The effects of fibronectin and the rgd binding sequence on gingival fibroblasts in an in vitro wound healing mode

Abstract

The purpose of this study was to ip.vestigate the influence of fibronectin or the tripeptide Arginine-Glycine-Aspartate (RGD) on human gingiyal fibroblast ceil migration and wound repopulation using an in vitro wound-healing model. The hypothesis was that increasing concentrations of .either fibroneetin or RGD would positively enhance the .. ability of human fibroblast ceiis to repopulate a wound over time. Fibronectin in concentrations of 5 ~tg, 10 )lg, arid- 15 Jlg/ ml and RGD concentrations of 1 TJg, 4 11g, 8 TJg, and 12 11g/ml were used in this study. The in vitro wound-healing model utilized 12-well culture plates in which gingival fibroblasts were seeded and grown to confluence. A 24-hour synchronization phase preceded the creation of a linear 3 mm wide cell-free zone across the diameter of each well. The cell-containing wells were replenished daily with a serum-free medium containing either fibronectin or RGD at the specified concentrations. Control samples received serum-free media only. Cell samples were then fixed and stained with either crystal violet or hematoxylin & eosin every 48-hours for eight days. Photomicrographs were taken of each stained well for the histomorphometric examination and measurements of wound fill using the Scion Image Analysis Software. In follow-up experiments, using the same procedures, the 3 mm wide cell-free zone was coated with a RGD-polyol substrate immediately after wounding. The polyols were mixed with RGD to concentrations of either 1 T]g, 4 TJg, 8 T]g, or 12 T]g/ml. Positive controls were coated with an RGD-free polyol substrate while the negative controls were coated with serum-free media only. All cells received daily nutrient replenishment and were subsequently fixed, stained, photographed, examined and measured as previously mentioned. Conclusions reached in this study were as follows: Both the fibronectin and RGD challenge appeared to result in positive dose-dependent responses in the early days of wound repopulation when compared to controls. 1. The serum-free defined media provides an excellent nutrition source for gingival fibroblast cells. 2. All experimental concentrations of fibronectin appeared to stimulate the migration of gingival fibroblasts into the wound area up to day two. Fibroblast cells exposed to fibronectin at 15ug/ml consistently displayed more wound fill. 3. All experimental concentrations of RGD also appeared to positively influence fibroblast migration through day two. 4. All RGD-polyol samples appeared to support better wound fill than the positive control (polyol only) but did not appear to improve fibroblast migration when compared to the negative control (serum-free media only).