Construction Of Tissue Engineered Corneal Transplantation With Rabbit Corneal Stromal Cells And Acellular Porcine Corneal Stroma

Objective:The primary rabbit CSCs were cultured in vitro and the tissue engineered corneal stroma was constructed using APCM as scaffold material.The growth of APCM in deep lamellar corneal transplantation after implantation of CSCs was investigated,which provided experimental basis for the later study of tissue engineered cornea.Methods:(1)Primary rabbit CSCs were isolated and cultured in vitro;(2)rabbit CSCs transfected with Lentivirus were used to determine the optimal infection time and MOI by inverted fluorescence microscopy and flow cytometry;(3)Using APCM as the scaffold material,the rabbit corneal deep layer was transplanted,and 24 New Zealand white rabbits were taken and divided into 3 groups.Experimental group:APCM+rabbit CSCs;control group A:APCM group;control group B:rabbit corneal in situ suture group;(4)1-4 weeks,8 weeks after operation,anterior segment photography and fluorescein staining;(5)Corneal thickness was measured by OCT at 1 week,1 month,and 2 months after operation;(6)2 months after operation,each group was subjected to immunofluorescence,HE staining,and GFP immunohistochemistry.Results:(1)Rabbit corneal stromal cells were successfully isolated and cultured;(2)Rabbit CSCs transfected with LV-EGFP were observed under inverted fluorescence microscope.After aspirating the virus solution for 24 hours,green fluorescence was observed.The fluorescence intensity gradually increased with time.Enhancement,72h was the best transfection time;flow cytometry showed that LV-EGFP had no effect on the survival rate of rabbit CSCs at MOI=400;(3)8 weeks after animal experiment,the experimental group had 8 points of cornea Azimuth neovascularization into the cornea about 2mm,the cornea is transparent,no obvious scar,fluorescein staining shows staining in the central area of the graft;control group A corneal 2 point orientation neovascularization into the cornea about 3 mm,fluorescein staining in the central region of the graft The control group B cornea 7 points azimuth neovascularization into the cornea about 1mm,the cornea is transparent,fluorescein staining shows corneal punctate coloration;(4)corneal matrix signal in the OCT experimental group is more uniform,the plant and the bed are closely fused The control group A corneal stromal layer signal is not uniform,it can be seen that there is a clear boundary between the transplanted APCM and the original matrix,the fusion is not good;the control group B matrix layer gray uniform,visible clear epithelial layer,stromal layer and endothelium(5)The central corneal thickness of each group was 323?m,166?m,324?m after operation;(6)Green fluorescence was observed in the immunofluorescence test group of frozen sections,and no fluorescence was observed in the control group A/B;immunohistochemistry experimental group It can be seen that GFP expression is positive,and the control group A/B is negative.Conclusions:APCM injected with rabbit CSCs can be used as scaffold material to construct rabbit tissue-engineered cornea in vitro.The graft fused well with the original cornea implant bed.The structure and function of APCM are closer to rabbit autologous cornea.It can provide good raw materials for artificial cornea transplantation.