Effect of Fibrin-binding Synthetic Oligopeptide on the Healing of Full-thickness Skin Wounds in Streptozotocin-induced Diabetic Rats

Abstract

Introduction Fibronectin(FN) has many other adhesive sites for various substances, including fibrin, heparin and collagen. The FN subunit contains two fibrin binding sites. Among these two sites, the major site close to the amino terminus serves as a transglutamination site for activated factor XIII, crosslinking FN to various other proteins including fibrin, fibrinogen. In blood clotting cascade, plasma FN-fibrin matrix is covalently cross linked, and then it promotes fibroblast adhesion, spreading, and migration into the clot. However, due to the common shortcomings of whole protein usage, including instability to enzymatic degradation, unexpected effects of other domains, nonspecific binding to other types of cells, difficulty in obtaining a stable attachment, and even integrin blocking effects, small synthetic peptides with active binding sequences of natural proteins have been proposed as alternatives. Based on these findings, we assumed that topical application of fibrin-binding oligopeptides from FN to the soft tissue wound site might compensate the impaired wound healing in diabetes individuals. The aim of this study was to investigate whether topical application of fibrin-binding oligopeptides derived from FN promotes wound healing in streptozotocin (STZ)-induced diabetic rats.

Materials and Methods Oligopeptides including fibrin-binding sequences (FF3 : CFDKYTGNTYRV, FF5 : CTSRNRCNDQ) of FN repeats were synthesized based on primary and tertiary human plasma FN structures (FINC-HUMAN: P02751). Each peptide was loaded in 15 x 15 mm fibrous alginate dressings, and the release kinetics of the peptides was evaluated using trinitrobenzene sulfonic acid (TNBSA) for 144 hours. Two full-thickness cutaneous wounds were prepared on the dorsal skin of each 75 diabetes induced rats. Each wound was divided into FF3-loaded alginate dressing group, FF5-loaded alginate dressing group, alginate dressing group and negative control group. Two rats were sacrificed at day 0, and 24 rats were sacrificed at day 3, day 7 and day 14, respectively. The wound closure rate, inflammation degree, expression of TGF-β1 and hydroxyproline content were evaluated.

Results Both FF3 and FF5 peptides were released rapidly within the first 24 hours. Regarding the wound closure rate, FF3-loaded dressing treated wounds closed significantly faster than other wounds at day 3. At day 7, both FF3- & FF5-loaded dressing treated wounds showed more wound closure than the alginate dressing treated group and the negative control group. At day 14, only negative control group showed delayed wound closure over the other groups. And at day 14, FF3- & FF5- loaded dressing treated wounds demonstrated less inflammatory cells infiltration than alginate dressing treated and negative group wounds. TGF-β1 positive cells were more abundant in FF3-, FF5-treated alginate dressing treated wound than negative control wounds at day 3 and 14. At day 7, FF3- & FF5- treated wounds showed more TGF-β1 positive cells than in alginate dressing treated wounds and negative control wounds. At last, the hyrdroxyproline contents in the FF3, FF5 group were higher than in the alginate dressing treated group and negative control group at day 7 and day 14.

Conclusions Topical application of fibrin-binding domain synthetic oligopeptides from FN resulted in acceleration of full-thickness cutaneous wound healing in diabetic rats. Enhancement of TGF-β1 expression in fibrin-binding domain synthetic oligopeptides treated wounds seems to be a contributing factor or possible mechanism for the improvement of diabetic skin wound healing.