Altered collagen metabolism and delayed healing in a novel model of ischemic wounds

Cellular mechanisms occurring in the healing wound have been well described in various animal models. However, the events associated with wound healing seen in ischemic skin have not been as thoroughly defined. In this series of experiments, we created a novel model of excisional skin wounds under gradient ischemia to study the cellular and extracellular events leading to delayed healing. We hypothesized that altered collagen metabolism accounts for delayed wound healing in ischemic skin. Three pairs of 4 mm punch wounds were made 4 days after bipedicle skin flaps were created on the dorsum of rats. Sham-operated control animals had the same punch wounds without flap creation. The kinetics of excisional wound healing were measured by means of computerized planimetry. In addition, wounds were excised with a 6 mm trephine, radiolabelled with ((3)H)-proline and in vitro collagen synthesis determined as collagenase digestible protein along with quantitation of DNA content. Total collagen deposition was determined as 4-hydroxy-L-proline by high-performance liquid chromatography, and wounds were histologically evaluated. Data was analyzed by means of two-way analysis of variance. Although control wounds healed by day 10, flap wounds consistently had greater surface area on days 2, 4, 6, 8, and 12 (p < 0.001). Relative collagen synthesis (% collagen/noncollagen protein), as measured by an in vitro synthesis method, showed no statistically significant differences between flap and controls wounds. However, the total collagen content (deposition) as measured by 4-hydroxy-l-proline was significantly lower in flap wounds compared with controls on days 7 (p < 0.05) and 9 (p < 0.001). In addition, a significant increase occurred in DNA content in the flap wounds on days 7 (p < 0.05) and 9 (p < 0.001) versus control wounds. These data indicate that, in ischemic wounds, significantly less collagen is deposited despite the inherent ability of the tissue to synthesize appropriate levels of collagen. Because the in vitro collagen synthesis technique only assesses the ability of the tissue to synthesize collagen in a well oxygenated environment, one cannot be assured that the tissue expresses this potential in vivo. However, these data are consistent with the hypothesis that the delay in wound closure is due to an alteration in collagen metabolism which results in a net decrease in collagen accumulation. Because of the observed increase in DNA within the ischemic wounds, we suggest that there is prolonged inflammation in these wounds which may enhance collagen degradation through the release of proteases. In addition, there may be an inability of the tissue to maintain appropriate levels of collagen in this inflammatory wound environment.