Improving corneal stroma repair with Silk Fibroin-derived membranes.

Abstract

Purpose : In recent years different biomaterials have been developed to improve corneal repair, among these, silk fibroin-derived biomaterials. In this study, we evaluated the corneal stroma cells response to silk fibroin-derived membranes (SFMs) following injury.

Methods : A stromal in vitro wound model, previously described by our group (Gallego Muñoz et al., 2017, 2018) was used to evaluate the effect of SFMs on the different processes occurring during stromal repair. Briefly, transparent SFMs were placed on the bottom of Petri dishes, on top of which human corneal stromal cells (HCSCs) were seeded and cultured. When HCSCs reached confluence a linear wound was made. Similar cultures without SFMs were used as control group. Wound closure time and cell organisation were evaluated by microscopy. Proliferation and myofibroblast differentiation were analysed by immunocytochemistry. Hepatocyte growth factor (HGF) secretion was measured by ELISA.

Results : SFMs induced a significantly faster wound closure than control showing complete closure at day 3 (Fig. 1A). HCSCs showed higher organisation on the substrate during the wound closure than in control (Fig. 1B). A peak of proliferation was observed at day 1 in both groups but it was significantly increased in HCSCs seeded on SFMs (p<0.001). On day 3, the percentage of proliferation decreased in both groups showing no differences. HGF secretion was significantly increased by SFMs at day 3 compared to control (p<0.05). No myofibroblast differentiation was observed in HCSCs seeded on SFMs at any study time compared to a low percentage of myofibroblasts observed in control at day 3.

Conclusions : We showed the positive effects of SFMs during the stromal wound closure. The SFMs could improve the corneal repair process and prevent the development of corneal opacities in view of the promotion of faster wound closure. The faster closure appears to be caused by an earlier proliferation peak in presence of SFMs, but may also indicate SFMs support of the migration process. The absence of myofibroblasts with SFMs may result from a higher HGF secretion, which has been shown to inhibit myofibroblast generation in the corneal stroma.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Fig 1. A. Effect of SFMs on wound closure. ****denote statistically significant differences at p<0.001 level between the two study groups. B. Representative micrographs show cell organisation in control and SFMs on days 0, 1, and 3. 100x magnification. Scale bars 100 µm.