| The cornea is the window of the eye, responsible for 70% of the eye's refractive power. Corneal blindness results when opacity occurs due to disease, aging and trauma, when irreversible and affects more than 10 million individuals worldwide (WHO, 2001), second only to cataracts in overall importance. To date, the only widely accepted treatment is transplantation with human donor corneal tissue. However there is an increasing need for human donor corneal tissue and a severe shortage of suitable cornea donors in many countries as the increase of the old-aged population and increased use of corrective laser surgery. Further more, many patients with a complex origin or with multiple graft failure will not benefit from further corneal transplantation. Research in corneal replacements, mainly keratoprosthesis (KPro), has been on-going for over a century. However, most KPros failed because of poor biocompatibility, non-biodegradability and inadequate interaction of artificial implant with host cornea. The best known KPro, AlphaCorTM designed with a porous skirt and a central optical core has shown some success, but it still has many disadvantages that limit its widespread application. Therefore, a tissue engineered cornea substitute structurally and functionally similar to human cornea and suitable for transplantation is in very high demand.A tissue engineered cornea should be ideally reconstructed from corneal cells and corneal extracellular matrix and biodegraded spontaneously after transplantation as new and functional host cornea is regenerated. The regenerated cornea will be theoretically permanent and have no rejection problem. In this proposal, Fresh porcine corneas are acquired from meat industry and then be decellularized with different methods. Morphological, histological examination and uniaxial tensile testing are carried out for examination of physical and chemical properties. An optimal method is selected and then the biocompatibility and biological safety of the acellular corneal matrix are to be examined. The matrix is biocompatible with cornea-derived cells and has potential for use in corneal transplantation and tissue-engineering applications.Part 1 Development of a full-thickness acellular porcine cornea matrix for tissue engineeringObjective: Our aim is to compare the effects of different decellularization methods on efficiency of cell removal from porcine cornea and optimize a protocol to produce an acellular porcine cornea scaffold and investigate its biologic properties for use in developing a tissue-engineered cornea.Methods: Fresh porcine corneas were decellularized with different detergents over a range of concentrations in the presence of protease inhibitors: Triton X-100 (concentration of 0.25%, 0.5%, 1%, 2%, 5% for 12h, 24h, 36h, 48h, 72h, 96h), sodium dodecyl sulfate (SDS) (concentration of 0.1%, 0.25%, 0.5%, 1% for 12h, 24h, 36h, 48h), sodium deoxycholate (SD) (concentration of 0.25%, 0.5%, 1% for 12h, 24h, 36h, 48h), and 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) (concentration of 0.5%, 1% for 12h, 24h, 36h, 48h). Snap freezing for 1h, 24h and 24hx3 was performed as physical method. Morphological and histological examinations were carried out at different intervals to detect the major structure of the cornea. Completely acellular cornea scaffolds were subjected to uniaxial tensile testing. The resulting acellular matrices were then characterized and examined specifically for completeness of the de-cellularization process and the potential effects on the biochemical composition, ultrastructure, and mechanical behavior of the ECM scaffold materials. Results: Treatment with 0.5% and 1% Triton X-100 solution for a period of 24 hours showed no apparent change in cellularity compared with a normal cornea. Increasing incubation times up to 96 h and the concentration to 5% resulted in only a slight decrease in corneal cells, with the epithelium, fibroblast cells and endothelium not being fully removed from the cornea. Only SDS at concentration of 0.5% and 1% for 24 h had adequate decellularizing properties without the disruption of the overall tissue histoarchitecture. With lower SDS concentrations and shorter incubation times showed inadequate removal of cells. Increasing SDS incubation times at concentrations of 0.5% and 1% resulted in apparent disruption of the tissue histoarchitecture, especially the intact Bowman's layer and the collagen fibers. Treatment with 0.5% and 1% SD solution for a period of 24 hours resulted in a significant decrease of whole cells and cell fragments. Increasing incubation times to 36 hours did not result in full loss of corneal cell nuclei and cell fragments. At incubation times up to 48 hours, full loss of corneal cells occurred but the tissue histoarchitecture, including the Bowman's layer and Descemet's membrane, was destroyed at the same time. Treatment with 0.5% and 1% CHAPS solution for a period of 24 hours showed little loss of corneal cells, with cell nuclei and fragments remaining. Increasing the incubation time to 36 h did not increase the apparent decellularization but destroyed the tissue histoarchitecture. Increasing the Snap freezing times to 24h×3 did not increase the apparent decellularization but destroyed the tissue histoarchitecture. Uniaxial tensile testing indicated that decellularisation by 0.5% SDS for 24h did not significantly compromise the ultimate tensile strength of the tissue (P>0.05). Histological analyses of decellularized corneal stromas (0.5% SDS for 24h) showed that complete cell and a-Gal removal was achieved, while the major structural proteins including collagen type I and IV, laminin, and fibronectin were retained. The rate of water contents and concentration of hydroxyproline in the decellularized cornea were 92.6805±0.3015 (%) and 