Construction And Characterization Of Tissue-Engineered Human Posterior Hemicornea In Vitro

Cornea, a transparent tissue located on the ocular surface, is an important structure to maintain normal vision. Due to the location, cornea is vulnerable to defeat injury and infection, resulting in decreased vision or even blindness, which is caused by corneal damage. The main therapy to restore the vision is keratoplasty. According to the survey, there are more than 3 million patients suffered with corneal diseases, and all of them need to be healed by corneal transplantation. The corneas from donors are limited and the quality decrease rapidly, therefore patients who could accept corneal surgery are less than 1,000 persons annually, leaving the majority without proper treatment due to the lack of donated corneas. In resent year, tissue-engineer human cornea (TE-HC) in vitro become alternative, and it is the main way to solve the shortage of donor cornea, which rising hope to blind patients. The core elements of are seeder cells, scaffold carrier and signaling molecules. The requirements of issue-engineering are seeder cells are able to maintain the cell morphology, strong proliferative capacity, genetic stability and the function, while the scaffold should have a similar three-dimensional network of natural cornea frame structure, good optical properties, mechanical strength and biocompatibility. The prerequisite of TE-pHC are obtaining sufficient quantities of seed cells and scaffolds with biocompatibility.The human cornea composed with five layers, epithelium, Bowman’s membrane, stroma, Descemet’ membrane and endothelium. The human cornea stroma (HCS). composed with 200-250 staggered collagen plates, and keratocyte scattered distributed between the plates. The thickness of stroma composed 90% of the whole thickness, and the stroma is important for maintenance of corneal thickness and transparency. Human cornea endothelium (HCE). located in the innermost layer of the cornea, is a natural barrier between the cornea and anterior chamber. The function of HCE are maintaining the transparency of the cornea and applying nutrition through the pump function, which play an irreplaceable role in maintenance of corneal thickness. When the serious cornea trauma and infection occurred, HCS and HCE are often getting hurt, causing corneal edema, corneal endothelial decompensation and even blindness. Currently corneal transplant treatment needs several times surgery, not only increases the risk of surgical trauma and infection, but also has many deficiencies in cornea transplant scar healing and residue. Thus, in vitro reconstruction of tissue-engineered human posterior hemicornea (TE-pHC) can be used for transplantation has become a hotspot. The purpose of this study is using nontransfected human corneal stromal and endothelial cell lines have been established by our lab. acellular porcine corneal stroma as scaffold and reconstructing the TE-pHC. The evaluation research focus on the morphology, tissues structure, expression of function proteins in cells. The establishment of TE-pHC reconstruction in vitro and TE-pHC obtaining pave the way to clinical application that TE-pHC become the alternative option of donated corneas for serious corneal stroma injury and infection patients.To construct TE-pHC in vitro, similar with in vivo, HCE cells and HCS cells morphology, proliferation activity, expression in vivo chromosomal properties, cell marker protein and function of proteins were identified. Observation of light microscopy showed monolayer hexagonal polygonal endothelial-like cell morphology at passage 65 of the HCE cells; the HCS cells at passage 63 show streamlined arrangement when covered with a single layer, that showed the 2 kind of cells still has their original cell morphology; growth and proliferation activity test showed HCE cells and HCS cell population doubling time was 48.54 h and 38.28 h. respectively, that is to say cells remain active proliferation in vitro; chromosome analysis found that the number of chromosomes characteristic of these two cells are 2n= 46. who are still have characteristics of human chromosome. Immunofluorescence results showed the cells. HCE cells and HCS cells were still maintain the expression of HCE cells marker proteins-vascular endothelial growth factor receptor-2 (FLK-1) and HCS cells marker protein-vimentin. which indicating that these two kinds of cells remained the inherent properties of HCE cells and HCS cells. Cell junction were found in HCE cells and HCS cells such as zonula occludens-1 (ZO-1), connexin -43 (CX-43), integrin (Integrin av/(35 and Integrinβ1). and membrane transport proteins --Na+/K+-ATPase were expressed, and HCS cell also expressed aldehyde dehydrogenase 3 (ALDH3). Both of the two cells still had the potential function of forming cells junction, the connection between the extracellular matrix and cells, membrane transport and of HCS cells maintain UV damage repair function-The cell evaluation above demonstrate that the HCE cells