Tissue engineered silk fibroin endothelial graft in a Descemet's membrane keratoplasty.

Abstract

Purpose : Develop an artificial endothelial graft using a silk fibroin (SF) film, aimed at treating corneal endothelial dysfunction in a Descemet's Membrane Endothelial Keratoplasty (DMEK).

Methods : Cocoons of Bombyx mori were degummed and SF was dissolved in 9.3M LiBr and dialyzed against distilled water for 3 days. Then, 580µL of the resultant 5% w/v SF solution was cast upon plastic Petri dish 5,8cm in diameter to give a 10 microns thickness film.
Cultures of corneal endothelial cells (CECs) were obtained from normal New Zealand white rabbits and human corneoscleral rims obtained during surgery after 8mm trephination of the graft. When cultures were confluent, human or rabbit CECs were subcultured onto SF films.
A DMEK surgery was performed in six New Zealand white rabbits. Rabbits were divided into three groups: SF films with or without cultured rabbit CECs and rabbits with only peeled off Descemet’s membrane. The exterior appearance of rabbit eyes was monitored by taking photographs, and six weeks after transplantation, corneal thickness was measured by anterior segment optical coherence tomography (AS-OCT) and a corneal endothelial count was performed. Finally, rabbits were euthanized and the transplanted corneas were histological and immunohistochemically analysed.

Results : We developed SF films with biological properties that supported the growth of CECs, which expressed its morphology and characteristically markers; and with mechanical characteristics that allowed its use in a DMEK surgery. In a rabbit model of corneal endothelial dysfunction, our artificial endothelial graft restored the corneal transparency and thickness, at 6 weeks of follow up, after a DMEK surgery. AS-OCT revealed SF film as a fully integrated component of the corneal tissue, displaying a similar corneal thickness when compared to its healthy contralateral cornea. In the same way, histological analysis showed that SF artificial endothelial graft was attached and integrated to the surface of the corneal stroma without a significant inflammatory reaction, and rabbit CECs consisted in a monolayer which showed their characteristics markers ZO-1 and Na⁺/K⁺ ATPase, suggesting proper intercellular junctions and cellular pump function.

Conclusions : It is possible to develop a human or rabbit artificial endothelial grafts using a SF film, aimed at treating corneal endothelial dysfunction in a DMEK surgery.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.