Human Amniotic Membrane Bioscaffold And Construction Of Tissue-engineering Corneal Epithelium

Ocular surface disorders, due to irreversibly destroy of corneal limbal stem cells (CLSC) maintaining corneal epithelial renewal, result from malcompensation of limbal stem cells, which was characteriqed by persistent epithelial defects, corneal stromal melting, corneal neovascularization with scarring and conjunctival epithelial ingrowth. The treatment for ocular surface disorders was arduous. Recently, human amniotic membrane (HAM) transplantation was a new operation performed for ocular surface disorders. According to documents, it had partial curative effects on some ocular surface disorders including recurrent pterygium, acute thermal/chemical-burned eye, severe burned eye with proliferating scarring and some chronic inflammation. The pure HAM tissue can not be operated easily. Cells on HAM can not be observed conveniently if Nitrocellulose(NC) membrane which wasn't been delt with is used to load HAM. The reported methods of disposing and conserving HAM are complex. And cells are difficult to adhere to HAM surface if it still contains basement membrane. For all these reasons, we try to make a new kind of HAM that can be easily and conveniently operated, prepared and preserved and is good for cells adhering. If the corneal epithelial cells successfully grow and form graft well in vitro on HAM carrier, the graft can be used into HAM-cornea. Autograft of corneal limbal epithelial cells carried by HAM can reconstruct injured cornea, but they can't repair the cornea injured by Stevens-Johnson syndrome (SJS) and Ocular Cicatricial Pemphigoid (OCP). In this research we first used HAM to culture corneal limbal epithelial cells to make HAM grafts, and succeed in curing the lack of corneal limbal epithelial cells. In this study, we removed epithelial cells, sponge layer and with basement membrane from HAM. HAM was frozen and stored at －80℃ in pure glycerol for 5 months. As a result, it was indifferent from fresh HAM and can also make cells grow well. The HAM scaffold with self-made NC membrane frame can adhere?to the scaffold closely, without shrivelling and floating and is good for cells` floating culture. Then we cultured cells on the scaffold and made them into HAM-cells grafts. Basiclly, the Animal models was made in similar methods of the general anaesthesia, settle, shear the eyelash, the ocular surface cleaning followed by eyeball anaesthesia. Rabbits were used for alkili-burn eye and disinfection model, while goat cornea limbal was ablated micro-operation under ophthalmoscope. In general, it was the successful phothological model of total CLSC deficiency that those corneal surface were haze with neovasculavization and conjunctivalizaion, as well as the corneal stroma were integrate, symblepharon (adhesions of lid conjunctiva to globe conjunctiva) were not observed, which could be used for limbal stem cells transplants. To create the pathological model of CLSC deficiency, 20 nomal rabbits were randomly divided into A,B,C and D groups. In group A, the rabbits corneal limbal epithelium lamella were excised, and the central corneal epithelia were burned with 1N NaOH. In group B, corneal limbal epithelium lamella were all excised, in group C only the upper–half of limbal epithelium as removed, the central corneal epithelium of the both groups were erased with a swab soaked in physiological saline. In the group D, the corneal and limbal epitheliums were burned with a cotton swab socked in 1N NaOH. After 4 weeks the results showed that 100 percent of the rabbits in group A could be used. In group B only two fifths of animals could be used. All corneas of rabbits in group C were transparent, with intact corneal epithelium, and so could not be used as model. In group D, 4 rabbits with total CLSC deficiency so could not be used because of serious stroma perforation and symblepharon, another rabbit recovered as group C. That corneal limbal epithelium lamella excised and the center corneal epithelium burned with 1N NaOH is applicable method to create the pathological model of total CLSC deficiency. Aft...