An Experimental Study Of Biomaterials Based On Chitasan For Reconstruction Of The Corneal Epithelia And Endothelium

The cornea is the most prominent part of the eye and connecting directly with the external environment. Thus it is vulnerable to the external damage. Human cornea consists of five layers of corneal epithelium, Bowman layer, stroma, Descemet's layer and the endothelium. Corneal epithelium is the outermost layer, so it is easy to be injuried by the the mechanical and chemical. After that, symptoms such as photophobia, tearing can caused, directly affecting the patient's normal life. Use of medication, eye drops, the permeability increases, which contains additives, preservatives will increase the side effects of drugs, easily leading to the dissolution of corneal stroma, secondary infection, or even perforation, corneal epithelial defect. Therefore, promoting wound healing quickly and completely and restoring the barrier function of corneal epithelial cells are the keys to maintaining good vision. So choosing rapid and effective methods for regeneration of corneal epithelium is an important role for the mechanical and chemical damage of corneal epithelium.Corneal endothelium plays an important role in the maintenance of corneal normal physiological function. Adult human corneal endothelial cells do not normally proliferate, and cell density decreased with age. The corneal endothelium is extremely sensitive to many external factors, such as mechanical trauma, chemicals, drugs and physical rays. After being injuried, corneal endothelial cells rely mainly on adjacent cell expansion, migration to fill the defect. When the corneal endothelial cell density less than the critical cell density (400~500/mm~2), the endothelium can not be compensated residual and lead to corneal stroma edema, corneal transparency decreased, or even blindness. Corneal transplantation is the main treatment methods to restore corneal transparency, but the source of corneal allograft donor is short, and it is also often accompanied by a certain degree of immune rejection, so the treatment of corneal disease is an international problem.With the development of tissue engineering, tissue engineering methods of treatment corneal diseases become a hot spot. Chitosan is a natural poly-positive polysaccharide, widely studied and applied in the pharmaceutical, biomaterials, food, chemical, bioengineering and agricultural fields. However, chitosan is water-insoluble, and some inflammatory responses will be accompanied in the course of degradation, so chitosan was modified by chemical modification in this paper to study the function and application of water-soluble chitosan-based biomaterials in corneal wound repair.This study has two parts: (a) hydroxyethyl chitosan (HECTS) was prepared first, its basic properties were studied; then with gelatin (Gel) and chondroitin sulfate (Cs) by a certain percentage of blend membrane were prepared. The membrane's physical and chemical properties, toxicity, compatibility with the corneal cells, degradation in vitro and in vivo, were studied, and the more excellent membrane was slected. The corneal epithelial cells were cultured on tne selected membrane, tissue engineering corneal epithelial was constructed, and then transplanted to mechanical damaged corneal epithelia of New Zealand rabbits, the repair effection was observed. (B) Hydrogel was prepared, and the cytotoxicity of the hydrogel, in vivo biocompatibility and degradation in rabbit anterior chamber were evaluated. With the hydrogel as a carrier, the cultured corneal endothelial cells were transplantated on the Descemet's membrane, the repair effection of corneal endothelial injuried model was observed.Get the following results:1, Prepared hydroxyethyl chitosan has good water solubility. Cell toxicity test results showed it is non-toxic biomaterials. In a certain concentration, HECTS could promote the growth of corneal epithelial and endothelial cells obviously.2, HECTS with CS and Gel were optimized for different mass ratios to obtain blend membrane 1# and 2#. They have high optical transparency, and 1# is even more transparent. The membranes have good ion permeability and their glucose permeability is higher than natural human cornea. Cytotoxicity meets the standards of biomedical materials. Stromal cells and endothelial cells can grow on the membranes well and form a good layer. The results of the muscle implantation test by the mice to evaluate the biocompatibility and biodegradability shows that the membrane could degrade in the muscle steadily and does not have a "cyst" around it, and compared to non-degradable sutures the inflammation was mild. Blend membrane implanted in rabbit anterior chamber can be slowly degraded and does not produce significant inflammatory responses.3, Cultured corneal epithelial cells seeded in the blend membrane could be adherent and connected to each other into a film layer. Using the membrane with cells to cure seriously mechanical damaged rabbit corneal epithelium, the results show that wound repair time of the experimental group is half of the control group's.4, Cell toxicity grade of hydrogel is 0. Hydrogel degradation in muscle indicates that there are certain inflammatory responses in the early stage. When most of the material degraded, inflammatory reaction subsided.5, With hydrogel as the carrier, the rabbit corneal endothelial cells were transplantated on the Descemet's membrane of rabbit eye. After surgery, observed by naked eyes, scanning electron microscopy, histological, and slit lamp, the results obtained are consistent. The experimental group observed has formed a good corneal layer until day 75, the cornea is thin and transparent, and the corneal endothelial cells transplanted are almost the same as normal rabbit corneal endothelial cells. On day 90, the experimental group had returned to normal rabbit eyes, damaged cornea of control group remained opaque and thick by slit-lamp. This indicated that this experiment created a very successful model of endothelial injury. Corneal endothelial cells transplanted by the composite hydrogel could survive and retain normal morphology on the Descemet's membrane.In brief, bimaterial based on Chitosan studied as the cell carrier could repair the damaged rabbit corneal epithelia and endothelium. These results provide an opportunity for corneal epithelia and endothelium reconstruction based on tissue engineering methods.