcome. Eye (Lond). 2011;25:1138–46.

    Article  Google Scholar 

  10. Ruberti JW, Zieske JD. Prelude to corneal tissue engineering—gaining control of collagen organization. Prog Retin Eye Res. 2008;27:549–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mahdavi SS, Abdekhodaie MJ, Mashayekhan S, Baradaran-Rafii A, Djalilian AR. Bioengineering approaches for corneal regenerative medicine. Tissue Eng Regen Med. 2020;17:567–93.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Barabadi Z, Azami M, Sharifi E, Karimi R, Lotfibakhshaiesh N, Roozafzoon R, et al. Fabrication of hydrogel based nanocomposite scaffold containing bioactive glass nanoparticles for myocardial tissue engineering. Mater Sci Eng C Mater Biol Appl. 2016;69:1137–46.

    Article  CAS  PubMed  Google Scholar 

  13. Yang CY, Song B, Ao Y, Nowak AP, Abelowitz RB, Korsak RA, et al. Biocompatibility of amphiphilic diblock copolypeptide hydrogels in the central nervous system. Biomaterials. 2009;30:2881–98.

    Article  CAS  PubMed  Google Scholar 

  14. Recouvreux DO, Rambo CR, Berti FV, Carminatti CA, Antônio RV, Porto LM. Novel three-dimensional cocoon-like hydrogels for soft tissue regeneration. Mater Sci Eng C Mater Biol Appl. 2011;31:151–7.

    Article  CAS  Google Scholar 

  15. Wright B, Mi S, Connon CJ. Towards the use of hydrogels in the treatment of limbal stem cell deficiency. Drug Discov Today. 2013;18:79–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Niu G, Choi JS, Wang Z, Skardal A, Giegengack M, Soker S. Heparin-modified gelatin scaffolds for human corneal endothelial cell transplantation. Biomaterials. 2014;35:4005–14.

    Article  CAS  PubMed  Google Scholar 

  17. Hayes S, Lewis P, Islam MM, Doutch J, Sorensen T, White T, et al. The structural and optical properties of type III human collagen biosynthetic corneal substitutes. Acta Biomater. 2015;25:121–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lawrence BD, Marchant JK, Pindrus MA, Omenetto FG, Kaplan DL. Silk film biomaterials for cornea tissue engineering. Biomaterials. 2009;30:1299–308.

    Article  CAS  PubMed  Google Scholar 

  19. Goodarzi H, Jadidi K, Pourmotabed S, Sharifi E, Aghamollaei H. Preparation and in vitro characterization of cross-linked collagen-gelatin hydrogel using EDC/NHS for corneal tissue engineering applications. Int J Biol Macromol. 2019;126:620–32.

    Article  CAS  PubMed  Google Scholar 

  20. Stafiej P, Küng F, Thieme D, Czugala M, Kruse FE, Schubert DW, et al. Adhesion and metabolic activity of human corneal cells on PCL based nanofiber matrices. Mater Sci Eng C Mater Biol Appl. 2017;71:764–70.

    Article  CAS  PubMed  Google Scholar 

  21. Yoeruek E, Bayyoud T, Maurus C, Hofmann J, Spitzer MS, Bartz-Schmidt KU, et al. Decellularization of porcine corneas and repopulation with human corneal cells for tissue-engineered xenografts. Acta Ophthalmol. 2012;90:e125–31.

    Article  PubMed  Google Scholar 

  22. Liu Y, Ren L, Wang Y. Crosslinked collagen-gelatin-hyaluronic acid biomimetic film for cornea tissue engineering applications. Mater Sci Eng C Mater Biol Appl. 2013;33:196–201.

    Article  CAS  PubMed  Google Scholar 

  23. Galis ZS, Khatri JJ. Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ Res. 2002;90:251–62.

    Article  CAS  PubMed  Google Scholar 

  24. Nichol JW, Koshy ST, Bae H, Hwang CM, Yamanlar S, Khademhosseini A. Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials. 2010;31:5536–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Shim K, Kim SH, Lee D, Kim B, Kim TH, Jung Y, et al. Fabrication of micrometer-scale porous gelatin scaffolds for 3D cell culture. J Ind Eng Chem. 2017;50:183–9.

