Reconstruction Of Tissue-engineered Corneal Endothelium In Vitro And Its Function In Animal Transplantation

The human corneal endothelium (HCE), comprised of a single layer of hexagonalcorneal endothelial cells in a well-arranged mosaic pattern, forms a selective barrierbetween the anterior chamber of the eye and the corneal stroma. HCE cells are themost metabolically active cells in the cornea expressing fluid pumps that activelymove fluid from the stroma back into the anterior chamber of the eye. The dynamicbalance between the ‘‘leaky’’ barrier and constant pump activity maintains cornealdetumescence, thereby keeping the cornea transparent, and the transparency isnecessary in maintaining the normal eye vision. Unfortunately, adult HCE cells losttheir proliferative capacity, and the numbers are decreasing yearly, can only rely onexpansion and migration of the adjacent HCE cells to maintain its integrity. However,a significant loss of corneal endothelial cells as the result of pathogen infection,mechanical damage and non physiological stress may result in permanent damage.Decompensation of the CE may occur, and eventually bullous keratopathy. Cornealblindness is often due to endothelial dysfunction and is the second leading cause ofvisual impairment. Currently, the only way to restore vision in eyes with endothelialdysfunction is transplantation of a full or partial-thickness donor cornea containing ahealthy corneal endothelium. Unfortunately, there is a global shortage oftransplant-grade donor cornea. Many patients with corneal blindness caused bycorneal endothelial dysfunction can not restore vision through corneal transplantation.The rise of tissue-engineered corneal endothelium (TE-HCE) brings hope to thepatients with blindness due to corneal endothelial dysfunction. But so far, there is nota product of TE-HCE successfully applied in clinical. Based on our previous work,this paper is intended to reconstruct a tissue-engineered corneal endothelium withnormal morphology and structure using the monoclonal HCE cells with a normalphenotype, karyotype and expression of functional protein as seed cells and thinnedmodified epithelium-denuded amniotic membrane with normal physicochemical properties, high safety and ideal biocompatibility as scaffold carrier, and evaluate itsstructural property and in vivo performance in cat and monkey models in this study.The purpose of this paper was to obtain a TE-HCE which can maintain long-termtransparency of transplanted animal cornea, and lay the foundation for its productionand clinical application.The seeder cells used for reconstruction of TE-HCE was passage38mcHCE cellsfrom the untransfected and nontumorigenic HCE cell line (utHCEC01). Before usedas seeder cells, we identified its properties, the expression of functional protein andlatent risk of tumorigenicity using growth properties, chromosome morphologicalobservation, immuno-cytochemistry analysis and tumorigenesis assay. The resultsshowed that the passage38mcHCE cells, with high transparency and polygonal orapproximate hexagon appearance in shape, proliferated actively and constantly with apopulation doubling time of41.07hours. Chromosome number of the mcHCE cellswas46. The passage38mcHCE cells expressed human type IV collagen and humanvascular endothelial growth factor receptor-2(Flk1) positively. It can conclud that thecell strain still had the intrinsic attribute of the HCE cells. Results ofimmunocytochemistry showed that the mcHCE cells expressed zonula occludens1,E-cadherin, connexin-43, and integrin αv/β5positively, which suggested that thepassage38mcHCE cells still had normal phenotypes and the potential to form normalHCE. Moreover, the passage38mcHCE cells still maintained positive expression ofhuman Na+/K+ATPase, chloride channel protein (hCLCN2), sodium bicarbonatecotransporter4(hSLC4) and aquaporin-1(AQP-1), which suggested that the passage38mcHCE cells still had the potential function of normal membrane transport.Besides, the mcHCE cells had no tumorigenicity. Therefore, the passage38mcHCEcells can be used as seeder cells for reconstruction of TE-HCE in vitro.To obtain thinned modified epithelium-denuded amniotic membrane (thinnedmdAM), the paper utilized