2.1510±0.1049μg/mg, while 81.1778±1.9123 (%) and 1.6134±0.0755μg/mg. There was no detectable DNA or soluble protein in the decellularized cornea.Conclusions: Only SDS at concentration of 0.5% for 24h had adequate decellularizing properties without the disruption of the overall tissue histoarchitecture. A full-thickness natural acellular matrix retaining the major structural components and native mechanical properties and biologic properties has been successfully developed. The matrix has potential for use in corneal transplantation and tissue-engineering applications.Part 2 Study on biocompatibility and biological safety of acellular porcine cornea matrixObjective: Our aim was to examine the biocompatibility and biological safety of the acellular porcine cornea matrix for use as a scaffold in developing a tissue-engineered cornea replacement.Methods: According to the biological evaluations of medical devices in ISO 10993 and GB/T16886 standards, the CCK-8 assay, systematic toxicity test, pyrogenic reactions test in rabbit model and implant test in vivo were performed.1. Effect of the extraction from acellular porcine cornea on cell viability was determined using the CCK-8 assay. The absorbance values of optical density (OD) at 450 nm were measured and then relative growth rate was calculated.2. Extraction of acellular porcine cornea matrix was injected into KM mice intravenously. The mice were observed for toxicity immediately after administration and at 24,48, and 72 hours.3. The body temperature of rabbits injected with extraction of acellular porcine cornea matrix intravenously was taken to determine the pyrogenicity.4. Thirty New Zealand White rabbits were divided into 3 groups randomly. Acellular and fresh porcine corneal stroma with diameter of 5mm and depth of 150μm was inserted into corneal stromal pocket of rabbits respectively. No stroma inserted served as control. Clinical observations of conjunctival congestion, cornea edema and neovascularization were performed at intervals postoperatively with a slit lamp. At each time point (2w, 4w, 8w, 12w and 24w), 2 corneas per group were excised to observe histological changes.5. The presence of porcine endogenous retrovirus (PERV) in fresh, acellular porcine cornea and peripheral blood of rabbits inserted with acellular porcine cornea stroma for 4w, 12 w and 24 w were determined by RT-PCR.Results: 1. After predetermined time points, the confluence of the monolayer and the change in cellular morphology after exposure to the extracts of acellular porcine cornea were similar to those of the negative control. There was no significant differences in cell proliferation of the rabbit keratocytes following incubation with the extracts from acellular porcine cornea compared to that of the negative control as determined by the CCK8 assay (P>0.05), the relative growth rate on 1, 3, 5 d is 108.36±1.89 (%) ,122.14±0.13 (%) , and 101.85±0.69 (% ) , respectively, which in turn excluded the presence of soluble toxins in the biomaterial. 2. There was no induced fever in rabbits injected with extraction of acellular porcine cornea matrix intravenously. 3. None of the mice tested with sodium chloride extracts of acellular cornea matrix or negative control with sodium chloride alone showed any mortality or evidence of systemic toxicity. 4. No obvious exudation and influx of inflammatory cells occurred in the cornea. After implantation, a corresponding inflammatory response including corneal opacification and neovascularization were noted, the extent of which in fresh porcine cornea stroma was more severe than that of acellular stroma. Transparency developed in all corneas gradually after cornea sac interstitial implant without rejection reaction. At 4 weeks postoperatively, acellular cornea stroma become transparent and a few rabbit keratocytes immigrated into the acellelar cornea stroma. PERV was not detected in the acellular porcine corneas and peripheral blood of rabbits inserted with acellular porcine cornea stroma for 4w, 12 w and 24 w by RT-PCR in which porcine kidney 15 (PK15) cells served as a positive control.Conclusions: Acellular porcine cornea stroma is a biocompatible material without extract cytotoxicity, systemic toxicity, pyrogenicity and immunogenicity. And PERV in porcine fresh cornea can be removed after decellularization. Thus, we have shown that porcine corneal matrix is suitable to serve as an ideal scaffold material in tissue engineering cornea. Part 3 Construction of cornea stroma based on acellular porcine cornea matrixObjective: To detect the possibility of corneal stroma construction based on acellular porcine cornea matrix and observe the re-cellularization of acellular porcine corneal matrix in vitro.Methods: Primary rabbit epithelial cells, stromal cells and endothelial cells were cultured through explant cell culture methods, and their morphology and phenotype were examined through immunohistochemistry. Cultured keratocytes suspended at 1×105～106 cells/mL will be inoculated on the surface or injected into ACM by a syringe. And then, the histological structure and cellular morphology was examined by HE staining.Results: Primary rabbit epithelial cells, stromal cells and endothelial cells prossess normal phenotype and express AE5, Viminin, and NF, respectively. Stromal cells can spread on the surface of the acellular porcine cornea matrix after inoculation and survive in the internal of the matrix, which gathered in the different part.Conclusions: We can develop 3 kinds of cells of cornea through primary cell culture methods; corneal stromal cells can survive and keep normal phenotype when cocultured with acellular porcine corneal matrix. We should improve the culture method to construct the ideal corneal stroma in the later study.
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