    Article  CAS  Google Scholar 

  26. Chen MB, Srigunapalan S, Wheeler AR, Simmons CA. A 3D microfluidic platform incorporating methacrylated gelatin hydrogels to study physiological cardiovascular cell-cell interactions. Lab Chip. 2013;13:2591–8.

    Article  CAS  PubMed  Google Scholar 

  27. Farasatkia A, Kharaziha M, Ashrafizadeh F, Salehi S. Transparent silk/gelatin methacrylate (GelMA) fibrillar film for corneal regeneration. Mater Sci Eng C Mater Biol Appl. 2021;120:111744.

    Article  CAS  PubMed  Google Scholar 

  28. Kilic Bektas C, Hasirci V. Cell loaded GelMA: HEMA IPN hydrogels for corneal stroma engineering. J Mater Sci Mater Med. 2019;31:2.

    Article  PubMed  Google Scholar 

  29. Venugopal B, Mohan S, Kumary TV, Anil Kumar PR. Peripheral blood as a source of stem cells for regenerative medicine: emphasis towards corneal epithelial reconstruction-an in vitro study. Tissue Eng Regen Med. 2020;17:495–510.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Carter K, Lee HJ, Na KS, Fernandes-Cunha GM, Blanco IJ, Djalilian A, et al. Characterizing the impact of 2D and 3D culture conditions on the therapeutic effects of human mesenchymal stem cell secretome on corneal wound healing in vitro and ex vivo. Acta Biomater. 2019;99:247–57.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Demirayak B, Yüksel N, Çelik OS, Subaşı C, Duruksu G, Unal ZS, et al. Effect of bone marrow and adipose tissue-derived mesenchymal stem cells on the natural course of corneal scarring after penetrating injury. Exp Eye Res. 2016;151:227–35.

    Article  CAS  PubMed  Google Scholar 

  32. Zakirova EY, Valeeva AN, Aimaletdinov AM, Nefedovskaya LV, Akhmetshin RF, Rutland CS, et al. Potential therapeutic application of mesenchymal stem cells in ophthalmology. Exp Eye Res. 2019;189:107863.

    Article  CAS  PubMed  Google Scholar 

  33. Rohaina CM, Then KY, Ng AM, Wan Abdul Halim WH, Zahidin AZ, Saim A, et al. Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane. Transl Res. 2014;163:200–10.

    Article  CAS  PubMed  Google Scholar 

  34. Van Den Bulcke AI, Bogdanov B, De Rooze N, Schacht EH, Cornelissen M, Berghmans H. Structural and rheological properties of methacrylamide modified gelatin hydrogels. Biomacromolecules. 2000;1:31–8.

    Article  CAS  PubMed  Google Scholar 

  35. Liu W, Deng C, McLaughlin CR, Fagerholm P, Lagali NS, Heyne B, et al. Collagen-phosphorylcholine interpenetrating network hydrogels as corneal substitutes. Biomaterials. 2009;30:1551–9.

    Article  CAS  PubMed  Google Scholar 

  36. He Y, Hou Z, Wang J, Wang Z, Li X, Liu J, et al. Assessment of biological properties of recombinant collagen-hyaluronic acid composite scaffolds. Int J Biol Macromol. 2020;149:1275–84.

    Article  CAS  PubMed  Google Scholar 

  37. Rizwan M, Peh GSL, Ang HP, Lwin NC, Adnan K, Mehta JS, et al. Sequentially-crosslinked bioactive hydrogels as nano-patterned substrates with customizable stiffness and degradation for corneal tissue engineering applications. Biomaterials. 2017;120:139–54.

    Article  CAS  PubMed  Google Scholar 

  38. Park SH, Kim KW, Chun YS, Kim JC. Human mesenchymal stem cells differentiate into keratocyte-like cells in keratocyte-conditioned medium. Exp Eye Res. 2012;101:16–26.

