A new tissue engineering approach for endothelial grafts.

Abstract

Purpose: To develop a tissue-engineered human corneal endothelium by culturing corneal endothelial cells on a human collagen type I membrane, both isolated from the same donor.

Methods: 1- Donors: Corneal tissue and fresh human cancellous bone were obtained from the same donor, stored and processed in Asturias Regional tissue bank according to Spanish laws.<br /> 2- Cellular culture: Peripheral endothelium (without trabeculum) was obtained from corneas during posterior endothelial preparation (DMEK). Descemet was stripped and trephined, the central part was used for graft and the peripheral ring was used for tissue engineering. Peripheral Descemet membrane was cut and the explants were seeded and cultured on a dish previously treated with fibronectine. Once confluent, endothelial cells were detached and subcultured on a collagen type I membrane.<br /> 3- Collagen type I isolation: Collagen isolation was performed as previously described in Sawkins et al. 2013. Briefly, bone granules were demineralized in HCl 0.5M and lipids were extracted with a solution of methanol-chloroform. Demineralized bone matrix was lyophilized and a pepsin digestion and acid solubilization technique was employed for the isolation of collagen type I. Collagen was characterized by western blot and quantified using a hydroxyproline assay.<br /> 4- Collagen membranes: 3 mg/cm² of isolated collagen type I was cast into a silicone mold and air-dried at room temperature. Collagen membranes were crosslinked using 254nm UV irradiation lamp to improve their resistance.<br /> 5- Cellular analysis: Human corneal endothelial cells growing on the collagen membranes were fixed and analyzed by phase contrast microscopy, scanning electron microscopy and immunocytochemistry against Na⁺/K⁺ and ZO-1.

Results: Type I collagen was isolated from human bone as proven by Western blot analysis. Concentration determined by hydroxyproline ratio was used to cast collagen into a membrane that was, later on, used as a scaffold. Human endothelial cells were able to attach and grow onto collagen type I membranes. Cells maintained their morphology and cellular markers, showing positive Na⁺/K⁺ and ZO-1 stain.

Conclusions: We have successfully obtained artificial lamellar endothelial grafts using cells from Descemet peripheral rings and collagen membranes from bone of the same donor. This strategy could supply extra endothelial tissue and compensate the deficit of cadaveric graft for endothelial transplantation.