Engineering of a curved corneal stromal substitute

Abstract

Purpose : The self-assembly approach of tissue engineering allows to reconstruct 3D corneal stromal substitutes (Proulx et al., 2009; Bourget et al., 2016). Presently, these substitutes are flat. The purpose of this study was to engineer a stromal substitute with the curved shape of a native cornea.

Methods : Curved plastics were formed using a thermoplastic elastomer (TPE) and sterilized. Their size fitted into a well of 6-well plates. Keratocytes, previoulsy isolated from a human cornea, were seeded on top of the TPE and cultured in the presence of serum and ascorbic acid. Flat 6-well culture plates were used as controls. Cells were cultured for 35 days and formed sheets of extracellular matrix. Two sheets were superposed to form a thicker stromal substitute, and cultured to allow for the sheets to adhere to each other (14 days for curved stromal substitutes; 8 days for flat stromal substitutes). The stromal substitutes were then characterized using macroscopic images. They were then fixed in 3.7% formaldehyde for histology (Masson’s Trichrome staining).

Results : The activated keratocytes secreted extracellular matrix and formed sheets on both the curved TPE and the flat plastic controls. When removed from the curved TPE surface, the stromal substitute maintained a more pronounced curved form. Thickness of the stromal substitutes were calculated using histology cross-sections. The curved stromal subsitutes had a mean thickness of 26.1±0.5µm (n=2 counts) and flat stromal subsitutes 15.4±2.7µm (n=3 counts).

Conclusions : This study demonstrates that the self-assembly approach can be used to reconstruct curved stromal substitute. Therefore, this brings us closer to a reconstructed cornea whose properties are similar to the native tissue.

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