| Objective:As we all known, inferior to cataract, keratinous is the second illness which leads to blindness in our country. There are 4 million patients suffered blindness caused by keratinous, and only 5 thousand patients can be treated by transplantation of donor corneas every year bacause donor corneas are insufficient.-With the development of tissue engineering and the methodological establishment of corneal cells culture, it would be advantageous by the way of corneal tissue engineering for the treatment of diseases caused by corneal endothelial disorders. Chitosan (Cts) is a partially deacetylated derivative of chitin, which shows a lot of interesting biological properties such as low immunogenicity, low cytotoxicity, biodegradability and biocompatibility. Recently, Cts is being taken more seriously in tissue engineering field, and it is reported that carriers made of Cts have been used in skin tissue engineering, bone tissue engineering, nerve tissue engineering, vessel tissue engineering and so on. In addition, blend membranes composed of Cts, gelatin (Gel), chondroitin sulfate (CS) or hyaluronic acid (HA) have been used for reconstructing artificial cornea. Aim to accelerate biodegradation and bring down the hardness of Cts(high deacetylation degree) based blend membrane, in this research the soluble Cts (D.D.≈50%) as main ingredient is crosslinked with Gel and CS, in order to obtain a kind of blend membrane which has excellent physicochemical property, good tissue compatibility, and is suitable for corneal endothelial cells.Methods:1.To improve the solubility of Cts, Cts with deacetylation degree at 50% was pepared in our lab. Strictly according to biomedical material and tissue engineering standard, aquiferous capacity, ash content, heavy metal content, pH, particle size, cytotoxicity of the soluble Cts were determined.2. The soluble Cts as main ingredient was crosslinked with Gel and CS to obtain three kinds of blend membranes at different proportions. Then transparency, water content, cytotoxicity, NaCl permeability, cytocompatibility with the rabbit keratocytes were tested, and Cts-Gel-CS blend membrane with the volume ratio at 9:1:0.1 was selected out.3. Biodegradability and biocompatibility of Cts-Gel-CS blend membrane were studied in vitro and in vivo respectively. The in vitro degradation was carried out by immersing the blend membrane in pH 7.4 buffered lysozyme solution at 37℃. While the in vivo biocompatibility and biodegradability were studied by implanting the blend membrane in the subcutaneous tissue, leg muscle of rats and in atria of rabbits.4. New Zealand white rabbit corneal endothelial cells were seeded on Cts-Gel-CS blend membrane to construct artificial cornea endothelium. And concave blend membrane was prepared through special equipment in our lab.Results:1. The soluble Cts prepared in our lab is in agreement with standard of biomedical material and tissue engineering. Cytotoxicity experiments showed that the soluble Cts has no cytotoxicity. The Cts solutions with different concentrations can promote L929 proliferation, significantly at 250μg/mL.2. Through Comparing the properties of three kinds of blend membranes, the blend membrane with the volume ratio at 9:1:0.1 of Cts-Gel-CS is selected out as the corneal tissue engineering carrier which has good optical transmittance, suitable water content, no cytotoxicity and better cytocompatibility with the rabbit keratocytes.3. Cts-Gel-CS blend membrane prepared not only degrades faster than Cts film in vitro, but also has good tissue compatibility. It is found that Cts-Gel-CS blend membrane can be degraded in less than 60 days in the subcutaneous tissue and leg muscle of rats and 2 months in rabbits' atria. HE staining results showed that the tissue reaction towards the blend membrane was very mild.4. Cts-Gel-CS blend membrane has good cytocompatibility with the rabbit corneal endothelial cells. In addition, the concave blend membrane has certain mechanical strength and good transparency.Conclusions:As we hoped, the soluble Cts prepared in our lab is in agreement with standard of biomedical material and tissue engineering. The blend membrane with the volume ratio at 9:1:0.1 of Cts-Gel-CS has good optical transmittance, suitable water content, no cytotoxicity and better cytocompatibility with the rabbit keratocytes and corneal endothelial cells. Animal tissue compatibility experiments showed that no remarkable inflammation appeared and the tissue reaction towards the blend membrane was very mild. The concave blend membrane prepared through special equipment has certain mechanical strength and good transparency. In summary, this blend membrane is a promising carrier of corneal endothelial cells to reconstruct tissue engineering cornea.
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