a method of reverse trypsin digestion and scraping by cellscraper to thin the dAM, and then modified its epithelial surface by coating withsignal molecules. Before the thinned mdAM was used as scaffold carriers forreconstructing TE-HCE, we detected and evaluated its physicochemical properties, biological properties and the biocompatibility with the passage38mcHCE cells. Theresults showed that the thinned mdAM with a smooth epithelial surface comprised ofbasement membrane and compact layer had uniform thickness, the average thicknessof about28.47μm; good transparency and light transmittance, above90%at thevisible light; and its heavy metal content met the requirements of national third kindof medical instrument, less than10μg/g; Bacterial endotoxin test and rabbit test werenegative and accord with each other, which implied that thinned mdAM had nopyrogen. Moreover, the thinned mdAM had an excellent biocompatibility with the thepassage38mcHCE cells. Therefore, the thinned mdAM can be used as scaffoldcarriers for reconstruction of TE-HCE in vitro.TE-HCE was reconstructed with the passage38mcHCE cells as seeder cells andthe thinned mdAM as scaffold carriers, and cultured in20%fetal bovineserum-containing DMEM/F12(1:1) medium and5%CO2at37°C on a24-wellculture plate. Light microscope and alizarin red staining observations every day ofTE-HCE showed that the mcHCE cells grew very well on the thinned mdAM, andcould form a confluent monolayer96h after be seeded with a cell density of3487±99.04cells/mm2. Paraffin section and HE staining, SEM and TEM showed thatthe mcHCE cells were in endothelial cell-like polygonal morphology and formed aconfluent monolayer. The ultra-structure of mcHCE cells was very similar to that ofnormal HCE. Immunofluorescence staining showed that TE-HCE maintained positiveexpression of cell junction proteins such as ZO-1, N-cadherin, Connexin-43andIntegrin av/β5, which was the guarantee of the barrier function of TE-HCE.In order to further evaluate the normal physiological functions maintaining normalcorneal thickness and transparency of the TE-HCE in vivo, we performed penetratingcorneal endothelial transplantation in cat and monkey whose corneal endothelial cellsalso had no proliferation. Six cats and six monkeys each were randomly divided intotwo groups, one was experimental group, transplanted TE-HCE; the other was control,transplanted mdAM; right eye of each animal’s was the experimental eye, left eye asnormal control. Firstly, we stripped the Descemet’s membrane and cornealendothelium, and then transplanted DiI labeled TE-HCE and mdAM. After lamellar keratoplasty surgery, no obvious edema was found in TE-HCE-transplanted catcorneas, which were transparent throughout the monitoring period. In contrast, intensecorneal edema developed in dAM-transplanted cat corneas, which were turbid. Thecorneal thickness gradually decreased to751.33±11.37μm on day104after TE-HCEtransplantation in cats and to717.33±142.77μm181days after transplantation inmonkeys, while that of dAM eye was over1,000μm in thickness during themonitoring period. A monolayer of endothelium consisting of TE-HCE-originatedcells at a density of2573.33±0.59cells/mm2in cats and2780.00±0.77cells/mm2inmonkeys, attached tightly to the surface of remnant Descemet’s membrane over104days in cats and181days in monkeys; this was similar to the normal eye control incell density. Moreover, the ultra-structure of corneal endothelium of TE-HCEtransplanted eye was similar to that of normal control eye in cat and monkey, but themcHCE cell had not produced new Descemet’s membrane at day181in monkeys.The proteins in aqueous humor of TE-HCE transplanted eye were in accordance withthat of normal control eye.These findings suggest that the TE-HCE reconstructed with the passage38mcHCE cells and thinned mdAM has normal morphology and structure, and is able toreconstruct a continuous monolayer of corneal endothelium and contributes to themaintenance of corneal thickness and clarity in cat and monkey models in vivo aftertransplantation. This TE-HCE can be used as an HCE equivalent, which might beuseful in the field of regenerative medicine for the treatment of diseases caused bycorneal endothelial disorders.