Preparation And Properties Of A Novel Chitosan Based Scaffolds For Corneal Stroma In Tissue Engineering

Purpose: Corneal stroma is the central part of cornea, which represents almost 90% of the total thickness. Corneal stroma is connected with epithelium through Bowman's membrane, and with endothelium through Descemet's membrane. Parallel collagen fibers formed corneal stromal lamellar structure is an essential element of normal corneal structure and its transparency. Keratocyte are the main cells of corneal stroma and locate between lamellar layers. The lack of corneal donors and immunological rejection after allograft transplantation both make it important for researchers to find a new source of cornea. And the construction of corneal stroma is the first step towards building full-layer cornea. In this dissertation, three biomaterials, namely hydroxypropyl chitosan, gelatin and chondroitin sulfate were chosen because of their outstanding biocompatibility and biodegradability, and then applied in preparation of a novel scaffold, which was hopefully able to replace human-sourced corneal stroma. The novel scaffold was examined and then compared with published data of normal cornea in some physical and chemical properties. Assessment of the scaffold's biocompatibility and biodegradability on the cell level and on the tissue level was also performed to evaluate its potential in tissue engineering application.Methods: Primary culture and subculture of rabbit corneal stroma cells were established to provide proper cells for scaffold evaluation. HPCTS, GEL and CS were blended and crosslinked when adjusted to proper pH, and then with freeze-drying treatment. The scaffolds were finally formed by heating and drying. The 7 groups of scaffolds were evaluated on the cell level using the cultivated rabbit corneal stroma cells to determine the most suitable one. The chosen scaffold was tested on light transmission values, moisture content, permeability, mechanical properties and structure. Cytotoxicity of the scaffold's extracted medium on L929 cells was measured by MTT assay. Distribution of human corneal stroma cells was analyzed by CFSE-PI double fluorescence stain. Meanwhile, to investigate the effects of CS on cells, especially on corneal stroma cells, CS group and CS-free group of scaffold were prepared and compared by means of SEM and LCSM. Muscle and subcutaneous implant tests were performed on rat. Scaffold was inserted into rabbit interlamellar stromal pocket to evaluate the compatibility in cornea. Daily nursing and taking pictures of the animals were performed with HE staining at a set interval.Results: Cells from the 1st and 2nd passages of subculture and from primary culture were chosen as the stable source of cells. Observation under light microscope and SEM helped select the most suitable material group for cultivation of rabbit corneal stroma cells. SEM and HE staining results showed that surface of the scaffold was in the form of flake structure and the inner was in loose structure. The light transmission values of scaffold in the visible spectrum was 83%~88%. The moisture content of the scaffold was 96%. The scaffold was NaCl and glucose permeable. The tensile strength was 1264KPa in dry state and 31.4KPa in wet state, while the breaking elongation rate were 5.9% and 11.0%, respectively. Elution test result demonstrated no cytotoxicity to L929 cells. LCSM images showed that human corneal stroma cells spread almost 1/4 depth of the scaffold. Biostatistics analysis results showed cell number in CS group was significantly more than in CS-free group. Muscle, subcutaneous implant tests on rat and interlamellar stromal pocket insertion test on rabbit demonstrated good compatibility of the scaffold. The scaffold degraded fast in rat muscle.Conclusion: HPCTS/GEL/CS crosslinked scaffold in this dissertation is demonstrated to have good compatibility with corneal stroma cells. But the scaffold has poor mechanical properties and degrades fast inside tissues,and so it needs further improvements.