Abstract
Human corneal endothelium in vivo demonstrates an age-related decrease in cell density and cannot be compensated due to its limited regenerative capacity. When the cell density is less than a critical level of 1000 cells/mm2, the endothelial monolayer no longer functions, causing corneal edema and loss of visual acuity. Penetrating keratoplasty (PK) is currently the common way to treat corneas that are opacified due to endothelial dysfunction. However, insufficient supplies of donor corneas and several complications associated with PK remain a worldwide problem. Therefore, transplantation of in vitro cultured human corneal endothelial cells (HCECs) to replace damaged corneal endothelium alone is a promising alternative to PK. In this study, we developed a novel therapy technique to fabricate and transplant cultured HCEC sheets for corneal endothelial reconstruction. On the basis of plasma chemistry, we have designed a two-step method to prepare a thermo-responsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted culture surface for controlling HCEC adhesion and detachment via a thermal stimulus. The results of surface characterization including energy-dispersive X-ray spectroscopy (EDX), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), atomic force microscopy (AFM), and static contact angle measurements show that an optimal grafting amount of PNIPAAm is 1.6 μg/cm2. We have also demonstrated that the introduction of AAc segment as short spacers onto the culture support can accelerate the cell detachment, which is beneficial to protect these harvested cells from functional damage. We further investigated whether the bioengineered HCEC sheets harvested from thermo-responsive culture supports could be used as biological tissue equivalents. Untransformed adult HCECs derived from eye bank corneas were cultivated on PNIPAAm-grafted surfaces for 3 weeks at 37°C. Confluent cell cultures were detached as a laminated sheet by lowering culture temperature to 20°C. In vitro characteristics of HCEC sheets were evaluated by viability, scanning electron microscopy, immunohistochemistry, and histology. Similar to the native corneal endothelium from eye bank donors, the fabricated HCEC monolayers having normal morphology, structure and viability are suitable to be used as tissue replacements for transplantation. Because of the soft and fragile nature of bioengineered HCEC sheets, we have designed and developed a multi-functional hydrogel carrier system for intraocular delivery of these sheet grafts. The functionality of gamma-sterilized cell carriers made from raw gelatins with a different isoelectric point (IEP = 5.0 and 9.0) and a molecular weight (MW) range from 3 to 100 kDa, was investigated by the determination of mechanical properties, water content, dissolution degree, and cytocompatibility. The results of our study indicate that the gamma-sterilized hydrogel discs consisting of raw gelatins (IEP = 5.0, MW = 100 kDa) are promising candidates as cell sheet carriers for effective corneal endothelial cell transplantation and therapy. In the in vivo tests, we evaluated the feasibility of HCEC transplantation by harvesting the cell sheets from the thermo-responsive culture supports and delivering with multi-functional gelatin hydrogel discs in a rabbit model. We have shown that the transplanted HCEC sheets could be integrated into rabbit corneas denuded of endothelium. Additionally, when endothelium alone was removed, the rabbit corneas became cloudy and remained opaque throughout the course of the experiment. Once receiving tissue-engineered HCEC sheets, the corneas have returned to a nearly normal thickness. These results imply the biological function of transplanted cell sheets. Our findings indicated that a well-organized and functional HCEC sheet is feasible to be used as tissue equivalents for replacing compromised endothelium. In the present study, we have demonstrated that the bioengineered human corneal endothelium fabricated from thermo-responsive culture supports and delivered by multi-functional hydrogel carriers can potentially offer a new therapeutic strategy for corneal endothelial cell loss. In addition, functional biomaterials have great potential for development of HCEC sheet engineering and regenerative medicine in ophthalmology. We hope this work will lead to insights into cell sheet-based therapy for corneal endothelial dysfunction and will open an exciting new door to the future.