TGF-beta1 alters the healing of cutaneous fetal excisional wounds.

BACKGROUND/PURPOSE: In a number of species, fetal wound healing differs from the adult in the absence of inflammation, fibrosis, scar formation, and excisional wound contraction. The lack of inflammation also may explain the relative absence of any cytokine levels at the wound site, such as transforming growth factor (TGF)-beta, and therefore the unique characteristics of fetal wound healing. The authors hypothesized that exogenous TGF-beta1 would induce contraction, inflammation, fibrosis, and scar formation in cutaneous excisional wounds in the fetal rabbit. METHODS: Cellulose discs (3 mm in diameter) were formulated with either 1.0 microg TGF-beta1 (n = 6) or bovine serum albumin (BSA; n = 7), as a control, for sustained-release over 3 days. Each disc was implanted into the subcutaneous tissue on the backs of fetal New Zealand White Rabbits in utero on day 24 of gestation (term, 31 days). A full-thickness, 3-mm excisional wound (7.4 mm2) was then made next to the implanted cellulose disc. All wounds were harvested 3 days later. RESULTS: At harvest, the excisional wounds in the TGF-beta1 group had contracted (5.6 +/- 2.0 mm2), whereas those in the control group had expanded (13.5 +/- 1.2 mm2, P< .01). The surrounding dermis in the TGF-beta1 group had 16.3 inflammatory cells per grid block compared with 12.4 cells in the control group (not significant). In addition, a greater amount of fibrosis was induced by the TGF-beta1 implant (1.7 +/- 0.3) than the control implant (0.4 +/- 0.2) on a scale of 0 to 3, P < .01. In situ hybridization analysis showed an increase in procollagen type 1alpha1 gene expression in the surrounding dermis of the TGF-beta1 group (36.7 +/- 3.6 grains per grid block) compared with the control group (7.1 +/- 0.9 grains per grid block, P < .001). CONCLUSIONS: These results demonstrate that the cytokine TGF-beta1 can induce fetal excisional wounds to contract, stimulate fibrosis, and increase procollagen type 1alpha1 gene expression. These findings further suggest that the absence of TGF-beta1 atthe wound site may be responsible in part for the lack of a postnatal healing response.

Inhibition of elastase by a synthetic cotton-bound serine protease inhibitor: in vitro kinetics and inhibitor release.

A cotton-bound serine protease inhibitor of elastase (fiber-inhibitor) has been formulated for in vitro evaluation in chronic wound fluid. As a model to understand the properties of the inhibitor in wound dressings, the kinetic profile and in vitro release of the fiber-inhibitor formulation have been examined. The elastase inhibitor N-Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone was modified onto cotton cellulose fibers and assayed as a colloidal system. Amino acid analysis and reversed phase high performance liquid chromatography were compared as semiquantitative methods to assess elastase inhibitor release from the cotton fibers. The kinetics of inhibition was assessed on treated fibers of synthetic dressings such that a colloidal suspension of the fiber-inhibitor and elastase was employed as an assay. A dose-response relationship was observed in the kinetics of substrate hydrolysis catalyzed by three elastases: porcine pancreatic elastase, which was employed to model this approach; human leukocyte elastase; and elastase in human chronic wound fluid. Both freely dissolved and fiber-bound inhibitors were studied. The initial rates of substrate hydrolysis were inversely linear with freely dissolved inhibitor dose. The apparent first order rate constants, kobs, for the elastase-inhibitor complex were calculated from the kinetic profiles. The kobs for inhibitor bound enzyme varied as a function of inhibitor vs. enzyme concentration and based on the order of mixing of substrate, inhibitor and enzyme in the assay. Enzyme inhibition by the fiber-inhibitor was measured as inhibitor concentration at 50% inhibition (I50). I50 values measured from the colloidal assay with fiber-released inhibitor were within the same range to those for freely dissolved inhibitor. Inhibition of elastase activity in chronic wound fluid was observed with 1-5 mg of fiber-inhibitor formulation. This approach constitutes an in vitro assessment of synthetic serine protease inhibitors on fibers and may be employed to evaluate structure vs. function of elastase inhibition in the modified fibers of wound dressing composites.