| This study made use of bone-derived mesenchymal stem cell cultured and screened in vitro to be seed cell, and then integrated to acellular corneal stroma. With morphologic observation in vitro and transplantation of rabit, it is inspected for its bio-characteristics and bio-compatibility; To explore culturing system of corneal epithelium cells and induce bone-derived mesenchymal stem cell different into corneal epithelium cells by Transwell system; Aim of study based on experiment about seed cells and scaffold is to make an effective and better method of preparation of tissue engineering cornea, and to supply evidence of construction and application of clinic tissue engineering cornea. Progress is designed as follows:(1) BMSC were obtained from rabits by density gradientcentrifugation and were tested the rate of adhesion. DMEM/F12 be foundation culture medium, the FBS density is 10%, add penicillin 100IU/ml and phytomycin 100μg/ml, PH value at 7.2～7.4. Cells inoculated into cell culture bottle and cultured under condition of 37℃, 5%CO2, was observed by phase-contrast microscope after cultured by 48h. Observation showed that lots of fibroblast-like cells adherenced and growed intensively in whirlpool. It could be passaged after origin culture 5 days and we chose 2 or 3 generation to be seed cell. Experiment showed clearly that bone-derived mesenchymal stem cell performed good ability of grow and differentiation, might be suitable seed cell for tissue engineering.(2) To take fresh swine's cornea, digested by pamcreatin and nuclease, into liquid nitrogen, freeze and melt again and again in order to break every cell of cornea, disinfection by 60Co, reserved after freeze-dry; bio-inspection showed that scaffold was in loose configuration and white, about 2mm thickness; it is in similar thickness, toughness and intension to normal cornea after immerge into DMEM for 3 hours; Observation by microscope present that epithelium and cells of stroma had removed and no remains, complete epithelium and collagen fiber no break, whole configuration on loose reticulodromous pattern; Scanning electron microscope showed same evidence; cells could grow well on scaffold; To transplant scaffold material to surface of eye of limber stem cell deficiency animal model so that do observation of bio-compatibility and recover of eye. Results showed that no imflammation and rejection could be observed, and cornea grew well. (3) Obtain whole cornea of rabbit under asepsis condition and washed by penicillin and streptomycin, then put it into Dispase II 1.2 IU/mL to digestion, stop progess by medium contain blood serum. Epithelium tissue removed from cornea was vicarianced into cells and inoculated into culture bottle with DMEM contains 10% FBS, 20ng/mL EGF, 5ng/mL insulin inside. Under Transwell system, transdifferentiation of BMSC into corneal epithelial cells were induced in vitro by co-cultured with corneal epithelium cells. By co-culture 15 days observation showed that fibroblasts-like bone mesenchyme stem cells had become epithelium-like cells, indicating that they had been transdifferentiated into corneal epithelial cells. |
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