| Purpose:Corneal endothelium, a monolayer of cells at the posterior side of the cornea, represents the most important part of the cornea. The barrier and"pump"functions of the endothelium are responsible for maintaining corneal transparency and thickness. Consequently, a disturbance of the endothelial functions can provoke corneal edema followed by partial or complete loss of transparency.Maintained in G1-phase, human corneal endothelial cells (CEN) do not normally proliferate in vivo. Following corneal wounding, endothelial cells expand or migrate over the Decement's membrane to close the wound. If the injury is severe enough, as in a pseudophakic bullous keratopathy, aphakic bullous keratophathy, or corneal endotheliopathy, the requirement for CENs is too much for eye to provide.Penetrating keratoplasty has ususlly been chosen to restore ocular integrity in this case.Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea, which has several drawbacks including limited availability of donor corneas, recurrent allograft rejection, and subsequent graft failure in certain cases. It would be advantageous if cultivated corneal endothelial cells could be transplanted for the treatment of diseases caused by corneal endothelial disorders. To achieve this, a matrix that can serve as a carrier for corneal endothelial cells is needed.Chitosan was a promising medical biomaterial in medical fields due to its marvelous special character. As the only kind of nature polysaccharide with cation, chitosan and its derivatives have successfully attracted more and more attentions. Many researchers have made it the main material of artifical bone, artifical skin and artifical saliva for its perfect biocompatibility and controllable biodegradation. Furthermore, chitosan could restrain the cornea tissue fibrosis, which makes it a much more promising medical biomaterial in clinical ophthalmology. However, few studies has been done on the use of chitosan to construct rabbit corneal endothelial cell carrier.The purpose of this study is to explore the applicable possibility of a cellular bioactive chitosan carrier in repairing of corneal endothelium defect. Using the biodegradable chitosan-chondroitin sulfate blend membrane (Ch-Cs BM) developed by our laboratory and cultured rabbit corneal endothelial cells, we studied the cell morphology, adherence and film intensity etc. after breeding cells onto the Ch-CsBM.Furthermore, some primary researches were explored on the transplantation of the rabbit corneal endothelial cell carrier.Methods: 1. In vitro cultivation of corneal endothelium cells: The method of cultivation rabbit corneal endothelial cells was established, which was also fit for the preparation of dog or cat corneal endothelial cells. Furthermore, the promotive effect of pelagi-active materials on the proliferation of descendant endothelial cells . 2. Study of the cellular and tissue biocompatibility of three kinds of chitosan blend carrier: Three kinds (63101, 63102, 63103) of chitosan blend carriers were used for corneal endothelium cells cultivation in vitro. Rabbit corneal endothelial cells cultured in vitro were plated onto the membrane. Cell shape, the strength of membrane and the attachment of the cells to the membrane were studied. In vivo, three kinds of chitosan blend carriers were implanted into the anterior chamber of 30 New Zealand rabbits respectively and and slit lamp, thickness meter , specular microscope and HE staining were performed at random time after operation to evaluate the biocompatibility. Inflammation in anterior, thickness of cornea, cell density, hexagonality and cell size of the surgical cornea were taken as the indexes of biocompatibility. 3. Experimental study on the transplantation of rabbit corneal endothelial cell carrier cultured in vitro: 15 New Zealand rabbits was separated into 3 groups at random. Test group: The cultured carrier was then transplanted into 6 rabbit's eyes to which the corneal endothelium was scraped off. Control group: Chitosan blend carriers without endothelial cells were implanted onto the inner surface of Descemet's membrane in 6 New Zealand rabbits of endothelium defect model. Blank group: 3 rabbit's eyes were scraped off corneal endothelium and then sutured in situ. The endothelium repairing capacity of 3 groups were observed. Results: 1. The method of cultivation rabbit corneal endothelial cells was established, which was also fit for the preparation of dog or cat corneal endothelial cells. And NAG,COS were found to have positive effect on the proliferation as well as EGF. These conclusion lighted a new pathway for the problem of how to get enough"seed cells"for tissue engineering. 2. Carrier 63103 has a favorable compatibility with the corneal endothelial cells and anterior chamber. The New Zealand rabbit CEN cells cultured in vitro could form a confluent monolayer on the film, which was proved similar to the nature one at 10 days after breeding. The results suggested the non-cytotoxicity and cytocompatibility of the film. Then the film was implanted into rabbit's anterior chamber of eyeball to examine the biocompatibility. The dilatation of the iris and conjunctial vessels, fibrinous exudates, flare in anterior chamber, cornea thickness, endothelial cell density, hexagonality and cell size were investigated as indexes. During first week after operation, although there was discrete dilatation of the iris and conjunctial vessels, slight edema of cornea, some fibrinous exudates in the pupillary area and slight flare in anterior chamber, the cornea thickness, cell density, hexagonality and cell size were observed no statistical significant difference compared with control group. 2 weeks after operation, the inflammatory reaction reached peak with moderate edema of cornea and moderate dilatation of the iris and conjuctial vessels. Meanwhile, the thickness of cornea and hexagonality exhibited statistical significance but not the cell density and cell size. Then these inflammations alleviate gradually until rehabilitation at 4 weeks after operation. The biocompatibility assay implicated that this chitosan blend film was fit for the reconstruction of cornea endothelium with satisfactory biocompatibility and non-toxicity. 3. Corneal endothelial cells on carrier 63103 showed good ultra-structure and steadier cellular membrane. Half of the experimental eyes with a bioactive cells carrier transplantation showed corneal transparency at the early post-operative period.Conclusions:This study shows the potential for using chitosan/chondroitin sulfate blend film as a carrier for corneal endothelial cell transplantation. Chitosan has a high affinity with corneal endothelial cells , it is nontoxic and stimulative for cell proliferation. Physical and chemic property of the carriers may influence its biocompatibility significantly. Shape and structure of corneal endothelial cells on the carrier with high biocompatibility were well preserved. The endothelium repairing capacity of this cellular bioactive carrier needs further study. |
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