ABSTRACT
The organization of the corneal stoma is modified due to different factors, including pathology, surgery or external damage. Here the changes in the organization of the corneal collagen fibers during natural healing after chemical burn are investigated using second harmonic generation (SHG) imaging. Moreover, the structure tensor (ST) was used as an objective tool for morphological analyses at different time points after burn (up to 6 months). Unlike control corneas that showed a regular distribution, the collagen pattern at 1 month of burn presented a non-organized arrangement. SHG signal levels noticeably decreased and individual fibers were hardly visible. Over time, the healing process led to a progressive re-organization of the fibers that could be quantified through the ST. At 6 months, the stroma distribution reached values similar to those of control eyes and a dominant direction of the fibers re-appeared. The present results show that SHG microscopy imaging combined with the ST method is able to objectively monitor the temporal regeneration of the corneal organization after chemical burn. Future implementations of this approach into clinically adapted devices would help to diagnose and quantify corneal changes, not only due to chemical damages, but also as a result of disease or surgical procedures.
ABSTRACT
The development of treatments that modulate corneal wound healing to avoid fibrosis during tissue repair is important for the restoration of corneal transparency after an injury. To date, few studies have studied the influence of growth factors (GFs) on human corneal fibroblast (HCF) expression of extracellular matrix (ECM) proteins such as collagen types I and III, proteoglycans such as perlecan, or proteins implicated in cellular migration such as α5ß1-integrin and syndecan-4. Using in vitro HCFs, a mechanical wound model was developed to study the influence of the GFs basic fibroblast GF (bFGF), platelet-derived GF (PDGF-BB) and transforming GF-ß1 (TGFß1) on ECM protein production and cellular migration. Our results show that mechanical wounding provokes the autocrine release of bFGF and TGFß1 at different time points during the wound closure. The HCF response to PDGF-BB was a rapid closure due to fast cellular migration associated with a high focal adhesion replacement and a high expression of collagen and proteoglycans, producing nonfibrotic healing. bFGF stimulated nonfibrotic ECM production and limited the migration process. Finally, TGFß1 induced expression of the fibrotic markers collagen type III and α5ß1 integrin, and it inhibited cellular migration due to the formation of focal adhesions with a low turnover rate. The novel in vitro HCF mechanical wound model can be used to understand the role played by GFs in human corneal repair. The model can also be used to test the effects of different treatments aimed at improving the healing process. Copyright © 2016 John Wiley & Sons, Ltd.