    Article  CAS  PubMed  Google Scholar 

  39. Sharifi E, Azami M, Kajbafzadeh AM, Moztarzadeh F, Faridi-Majidi R, Shamousi A, et al. Preparation of a biomimetic composite scaffold from gelatin/collagen and bioactive glass fibers for bone tissue engineering. Mater Sci Eng C Mater Biol Appl. 2016;59:533–41.

    Article  CAS  PubMed  Google Scholar 

  40. Du YA, Lo E, Ali S, Khademhosseini A. Directed assembly of cell-laden microgels for fabrication of 3D tissue constructs. Proc Natl Acad Sci U S A. 2008;105:9522–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Elsheikh A, Geraghty B, Rama P, Campanelli M, Meek KM. Characterization of age-related variation in corneal biomechanical properties. J R Soc Interface. 2010;7:1475–85.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Garcia-Porta N, Fernandes P, Queiros A, Salgado-Borges J, Parafita-Mato M, González-Méijome JM. Corneal biomechanical properties in different ocular conditions and new measurement techniques. ISRN Ophthalmol. 2014;2014:724546.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Luo LJ, Lai JY, Chou SF, Hsueh YJ, Ma DH. Development of gelatin/ascorbic acid cryogels for potential use in corneal stromal tissue engineering. Acta Biomater. 2018;65:123–36.

    Article  CAS  PubMed  Google Scholar 

  44. Arica TA, Guzelgulgen M, Yildiz AA, Demir MM. Electrospun GelMA fibers and p(HEMA) matrix composite for corneal tissue engineering. Mater Sci Eng C Mater Biol Appl. 2021;120:111720.

  45. Sun M, Sun X, Wang Z, Guo S, Yu G, Yang H. Synthesis and properties of gelatin methacryloyl (GelMA) hydrogels and their recent applications in load-bearing tissue. Polymers (Basel). 2018;10:1290.

    Article  Google Scholar 

  46. Dragusin DM, Van Vlierberghe S, Dubruel P, Dierick M, Van Hoorebeke L, Declercq HA, et al. Novel gelatin-PHEMA porous scaffolds for tissue engineering applications. Soft Matter. 2012;8:9589–602.

    Article  CAS  Google Scholar 

  47. Yue K, Trujillo-de Santiago G, Alvarez MM, Tamayol A, Annabi N, Khademhosseini A. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials. 2015;73:254–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Aldana AA, Rial-Hermida MI, Abraham GA, Concheiro A, Alvarez-Lorenzo C. Temperature-sensitive biocompatible IPN hydrogels based on poly (NIPA-PEGdma) and photocrosslinkable gelatin methacrylate. Soft Mater. 2017;15:341–9.

    Article  CAS  Google Scholar 

  49. Ma J, Wang Y, Wei P, Jhanji V. Biomechanics and structure of the cornea: implications and association with corneal disorders. Surv Ophthalmol. 2018;63:851–61.

    Article  PubMed  Google Scholar 

  50. Shin YN, Kim BS, Ahn HH, Lee JH, Kim KS, Lee JY, et al. Adhesion comparison of human bone marrow stem cells on a gradient wettable surface prepared by corona treatment. Appl Surf Sci. 2008;255:293–6.

    Article  CAS  Google Scholar 

  51. Meek KM, Knupp C. Corneal structure and transparency. Prog Retin Eye Res. 2015;49:1–16.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Choong PF, Mok PL, Cheong SK, Then KY. Mesenchymal stromal cell-like characteristics of corneal keratocytes. Cytotherapy. 2007;9:252–8.

    Article  CAS  PubMed  Google Scholar 

  53. Espana EM, He H, Kawakita T, Di Pascuale MA, Raju VK, Liu CY, et al. Human keratocytes cultured on amniotic membrane stroma preserve morphology and express keratocan. Invest Ophthalmol Vis Sci. 2003;44:5136–41.

