In Vitro Reconstruction Of Tissue-Engineered Human Corneal Endothelium And Research Of Its Function By Rabbit Corneal Endothelial Transplantation

Integrative monolayer of human corneal endothelial cells (HCE cells) is the key factor of maintaining the normal thickness and transparency for cornea, and the transparency is essential to maintain normal function of eye vision. HCE cells in adults have lost their ability in cell division. The cell damaged area in endothelium could only be mended by the neighboring HCE cells via enlargement or extension. Once cell density is lower than the critical density needed to sustain endothelial functions, the endothelium will undergo un-reversible pathological changes and cause eye blindness which is called primary corneal endotheliopathy. The number of this kind of patients is more than 0.8 million in China, and 11 millions in the whole world, and the number is still growing with year. The corneal endotheliopathy suffers can almost all be recuperated by corneal transplantation, but most of them have no opportunity to receive keratoplasty therapy because of the extremely limited number of donor corneas. Recently, corneal tissue engineering opens a new path for in vitro reconstruction of tissue-engineered HCE which will cure the corneal endotheliopathy by clinical corneal transplantation. Establishment of the techniques for reconstruction of tissue-engineering HCE in vitro will lay solid foundations of large scale reconstruction of tissue-engineering HCEs and their clinical applications. Therefore, this paper is intended to reconstruct tissue-engineered HCE, monoclonal cell lines with normal karyotype which is based on the molecular identification of the HCEC cell lines as seed cells and modified epithelium-denuded amniotic membrane as scaffold carrier, and examine their functions by Rabbit corneal endothelial transplantation.To identify the properties and functions of HCEC cell line, Results of immunocytochemical staining, Western blot and morphology recovery showed that the HCE cells had positive expression of typeⅣcollage and human vascular endothelial growth factor receptor-2 (FLK1),and negative expression of human von Willebrand Factor (vWF) and keratin.it can be concluded that the HCE cells were indeed human corneal endothelial cells, but not vascular endothelial cells or corneal epithelial cells or corneal stromal cells. Results of immunocytochemistry showed that HCE cells stably expressed zonula occludens protein 1 (ZO-1,tight junction protein), connexin-43 (gap junction protein), N-cadherin (anchoring junction protein), and integrinαv/β5 (cell-matrix anchoring junction protein), which suggested that the established HCE cell line still had the potential to form normal cell-cell and cell-extracellular matrix junctions. Real-Time PCR of membrane transport proteins showed that the HCE cells still maintained the positive expression of human aquaporin 1 (AQP-1),Na+/K+ ATPaseal polypeptide, voltage-dependent anion channels (hVDAC2, hVDAC3), human chloride channel proteins (hCLCN2, hCLCN3), sodium bicarbonate cotransporter 4 (hSLC4), and cystic fibrosis transmembrane conductance regulator (CFTR), which indicated-that the established HCE cell line still had the potential to carry out normal functions of transmembrane transport.The HCE cell cloning was performed by limiting dilution methods, and 13 monoclones were obtained. According to the chromosome analysis,7 cell monoclones with normal karyotype (2n=46). The strain of C3B monoclone was largely proliferated, which can be used as HCE seed cells for in vitro reconstruction of tissue-engineered HCE.To denude the epithelial cells of amniotic membranes (AMs), a reverse trypsin digestion and scraping by cell scraper were utilized.The AMs had a slick surface without any residual epithelial cells. Besides, the AMs were further modified by coated with signal molecules. The examination results showed that the modified denude AMs (dAMs) had excellent biocompatibility with HCE cells, which implied that the dAM can be used as the scaffold carrier of tissue-engineering HCE.By using C3B strain HCE cells as seed cells and dAMs as scaffold carries, tissue-engineered HCE was reconstructed and cultured in DMEM/F12 medium containing 20% BCS at 37℃with 5% CO2.Light microscope observations of reconstructed tissue-engineered HCEs showed that the HCE cells grew very well on dAM, and could form a confluent monolayer 116 h later with a cell density of 3611 cells/mm. Results of Alizarin red staining and cryosection staining also confirmed that the HCE cells could form a confluent monolayer on dAM.Scanning electron microscope(SEM) and transmission electron microscope (TEM) observations showed that most of the HCE cells were in endothelial cell-like polygonal morphology and formed a confluent monolayer. The ultra-structure of HCE cells was very similar to that of normal HCE cells, and a lot of extensive cell-cell and cell-dAM junctions. Immunofluorescence staining showed that the HCE cells still had positive expression of cell junction proteins such as ZO-1,N-cadherin, Connexin43 and Integrinαv/β5, which implied that the HCE cells had the potential to form cell-cell and cell-dAM junctions.To examine the functions of reconstructed tissue-engineered HCEs, animal corneal transplantation were performed with New Zealand white rabbits whose corneal endothelium and Descemet's membrane were ripped off before penetrating corneal endothelium transplantation.After transplantation, no adverse reaction such as edema and rejection were observed in transplanted rabbits, and the rabbit corneas kept-transparency for more than 280 days. The cells in-the corneal endothelia of transplanted rabbits were all with DiI labels, indicated that they are all from reconstructed tissue-engineered HCEs. Results of Alizarin red staining and cryosection staining showed that the thickness of corneas from transplanted eyes was similar to that of untransplanted eyes, and HCE cells, in hexagon shape, formed confluent monolayers with a cell density of 2307 cells/mm2.After transplantation. SEM and TEM observations showed that the endothelium of transplanted cornea was integrity by forming a lot of cell junctions, and most of them were in hexagon shape. The HCE cells contained large numbers of rough endoplasmic reticulums and mitochondria, and a new Descemet's membrane was formed 1 month later. The morphology and structure of HCE cells were similar to those of rabbit corneal endothelial cells in vivo.In addition, in order to simulate the clinical treatment of primary corneal endotheliopathy, we use the method of scraping the corneal endothelial cells to establish a New Zealand rabbit model of corneal endotheliopathy, animal corneal transplantation were performed with New Zealand white rabbit. After transplantation, the adverse reaction such as edema and rejection were gradually disappeared,and the rabbit corneas kept transparency for more than 198 days.The results of corneal endothelial transplantation to New Zealand model of corneal endotheliopathy show that the TE-HCE transplantation can achieve the purpose of healing corneal endotheliopathy.In this paper, in vitro reconstructed tissue-engineered HCEs, with the normal structure and function of HCEs, can be used as the HCE substitutes for clinical corneal transplantation.The tissue-engineered HCEs provide a promising path for restoring sight of corneal endotheliopathy suffers and a radical solution for the lack of donor corneas and immune rejection after transplantation. Therefore, successful in vitro reconstruction of tissue-engineered HCEs and their future clinical applications have not only great theoretical significance, but also large economic benefits and enormous social effects.