Preparation And Properties Of Chitosan-based Carriers Of Corneal Endothelial Cells In Tissue Engineering

The corneal endothelium represents the most important part of the cornea. Only an intact endothelium with a sufficient cell density can function properly and maintain clarity of the cornea by its dehydrating pump function. Various conditions including Fuchs corneal endothelial dystrophy or increased intraocular pressure (IOP) after keratoplasty can also increase or accelerate irreversible endothelial cell loss. Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea. However, this treatment has several drawbacks, e.g., insufficient donor corneas, recurrent allograft rejection, and subsequent graft failure in certain cases.With the development of tissue engineering and the methodological establishment of isolation and culture of corneal cells, it would be advantageous if cultured corneal endothelial cells could be transplanted for the treatment of diseases caused by corneal endothelial disorders. So far, the carriers used for corneal endothelial cells include allograft cornea, thin gelatin membrane, hydrogel membrane, collagen matrix, collagen sheet, biodegradable polymers, amniotic membrane, Descemet membrane, and so on. However, the shortages such as immunological rejection, poor mechanical properties and uncontrolled degradation rate have limited their clinical application. Therefore, the present study aimed at searching for carrier materials that may have better cell attachment promoting properties and implantation applicability for corneal endothelial cells.Chitosan (CTS) is a partially deacetylated derivative of chitin, being composed mainly of (1-4)-2-amino-2-deoxy-D-glucopyranose repeating units. It shows a lot of interesting biological properties such as low immunogenicity, low cytotoxicity and biodegradability. Therefore, it has been extensively used in tissue engineering. But its biocompatibility in cornea can not satisfy the need of transplantation. In this study, we prepared hydroxypropyl chitosan (HPCTS) to improve the biocompatibility of chitosan. Chitosan-based blend membranes were developed and used as carriers of corneal endothelial cell. The studies develop four contents as below:1. To improve the biocompatibility of CTS, a water-soluble derivative of CTS-HPCTS was prepared. It was characterized by Fourier-transform infrared(FTIR), ~1H-NMR and X-Ray diffraction(XRD). The substitution degree of HPCTS was measured by Elemental analysis. The effect of HPCTS on growth of rabbit corneal stromal cells was also evaluated by using MTT assay. The structure characterization results showed that hydroxypropyl group was successfully grafted on the chitosan chain; MTT testing suggested that HPCTS had no obvious cytotoxicity, and had good cytocompatibility, which offers a theoretical basis for its application in tissue engineered cornea.2. Three kinds of blend membranes labeled hydroxypropyl chitosan/ chondroitin sulfate, hydroxypropyl chitosan/gelatin /chondroitin sulfate and hydroxypropyl chitosan/ oxidized hyaluronic acid/chondroitin sulfate were prepared, respectively. To evaluate the cytocompatibility of the blend membranes with corneal epithelial cells, the rabbit corneal epithelial cells were cultured on the surface of the carrier membranes. The morphological characteristics, cell adhesion, cell proliferation and the activity of lactate dehydrogenase (LDH) in the media were investigated. The results showed that less injury was made to corneal epithelial cells by the hydroxypropyl chitosan/gelatin/chondroitin sulfate blend membrane than the others. This kind of membrane benefited the growth and adhesion of corneal epithelial cells. The hydroxypropyl chitosan/gelatin/chondroitin sulfate blend membrane is a promising carrier of corneal cells, which can be used in reconstruction of tissue engineered cornea.3. The transparency, equilibrium water content, permeability, mechanical properties, protein absorption ability, hydrophilicity and surface morphology of the blend membrane were determined. The biodegradability and biocompatibility of the blend membrane in vivo were also evaluated. The in vivo biocompatibility and degradability were assessed by implanting the blend membranes into the skeletal muscle of rat and in the anterior chamber of rabbits. HE staining was performed at predetermined time after operation to evaluate the biocompatibility. It was found that hydroxypropyl chitosan-chondroitin sulfate-gelatin blend membrane has excellent physical and biological properties. The results indicate that the hydroxypropyl chitosan-chondroitin sulfate-gelatin blend membrane can be degraded in 90 days in muscle and 60 days in rabbit corneal anterior chamber. The tissue reaction towards the blend film was very mild. Thereby, it has good biocompatibility and biodegradability. This study shows the potential for using hydroxypropyl chitosan-chondroitin sulfate-gelatin blend membrane as a carrier of corneal endothelial cell transplantation.4. To reconstruct the corneal endothelium in vitro, New Zealand white rabbit corneal endothelial cells were cultured and seeded on the surface of hydroxypropyl chitosan-chondroitin sulfate-gelatin blend membrane. After several days, the corneal endothelial cells on the blend membranes formed a monolayer, which demonstrates that this membrane is suitable for corneal endothelial cells to attach and grow.In summary, the hydroxypropyl chitosan-chondroitin sulfate-gelatin blend membrane had excellent physical properties and favorable biocompatibility and biodegradability. The blend membrane showed to be a good carrier for attachment and growth of corneal endothelial cells. All of the results demonstrated the potential for using hydroxypropyl chitosan-chondroitin sulfate-gelatin blend membrane as a carrier of corneal endothelial cell transplantation.