Experimental Study Of The Combination Of Autologous Cultured Rabbit Limbal Epithelial Cell Grafting Along With Amniotic Membrane Transplatation

Objective: To study the combination of autologous in-vitro cultured limbal stem cell grafting along with human amniotic membrane transplantation in the treatment of rabbit eyes with total limbal stem cell deficiency.Methods: The rabbit-eye models with limbal stem cell deficiency in the right eyes were made from 20 selected healthy New Zealand white rabbits by operations under the microscope. A few limbal tissues from the left eyes of the 12 models were cultured in vitro, expanded for about one week, then generated and planted on the human amniotic membrane without epithelium. Continuously cultured for 20 days, synthetic corneal epithelial grafts were made. They were transplanted autologously to the corresponding 12 models with total limbal stem cell deficiency. On the contrary, we only practised amniotic membrane transplantation without epithelial cells on the other 8 rabbits.Result: For the rabbits which received amniotic membrane with autologous corneal epithelial cells, Corneal epithelialization and distinct inhibition of neovascularization were observed at the early stage of the post-operation period. It was proved that there was no distinct differences between cultured and transplanted corneal epithelium and normal one though the observation of corneal fluorescein, hematoxylin-eosin stain,Cell Keratin AEl monoclone immunohistochemistry, scanning electronic microscope and transmitting electronic microscope. On the contrary, for the rabbits which only received amniotic membrane transplantation, corneal opacification and distinct neovascularization could be observed on their diseased eyes after the operations. In the hematoxylin-eosin stained cornea, we can observe disorganized laminated epithelial cells with vacuoloid goblet cells among them, under which there are inflammatory cells, red blood cells and neovascularization in the medium. This indicates that the corneal surface has been covered by conjunctival epithelium.Conclusion: By using amniotic membrane without epithelial cells as a carrier to autologously transplant in-vitro cultured limbal stem cells, restoration of the epithelialization, inhibition of neovascularization, maintenance of the corneal transparency can be achieved at the early stage of the post-operation period. The immunorejection can be avoided as well. Animal experiment has proved that this technology is effective and practicable.