| BackgroundOnce corneal tissue is damaged by trauma, inflammation, or degeneration, the cornea loses its normal function, as the major element in the optical pathway of the eye and as a physical barrier to the outside environment, leading to visual impairment and even blindness. Currently, corneal blindness ranks second in eye diseases that cause blindness, corneal transplantation, using human donor tissue, is the most effective treatment of irreversible corneal blindness. But a severe shortage of corneal donors and high risk for rejection after transplantation has led to the lack of timely and effective treatment for the majority of patients. Therefore, efforts have been made to fabricate biological human corneal equivalents by employing tissue engineering principles to overcome the present disadvantages of allografts.The construction of tissue engineering of the cornea is a promising opportunity for the treatment of corneal blindness. Many researchers have constructed full thickness corneal substitutes similar to the native cornea by using natural or synthetic polymers with corneal cells.Our present study explored that human corneal epithelial cells and fibroblasts could be cultured with the acellular porcine cornea matrix in vitro to construct a corneal anterior lamellar replacement for further human clinical trials such as lamellar keratoplasty (LKP).AIM:To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix(APCM) in vitro.METHODS:Limbal epithelial cells were obtained from human donor limbal rims which were not adequate for transplantation. Excessive sclera, iris, corneal endothelium, conjunctiva, and Tenon’s capsule was separated from the corneal stroma tissue. Dispase Ⅱ incubated the remaining tissue for 1 hour at 37℃, and separating the corneal epithelium under a dissecting microscope using two fine forceps, then further digestion with 0.25%Trypsin/0.02% EDTA at 37℃ for 5 minutes to isolate single cells.To obtain the corneal fibroblasts., the remaining corneal stroma was cut into pieces, were digested by 0.5mg/ml Collagenase I at 37℃ for 3hours to isolate single cells, and then incubated with fresh DMEM/F12 medium containing 10% fetal bovine serum. The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by H&E staining,DAPI staining and transmission electron microscopy(TEM).To determine whether the extracts from the APCM would cause cytotoxicity, each scaffold (10 mm diameter) was extracted using 3 ml 1:1 mixture of Dulbecco’s minimal essential medium and Ham’s F12 medium containing 10% fetal bovine serum at 37℃ for 48 h. And human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, then cell proliferative ability was evaluated by MTTassay. Before cell seeding, the APCM was incubated in DMEM/F12 for 24 h. Cultured human corneal fibroblasts from passage 3 were trypsinized and re-suspended at a final concentration of 5×105cells/ml. Parallel to the surface of the scaffold,1 ml of cell suspension was gently injected into the prepared APCM at eight different sectors using a 1 ml insulin syringe, and cultured for 3 days. After 3 days, cultured human corneal epithelial cells from passage 1 for each scaffold were gently seeded onto the surface of the reconstructed stroma, and cultured for anther 10 days.The corneal replacement was analyzed by HE staining, immunofluorescence staining and immunohistochemistry staining.RESULTS:Histological examination indicated that there were no cells in the APCM by H&E staining, and DAPI staining did not detect any residual DNA. The TEM examination showed that cell components were removed and the collagen fibrils were regularly arranged.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells cells.H&E staining showed that, corneal epithelial cells formed a mono-layer covering the surface of the APCM at 3 days, formed closely arranged 2 or 3 layers of cells at 5 days, and then formed arranged 3 to 5 layers of cells at 10 days.There were a growing number of fibroblasts within the acellular porcine cornea matrix at 3,5,7 days.The human corneal anterior lamellar replacement was efficiently constructed with the scaffold of the APCM. After the epithelial cells were seeded on top of the stromal substitutes within fibroblasts, a number of corneal cells were observed at 3,5,10 days by H&E staining.The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma, as demonstrated by immunofluorescent staining and immunohistochemistry staining.CONCLUSION:Human corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo. |
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