Ultrathin Poly-DL-Lactic Membranes for Corneal Endothelial Transplantation

Abstract

Purpose : Corneal endothelial transplantation is limited by a donor shortage, making transplant lists long. As hypothesized in 1978, tissue-engineering could overcome this hurdle by growing corneal endothelial cells (CEnCs) on transplantable scaffolds. Despite the global interest, no tissue-engineered graft has made it yet to the clinic in part due to challenging properties scaffolds must fulfil. This study investigates the use of Poly-DL-lactic (PDLLA) membranes as scaffolds to aid in production of corneal endothelial grafts.

Methods : Membranes were synthesized by spincoating a 10 w/v% solution of gelatin type A on glass as a sacrificial layer (Fig 1A). PDLLA was spincoated on top and plasma treated for surface activation. Gelatin B derivatives, gelatine-methacrylamide (gel-MOD) and gel-MOD-2-aminoethyl methacrylate (gel-MOD-AEMA) were analyzed using Nuclear Magnetic Resonance (NMR) spectroscopy, spincoated on the PDLLA and UV crosslinked (Fig 1B). The membranes were characterized with x-ray photoemission spectroscopy (XPS), optical profilometry (OP) to determine thickness and glucose diffusion. Also, cytocompatibility was investigated using primary and immortalized CEnCs followed by immunocytochemistry.

Results : NMR spectra confirmed the modification of gel-MOD(-AEMA). The presence of stable gelatin layers during the synthesis was confirmed using XPS. The developed membranes exhibited thicknesses ranging from 1.1 - 2.6 µm as obtained via OP and the PDLLA showed a diffusion glucose coefficient of 0.0161cm/s. Finally, the membranes were seeded with primary and immortalized CEnCs and exhibited the characteristic hexagonal shape. Immunocytochemistry showed expression of Na⁺/K⁺ ATPase and ZO-1 along the cell membranes of primary and immortalized CEnCs cultured on both culture plastic and gel-MOD(-AEMA) membranes.

Conclusions : In this study, membranes were constructed using PDLLA for mechanical strength and functionalized gelatins to mimic the extracellular matrix. The membranes displayed glucose permeability and because of the ultrathin dimensions they qualify as scaffolds for endothelial keratoplasty. Cultured endothelial cells expressed hallmark proteins representing their pumping and barrier function. Therefore, these novel ultrathin membranes are potentially a suitable scaffold for corneal endothelial tissue engineering required to alleviate the donor shortage.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

(A) Principle of spincoating (B) A diagram of the PDLLA membranes