Human corneal stromal cells response to silk fibroin-based membranes cross-linked with polyethylene-glycol.

Abstract

Purpose : Silk-Fibroin (SF) based biomaterials hold promise to replace amniotic membrane (AM) treatment in severe corneal injury due to their favorable properties (transparency, strength), possibility to be loaded and release with growth factors (GF) and ease of handling. We investigate the potential use of SF cross-linked with polyethylene-glycol (PEG) membranes (SFM) as a corneal dressing and as a biomaterial in regenerative medicine. Here we demonstrate the SFM`s biocompatibility, interactions with human corneal stromal cells (HCSC), and capacity to maintain the keratocyte phenotype.

Methods : Keratocytes from corneal donors were cultured in DEMF12+10% FBS on plates covered on 3%SF-5%PEG and on 0.1% collagen I (Col) as a control. To reduce serum-induced activation, serum-free medium was added after cells reached confluence. At days 3 and 6 after removing serum, cultures were used to be imaged in Scanning Electron Microscopy (SEM). At the same study times, keratocyte phenotype markers Keratocan and ALDH1A1 were detected by immunocytochemistry, and their mRNA expression levels were analysed by qRT-PCR.

Results : SEM images show that HCSC grown on SFM have very thin and fusiform shape with multiple nucleolus, consistent with enhanced biosynthetic activity. In contrast, HCSC grown on Col show a broad cytoplasm with wide extensions. Cells seeded on both substrates were Keratocan and ALDH1A1 positive, showing similar distribution of both markers. The Keratocan mRNA expression levels were upregulated in both substrates, but significantly higher in cells grown on Col at days 3 and 6. ALDH1A1 mRNA expression levels were also upregulated, and at day 6 those were significantly higher in HCSC seeded on Col.

Conclusions : HCSC grown on SFM can reach confluence and express normal keratocyte characteristic proteins. The mRNA overexpression found with Col substrate stands in contrast to the lower Keratocan and ALDH1A1 levels found with SFM suggesting that SFM may require to increase the serum withdrawal time, as a result over-anchoring of GF from the culture medium to the membrane. In addition, the morphological changes observed in the HCSC grown on both Col and SFM suggest specific interactions between the cytoplasmic membrane and the substrates.
Our findings show that the higher transparency, biocompatibility, and ease handling of SFM over Col makes the latter a promising surrogate of AM in corneal treatment.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.