at passage 65 and the HCS cells at passage 63 cultured in vitro maintain the normal chromosome, active proliferative capacity, and still maintain a connection with the potential to perform the function of cells to form membrane transport, which can be used in TE-pHC reconstruction.To obtain TE-pHC scaffold. this study chose porine cornea stroma for the similarity of native cornea. Joint use of freeze-thaw cycles and nuclease digestion method to decellularized porine cornea stroma. crosslinking treatment by riboflavin and air-dried. Acellular porcine corneal stroma (aPCS) posterior lamellar were evaluated on their physical and chemical properties and tissue structure. Physical and chemical properties of the test results showed aPCS transparency and light transmittance are similar to the native cornea, slightly higher than the natural moisture content of the cornea, having a good optical performance. Histological examination showed that, after decellularization treatment, aPCS remain compact structure, collagen lamellar aligned arranged, the surface of Descemet remained intact and smooth, heterogeneous cells were completely removed during the process, low DNA content and in accordance with standard medical biomaterials, glycosaminoglycan (GAG) distribution is alike native cornea. These results showed aPCS in complete cellular removal processes retained intact extracellular matrix and glycoprotein structure. These results suggest that acellular method established in this work can effectively remove the heterogeneous cell components from porine cornea, the structure and physicochemical properties of aPCS are similar with natural cornea, which can be considered as excellent scaffold in TE-pHC reconstruction.To further evaluated the biocompatibility as cell scaffold, aPCS cytotoxicity assessed and histological were examination after seeding cells. MTT assay using HCE cells and HCS cells results showed, aPCS have no toxic effect on cells. Immunofluorescence of HCS cells and HCE cells seeding on aPCS showed, both the HCS cells inside scaffold and HCE cells on Descemet surface can express marker proteins, connect proteins and functional proteins. These results indicate that aPCS have no cytotoxicity, with good biocompatibility, provide the three-dimensional structure for cell adhesion, proliferation, migration and functional expression, which is an ideal scaffolds for TE-pHC in vitro.On the condition of preparing ideal seeder cells and excellent scaffold. this study was intended to construct functional TE-pHC and evaluate the characteristics. Injected into the stromal layers, HCS cells cultured for 1 day.3 days and 5 days, cells were fluorescently labeled and HE staining results showed HCS cell located around the injection site, and with the increase of incubation time HCS cells had the trend to spread around. HCE cells inoculated on the surface of Descemet" s membrane showed alizarin red positive stain after cells were cultured 4 day, cell Dil labeling and H&E staining showed integrity monolayers formed on the scaffold. By comparison of HCS cells and HCE cells different seeding strategies, TE-pHC reconstructive protocol was established:by microinjection methods HCS cells were seeded on the aPCS scaffold matrix and cultured for 24 h, after that HCS cells were directly seeding over Descemet membrane, then cultured for 4 days in order to reconstruct the TE-pHC. Alizarin Red staining demonstrated HCE cells form a continuous monolayer; the cell density was 2910 ± 420 cells/mm2 that equal to the cell density of 30 years old healthy adults. H&E staining showed that the HCS cells adhere around the injection point and distribute to surrounding, HCE cell continuous monolayers tightly combined with scaffold:electron microscopy of TE-pHC observation showed HCE cell monolayers on the surface formed cell-cell, cell-extracellular matrix connections and HCS cell form the cell-extracellular matrix connections. Immunofluorescence showed that the HCE cells and HCS cells on reconstruction of TE-pHC. marker proteins, connect proteins and functional proteins were positive. These results indicate that the reconstruction of TE-pHC are similar on structure organization and has the potential to exert its biological function.In summary. The purpose of this work is using human corneal stromal and endothelial cell lines maintain the normal chromosome and active proliferative capacity, acellular porcine corneal stroma with good biocompatibility as scaffold and reconstructing the TE-pHC in vitro. After culture 5 days TE-pHC with the similar structure and biological properties of native cornea tissue have the potential biological functions. These findings suggest that the successfully reconstructive TE-pHC in vitro, similar in structure and function of human cornea posterior hemicornea, can serve as a good experimental model of the cornea in vitro and may have the potential of posterior hemicornea equivalents for the clinical application laid the experimental foundation of tissue engineering cornea in vitro.