Preparation And Functional Reconstruction Of Tissue Engineered Lamellar Corneal Matrix

Chapter1Induction of UC-MSCs to differantiate to corneal epithelial cellsObjective: To investigate the feasibility of inducing the UC-MSCs to differentiate into the corneal epithelial cells in vitro.Methods:The UC-MSCs were isolated and cultured by means of enzyme digestion. The flow cytometry was employed to analyse cell phenotype and cell cycle. The cell proliferation was measured by MTT assay. The UC-MSCs were cultured in different concentrations of EGF to observe whether the cells could differentiate into corneal epithelial cells. The UC-MSCs were cocultured with rabbit corneal epithelial cells by using Transwell culture system. Then we analyse the expression of corneal epithelial marker protein by using immunofluroscent staining.Results:The UC-MSCs can be isolated and cultured successfully in vitro. The cells express CD29and CD34, maintain strong proliferative capacity.The cells could express K3under15ng/ml EGF for30days. After coculture with rabbit corneal epithelial cells for2weeks, the cells could differentiate into corneal epithelial cells.Conclusion:The UC-MSCs can be induced to differentiate into corneal epithelial cells by co-culturing with rabbit corneal epithelial cells in vitro. Chapter2The use of nucleic acid enzyme and electrophoresis at low temperature to prepare decellularized porcine corneal matrixObjective:To explore a method using nucleic acid enzyme and electrophoresis at low temperature to prepare decellularized porcine corneal matrix for tissue engineering.Methods:After removing the corneal epithelial and corneal endothelial cells, the native porcine corneas (NPC) were treated with17U/mL DNase I for12h, and then the samples were subjected to electrophoresis at4℃to remove residual cells and DNA fragments. The physical and biological characteristics were conducted in both NPC and DPCMs. The morphology, histological features, decellularized efficiency, DNA content, proteoglycan content, ultrastructural changes, and extract cytotoxicity of DPCM were analysed.Results:The removal of DNA content was reached to96.43%, which represents decellularization efficiency. And92%of glycosaminoglycan was retained in the DPCM when compared with NPC. The ultrastructure of collagen fibrils were not changed in statistically. The DPCM exhibited similar light transmittance to NPC over the wavelength range of300-800nm. The DPCM retains adequate biomechanical intension. There was no sign of infiltration of neutrophilic leukocytes or leukomonocytes of DPCM after subcutaneous implantation in vivo for2weeks. The extracts of DPCM had no inhibitory effects on proliferation of corneal epithelial and endothelial cells as well as keratocytes.Conclusion:Our findings demonstrate that the decelluarization method using using nucleic acid enzyme and electrophoresis at low temperature could remove almost all of the cells, retained the fine ultrastructure of native corneal matrix, maintain the collagen components, preserved92%proteoglycans. The DPCM possessed high transparency,low immunogenicity, favorable biocompatibility, adequate biomechnical intension, and long-term biosability.Chapter3The reconstruction of tissue engineered lamellar cornea and transplantation in vivoObjective:To reconstruct rabbit cornea using DPCM and UC-MSCs for tissue engineering.Methods:The UC-MSCs were cultured on the DPCM for3weeks. During the culture, tissue engineering lamellar cornea (TELC) wered dislodged for invert microscope to observe the epithelium morphology and HE staining to examine the cornea construction at1,2,3weeks. The ultrastructure of tissue engineering cornea was detected at2weeks, immunofluorescent staining of K3, ZO-1, Cadherin, ntegrinβ4, LN, and FN were determined. Then, the rabbit lamellar keratoplasty (LKP) using the TELC and DPCM was carried out. Epithelization, immunogenicity, transparency, biostability and wound healing process of TELC and DPCM grafts were evaluated.Results:UC-MSCs could grow well on DPCM, and could differentiate into corneal epithelial cells. The TELC could form3-4layers epithelial cells after2weeks construction in vitro. In the srafified epithelium, the cell-cell and cell-ECM connenctions were formed, the tight junction, desmosome, and hemidesmosome can be observed. The membrane structure and proteins related to cell junctions such aas ZO-1, Cadherin, ntegrinβ4, LN, and FN had expressed obviously. Rabbit LKP showed that the grafts of DPCM were epithelialized completely7±2days, and their transparency was restored in30±5days. The TELC can epithelialize immidiately and keep transparency after surgery. In60days, the light tansmittance of TELC-transplanted corneas displayed no significant difference compared with native corneas. However, the DPCM exhibited decreased slightly.Conclusion:It can at be concluded the cornea engineerd from the DPCM can mimic the nature cornea well in the structure. The TELC possesses physical functions and can keep favorable biocompatibility, high transparency, and favorable barrier function.