In Vitro Corneal Regeneration Based On Photo-crosslinked Hydrogels

Corneal blindness as the second leading cause of blindness,the traditional treatment is to transplant healthy donor cornea,or in severe cases,to implant artificial cornea without bioactivity.However,due to the severe shortages of donor corneas and the risk of complications associated with artificial corneas,it is particularly important to use corneal tissue engineering techniques to construct viable corneal substitutes.The selection of appropriate bio-scaffold materials and seeded cells are two key issues that urgently need to be resolved in corneal tissue engineering.In this thesis,firstly,gelatin was modified by methacrylic anhydride(MA)to improve its thermal stability to get gelatin methacrylate(GelMA).Four different concentrations(7%,10%,15% and 30%)of GelMA hydrogel were prepared,and the physicochemical properties,optical properties and biocompatibility were characterized.The results showed that the lower the concentration of GelMA,the smaller the Young’s modulus of the hydrogel,the better the hydrophilicity and optical properties.Meanwhile,in order to study the cell adhesion,viability and proliferation of rat bone marrow mesenchymal stem cells(rBM-MSCs)cultured in different concentrations of GelMA hydrogel,MTT assay,live/dead staining and cell morphology analysis were performed.The ability of rBM-MSCs to differentiate into corneal stromal cells was characterized by immunofluorescence staining and the expression of marker genes of corneal stromal cells,including Keratocan,Lumican,ALDH1A1,and α-SMA.Based on the in vitro cell experiment,it was found that 7% GelMA hydrogel was more conducive to the growth and proliferation of rBM-MSCs,while 30% GelMA hydrogel provides a better microenvironment for rBM-MSCs to differentiate into corneal keratocyte-like cells.Secondly,MA was also employed to modify hyaluronic acid in order to synthesis hyaluronic acid methacrylate(HAMA)hydrogel.GelMA/HAMA dual network(DN)hydrogel was prepared by mixing GelMA(10% w/v)and HAMA(0.5% w/v).DN hydrogels were compared with GelMA and HAMA hydrogels through characterization experiments of mechanical properties,optical properties,hydrophilicity and in-situ degradation in vitro,and it was found that DN hydrogels were optically transparent in the visible light region,with good mechanical properties beyond GelMA and HAMA hydrogels,and hydrophilicity was similar to that of normal human corneas.The culturing results of rabbit corneal epithelial cells(CEp Cs)in vitro showed that the proliferation effect of CEp Cs inoculated on DN hydrogel was obvious,which indicated that the hydrogel had good cell adhesion and excellent biocompatibility.The expression of specific marker cytokeratin 3(CK3)in CEp Cs cultured in vitro was characterized by immunofluorescence staining.The experimental results showed that compared with GelMA and HAMA hydrogels,DN hydrogels were more favorable for CEp Cs to maintain the expression of CK3 in vitro.The experimental results showed that DN hydrogel was more conducive to the growth and proliferation of CEp Cs and could maintain the higher expression of CK3 of CEp Cs in vitro,which all proved the potential of GelMA/HAMA DN hydrogel as a corneal epithelial tissue engineering bio-scaffold.