Preparation, Characterization And Transplantation Of Tissue Engineered Corneal Endothelium

Chitosan is a biodegradable, semicrystalline polysaccharide obtained by N-deacetylation of chitin, which is harvested from the exoskeleton of marine crustaceans. Chitosan has numerous applications in various fields. Due to its biocompatibility, biodegradability and bioactivity, it is more and more considered as a very interesting substance for diverse applications in biomaterial and tissue engineering.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.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 reconstructed corneal endothelium by tissue engineering is needed. In previous, many progress have been reported in this field, however, no research choose chitosan to reconstruct corneal endothelium.In the study, a new chitosan-chondroitin sulfate blend film was developed and used as a carrier for corneal endothelial cell transplantation. The studies develop five contents as below:1. Chitosan with different degree of acetylation was obtained from N-deacetylation of chitin; carboxymethyl-chitosan and carboxymethyl-chitin were obtained for carboxymethylation of chitosan and chitin respectively. Chitosan, carboxymethyl-chitosan and carboxymethyl-chitin were purified and were validated as germfree and no pyrogen. The degradation of chitosan films with different degree of acetylation was studied by lysozyme action in vitro and by rat muscle in vivo, the result indicated that the degradation rate in vivo was quicker when as the degree of deacetylation increased. Interestingly, mild tissue reaction was observed for all the deacetylated chitosan. Cell culture assay show that carboxymethyl-chitosan and carboxymethyl-chitin had no cytotoxicity effect on L929 mouse fibroblasts.2. Three kinds of blend films were prepared by chitosan and chondroitin sulfate (Ch-Cs ); chitosan and carboxymethyl-chitosan(Ch-Cm ) and chitosan and hyaluronic acid(Ch-Ha ). Transparence, water absorption percentage, permeability, mechanical properties, surface structure, adsorption of protein and FTIR of the blend films were analyzed. The results indicated that Ch-Cs blend films has excellent transparence, uniform structure, and appropriate hole size. Chitosan and chondroitin sulfate can disperse to molecules and strong effects exits between molecules. The rabbit corneal epithelial, stromal and endothelial cells were cultured on three kinds of blend films respectively. The cells on the membranes were observed in order to study the biocompatibility of the corneal cells. The cells can overspread the whole surface of the blend films, which indicate that they have excellent compatibility. Ch-Cs blend film was screened as the best scaffold for the attachment and growth of corneal endothelial cell.3. Biodegradability and biocompatibility of Ch-Cs blend film were studied in vitro and in vivo respectively. The in vitro degradation was carried out by immersing the blend films in pH 7.4 bufferd lysozyme solution at 37℃, the results indicated that the blend film can be degradated by lysozyme in 21 days.While the in vivo biocompatibility and degradability were studied by implanting the blend film in the leg muscle of rat and in the stromal of cat, HE staining were performed at fixed time after operation to evaluate the biocompatibility. It was found that the Ch-Cs blend film can be degradated in 60 days in muscle and 35 days in cat corneal stromal. The tissue reaction towards the blend film was very mild. Especially after transplantation, the experimental cornea of cat keep absolutely transparent during the period.4. In order to improve the permeability of Ch-Cs blend film, KAc was screened as the best porogen. Different dosage of KAc was added during the preparation of film, the results indicated that the permeability was improved when certain dosage of KAc was added. SEM indicated that the blend film treated by certain dosage of KAc has appropriate surface structure.5. New Zealand white rabbit corneal endothelial cell and cat corneal endothelial cell were seeded on concave Ch-Cs blend film, respectively, the concave blend film was prepared through special equipment in our lab. After several days, the blend film with a monolayer of rabbit corneal endothelial cells was transplanted onto rabbit corneas and cat corneas respectively, whose endothelial cells had been completely removed. In our study, 80% of the experimental eyes of rabbit with a bioactive cells carrier transplantation showed corneal transparency at the early post-operative period, and 46% showed corneal transparency at the long post-operative period. The result indicate that the tissue engineered corneal endothelium has perform the activity of corneal in rabbit eyes.In summary, the Ch-Cs blend film had excellent physical properties, Chitosan and chondroitin sulfate can disperse to molecules and strong effects exited between molecules. The blend film had favorable biocompatibility and biodegradability. Its'permeability was improved when certain dosage of KAc was added. SEM indicate that the blend film treated by certain dosage of KAc has appropriate surface structure. The blend film showed to be good carrier for attachment and growth of corneal endothelial cell. Corneal endothelial cell-loaded blend film was transplanted into rabbit eyes with endothelium scraped. The experimental eyes remained transparent in long time, shows the potential for using Ch-Cs blend film as a carrier for corneal endothelial cell transplantation.