    Article  PubMed  Google Scholar 

  54. Calonge M, Pérez I, Galindo S, Nieto-Miguel T, López-Paniagua M, Fernández I, et al. A proof-of-concept clinical trial using mesenchymal stem cells for the treatment of corneal epithelial stem cell deficiency. Transl Res. 2019;206:18–40.

    Article  PubMed  Google Scholar 

  55. Yun YI, Park S, Lee HJ, Ko JH, Kim MK, Wee WR, et al. Comparison of the anti-inflammatory effects of induced pluripotent stem cell-derived and bone marrow-derived mesenchymal stromal cells in a murine model of corneal injury. Cytotherapy. 2017;19:28–35.

    Article  CAS  PubMed  Google Scholar 

  56. Chen J, Wong-Chong J, SundarRaj N. FGF-2-and TGF-beta 1-induced downregulation of Lumican and Keratocan in activated corneal keratocytes by JNK signaling pathway. Invest Ophthalmol Vis Sci. 2011;52:8957–64.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Shin SR, Zihlmann C, Akbari M, Assawes P, Cheung L, Zhang K, et al. Reduced graphene oxide-GelMA hybrid hydrogels as scaffolds for cardiac tissue engineering. Small. 2016;12:3677–89.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Kwak BS, Choi W, Jeon JW, Won JI, Sung GY, Kim B, et al. In vitro 3D skin model using gelatin methacrylate hydrogel. J Ind Eng Chem. 2018;66:254–61.

    Article  CAS  Google Scholar 

  59. Chen J, Backman LJ, Zhang W, Ling C, Danielson P. Regulation of keratocyte phenotype and cell behavior by substrate stiffness. ACS Biomater Sci Eng. 2020;6:5162–71.

    Article  CAS  PubMed  Google Scholar 

  60. Zakirova EY, Valeeva AN, Aimaletdinov AM, Nefedovskaya LV, Akhmetshin RF, Rutland CS, et al. Potential therapeutic application of mesenchymal stem cells in ophthalmology. Exp Eye Res. 2019;189:107863.

    Article  CAS  PubMed  Google Scholar 

  61. Hong H, Kim H, Han SJ, Jang J, Kim HK, Cho DW, et al. Compressed collagen intermixed with cornea-derived decellularized extracellular matrix providing mechanical and biochemical niches for corneal stroma analogue. Mater Sci Eng C Mater Biol Appl. 2019;103:109837.

    Article  CAS  PubMed  Google Scholar 

  62. Urbanczyk M, Layland SL, Schenke-Layland K. The role of extracellular matrix in biomechanics and its impact on bioengineering of cells and 3D tissues. Matrix Biol. 2020;85–86:1–14.

    Article  PubMed  Google Scholar 

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (Nos. 51975400, 61703298, 61501316, 51505324), National Key Research and Development Program (2019YFB1310200), Science and Technology in Novation Project for Outstanding Talent of Shanxi Province (201805D211020) and Beijing Natural Science Foundation (7202190). The authors would like to thank all the anonymous referees for their valuable comments and suggestions to further improve the quality of this work.

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Authors and Affiliations

  1. MicroNano System Research Center, College of Information and Computer & Key Lab of Advanced Transducers and Intelligent Control System, Ministry of Education, Taiyuan University of Technology, Taiyuan, 030024, China

    Yayun Yan, Yanyan Cao, Rong Cheng, Zhizhong Shen, Yajing Zhao & Shengbo Sang

  2. College of Information Science and Engineering, Hebei North University, Zhangjiakou, 075000, China

    Yanyan Cao

  3. College of Biomedical Engineering, Taiyuan University of Technology, Taiyuan, 030024, China

    Yixia Zhang

  4. Department of Lacrimal Duct Diseases, Shanxi Eye Hospital, Taiyuan, 030002, China

    Guohong Zhou

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  1. Yayun Yan
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Yan, Y., Cao, Y., Cheng, R. et al. Preparation and In Vitro Characterization of Gelatin Methacrylate for Corneal Tissue Engineering. Tissue Eng Regen Med 19, 59–72 (2022). https://doi.org/10.1007/s13770-021-00393-6

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