The Preparation And Application Of Porous Hydrogel Based On GelMA As A Corneal Stromal Replacement

Corneal blindness is the second common blindness in China and corneal transplantation is an effective treatment to help patients.However,the lack of donor sources for corneal transplantation makes it possible to develop alternative materials for corneal regeneration and repair.The stroma is the thickest layer in the cornea.Tissue engineering was widely observed by producing biomimetic corneal substitute.Collagen is the main component of the matrix layer.The hydrolysate of collagen,gelatin,can be chemically modified using methacrylic anhydride to form methacrylated gelatin(GelMA)which can be used as the materials of extracellular matrix(ECM).At present,the internal structure of traditional hydrogels only contains inherent nanopores formed by cross-linked polymer chains,which hinder the spread of cells.Freeze-drying is often used to prepare microporous hydrogels,but it fabricates at low temperature is not suitable to encapsulate living cells directly in the manufacturing process.In this paper,a Construction of a corneal stromal replacement using light-curing porous hydrogel based on the two-phase aqueous emulsion is prepared for corneal repair.In this study,gelatin was used to prepare GelMA and the GelMA solution containing photoinitiator 2959 was mixed with aqueous polyethylene oxide(PEO,600000 Da)solution to form an immiscible two-phase emulsion.After light curing,hydrogels are obtained and remove the PEO phase,forming a connected and porous hydrogel structure.First of all,this study characterizes the physical and chemical properties of GelMA-PEO emulsion and porous hydrogel.We characterize some properties,containing the pore size,mechanical properties,degradation properties,and swelling properties,to choose the preparation process of porous hydrogel.The results show that the saturated solubility of PEO is 1.4 wt% and the volume ratio of GelMA solution and PEO solution has a greater influence on the pore size.As the solid content of GelMA decreases,the strength,and stiffness of porous hydrogel decrease,while the pore size and swelling ratio,degradation rate,and equilibrium moisture content of porous hydrogel increase.GelMA-PEO emulsion with a volume ratio of 1:1 is selected to prepare porous hydrogels due to the pore size,which meets the need of cell proliferation and proliferation.With low stiffness,the cells encapsulated in the porous hydrogels are allowed to spread and maintain a better cell morphology.In addition,in this study,rabbit corneal stromal cells were extracted from the tissue explants.The corneal stromal cells were dispersed in GelMA solution containing photoinitiator 2959 and the GelMA/cell mixture is mixed with PEO solution to form an emulsion.After light curing,the hydrogels are immersed in the culture medium to remove the PEO phase,and the porous hydrogel encapsulating corneal stromal cells form.The nonporous GelMA hydrogel encapsulating corneal stromal cells is set as the control group and two groups of corneal stroma replacement were obtained in vitro.To verify its cytocompatibility,immunofluorescence staining of the corneal stromal replacements on Day 14 and Day 30 evaluated the synthesis of ECM.Moreover,the implantation of corneal stromal replacements in a mid-stromal pocket created by minor surgery confirms their histocompatibility and application in corneal repair.The results showed that the cells extracted in this study were identified as corneal stromal cells.The porous hydrogels have good biocompatibility and the specific matrix secreted by corneal stromal cells gradually increased,so the porous hydrogels promote the secretion of extracellular matrix.The corneal stromal replacement of porous hydrogel with r CSCs was well integrated with the host tissue and minimal inflammation.The immunofluorescence staining both revealed that collagen type V(COL V)and decorin were highly expressed in the corneal stromal replacement of porous hydrogels with r CSCs,while no evident expression of α-SMA was observed.The TEM revealed that the fibrils were orderly in the scaffolds of porous hydrogels with r CSCs.The ECM molecules regulated the fibril assembly and induced the regeneration of the stroma for secreting components of native human corneal stromal tissue.In summary,this thesis prepared a light-curing porous hydrogel based on the immiscible two-phase emulsion as a corneal stromal replacement for regeneration and repair.The porous hydrogels are prepared by mixing an equal volume of GelMA solution(10 wt%)containing corneal stromal cells and PEO solution(1.4 wt%).We study their physical,chemical,mechanical properties,degradation properties,swelling properties and biological properties.We extract and identify the rabbit corneal stromal cells and study the extracellular matrix secreted by corneal stromal cells in corneal stromal replacements.Furthermore,the implantation of corneal stromal replacements in a mid-stromal pocket created by minor surgery confirms their histocompatibility and application in corneal repair.The fibrils were mainly in the same direction in the scaffolds of porous hydrogels with r CSCs.Our study provides a theoretical reference for corneal regeneration and repair and develops corneal stromal replacement in the field of tissue engineering.