Silk Fibroin Films for Corneal Endothelial Regeneration: Transplant in a Rabbit Descemet Membrane Endothelial Keratoplasty.

Purpose: Develop a silk fibroin (SF)-based artificial endothelial graft for its use in a rabbit Descemet membrane endothelial keratoplasty (DMEK). Methods: Human and rabbit artificial corneal endothelial grafts were developed through the culture of human and rabbit corneal endothelial cells (CECs) on SF films. Rabbit artificial SF endothelial grafts were transplanted in a DMEK surgery into a rabbit in vivo model. Results: SF artificial endothelial grafts showed the characteristic endothelial markers: zonula occludens (ZO-1) and Na+/K+ ATPase. In a rabbit model of DMEK surgery, SF artificial endothelial graft restored the corneal transparency and thickness at 6 week of follow-up. Anterior segment optical coherence tomography revealed the SF graft as a fully integrated component in the corneal tissue, displaying a similar corneal thickness and endothelial cell count when compared with its healthy contralateral cornea. Histologic analysis showed that the SF artificial endothelial graft was attached and integrated on the surface of the corneal stroma without a significant inflammatory reaction, and rabbit CECs consisted in a monolayer that showed their characteristic markers ZO-1 and Na+/K+ ATPase, suggesting proper intercellular junctions and cellular pump function. Conclusions: We have developed SF films with biological properties that supported the growth of rabbit and human CECs, which showed normal morphology and characteristic markers; and with mechanical properties that allowed its use in a DMEK surgery, proving its in vivo functionality in a rabbit model of endothelial dysfunction.

Human Bone Derived Collagen for the Development of an Artificial Corneal Endothelial Graft. In Vivo Results in a Rabbit Model.

Corneal keratoplasty (penetrating or lamellar) using cadaveric human tissue, is nowadays the main treatment for corneal endotelial dysfunctions. However, there is a worldwide shortage of donor corneas available for transplantation and about 53% of the world's population have no access to corneal transplantation. Generating a complete cornea by tissue engineering is still a tough goal, but an endothelial lamellar graft might be an easier task. In this study, we developed a tissue engineered corneal endothelium by culturing human corneal endothelial cells on a human purified type I collagen membrane. Human corneal endothelial cells were cultured from corneal rims after corneal penetrating keratoplasty and type I collagen was isolated from remnant cancellous bone chips. Isolated type I collagen was analyzed by western blot, liquid chromatography -mass spectrometry and quantified using the exponentially modified protein abundance index. Later on, collagen solution was casted at room temperature obtaining an optically transparent and mechanically manageable membrane that supports the growth of human and rabbit corneal endothelial cells which expressed characteristic markers of corneal endothelium: zonula ocluddens-1 and Na+/K+ ATPase. To evaluate the therapeutic efficiency of our artificial endothelial grafts, human purified type I collagen membranes cultured with rabbit corneal endothelial cells were transplanted in New Zealand white rabbits that were kept under a minimal immunosuppression regimen. Transplanted corneas maintained transparency for as long as 6 weeks without obvious edema or immune rejection and maintaining the same endothelial markers that in a healthy cornea. In conclusion, it is possible to develop an artificial human corneal endothelial graft using remnant tissues that are not employed in transplant procedures. This artificial endothelial graft can restore the integrality of corneal endothelium in an experimental model of endothelial dysfunction. This strategy could supply extra endothelial tissue and compensate the deficit of cadaveric grafts for corneal endothelial transplantation.