Tissue-engineered Repair Of Rabbit Corneal Endothelial Injury

The transparent disc-shaped structure, one-sixth of the outer wall of the eyeball,is the cornea. It plays the role of protection and as a refractive medium. Cornea is afive-layers structure: epithelium, Bowman layer, stroma, Descemet’s layer andendothelium. Endothelium is composed of single-layer endothelial cells to maintainthe dehydrated state of cornea by the Na+-K+ATPase pumps on the cell membrane.The adult corneal endothelial cells lose the proliferative ability, so the cell loss isfilled by expansion and migration of neighboring cells. However, once the cell densitydecreased to its critical value (700/mm), it might cause corneal endothelium blindness.At present,11million patients all over the world suffers from the corneal endotheliumblindness and the only effective clinical treatment measure is penetrating keratoplasty.However, allogeneic cornea transplantation often cause immune rejection and islimited by the lack of donor. Therefore, the researchers are committed to theconstruction of tissue-engineered corneal endothelium. Now, corneal endothelial cells,embryonic stem cells, adult stem cells and adult cells with stem cells’ characteristicsare often used as seed cells while allogeneic or xenogeneic corneal lamellar andnatural or synthetic polymer materials are often used as carriers. This study isdesigned to construct tissue-engineered corneal endothelium by rabbit cornealendothelial cells and chitosan-based hydrogel and to repair the corneal endothelialinjury.The research is composed of the following aspects:(1)Evaluate the toxicity of the chitosan-based hydrogel: after long-termintraperitoneal injection of the hydrogel to rats, their general clinical conditions aremonitored, serum biochemical indices and organ coefficients are tested and organhistological structure are observed to evaluate the hydrogel’s subchronic toxicity onthe main organs of rats; rabbit corneal endothelial cells are cultured in the medium with the leaching liquor of the hydrogel to evaluate its cytotoxicity.(2) Optimize themedia: rabbit corneal endothelial cells are cultured in media with different chitosanderivatives to filter the ones which can promote the cells growth significantlly.(3)Repair the injured corneal endothelium of rabbits by tissue engineering: the injurymodel is build by removal of Descemet’s layer and endothelium, after that, thechitosan-based hydrogel and the rabbit corneal endothelial cells cultured in optimizedmedia are used for the reconstruction of the corneal endothelium in the model animals.The eyeball photos, the cornea pictures by SEM and the histological structure in thecornea of model rabbits are parameters to evaluate the feasibility of the above method.The results are as follows:(1) The hydrogel shows no significant toxicity in rats’ liver and spleen, whilecauses minor injury on rats’ kidney at first, but the injury recover later. And it isnon-cytotoxicity.(2) CM-CT and CS could promote the growth of rabbit cornealendothelial cell both separately and together.(3)12weeks after cells transplantation,the corneas of half of the experimental group rabbits recover to be transparent. Asingle layer of corneal endothelium were observed under microscope and the cellswere typically polygonal in the pictures by SEM.In summary, it is conclusive that: this chitosan-based hydrogel is suitable to beused as scaffold in the construction of tissue-engineered corneal endothelium; thegrowth of the rabbit corneal endothelial cells could be promoted significantly in themedia mixed with CM-CT and CS; the treatment measure for the corneal endothlialinjury by tissue engineering method is feasible but further research are needed toincrease the success rates.