Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers

The purpose of this study was to determine the biochemical composition of fluid taken from chronic wounds, to compare these values with that of serum, and therefore to assess whether the wound fluid is representative of the extracellular environment of the wound. Paired wound fluid and blood samples were collected from eight patients with chronic leg ulcers in a nonhealing and healing phase. Wound fluid and serum samples were screened for a profile of general biochemical analyses, including electrolytes, lactate, glucose, and protein analyses. Electrolyte levels were essentially identical in wound fluid and serum samples. Lactate and lactate dehydrogenase levels were significantly greater and glucose and bicarbonate levels were significantly lower in wound fluid when compared with the paired serum samples. Albumin and total protein levels in wound fluid were on average half those of serum levels. In this small sample of eight patients, wound fluid collected from chronic leg ulcers is an exudate with the biochemical composition expected in extracellular fluid. In addition, bicarbonate and glucose levels increase and C-reactive protein levels decrease in wound fluid, but remain unchanged in serum, during healing. These results suggest changes in the state of hypoxia and the inflammatory process in the healing wound.

It has been proposed that occlusive wound dressings may enhance chronic wound repair by the stimulatory action of the fluid accumulating beneath the dressings. In this report, we investigated the in vitro proliferative effects of chronic wound fluid obtained from under a polyurethane membrane applied for 24 hours to venous ulcers in the ambulatory setting. By measuring cell counts and DNA synthesis, we found that chronic wound fluid inhibited the proliferation of human dermal fibroblasts (p = 0.008) and failed to stimulate the proliferation of microvascular endothelial cells (p = 0.03) and keratinocytes (p = 0.03). The inhibitory activity of chronic wound fluid on fibroblast proliferation was blocked after the fluid was heated to 100 degrees C, but not 56 degrees C, and was mainly restricted to a fraction of chronic wound fluid enriched in components less than 30 kd in molecular weight (p = 0.028). At concentrations ranging from 1% to 4% and in the presence of serum, chronic wound fluid decreased the viability of fibroblasts, as shown by a decreased ability of the cells to exclude trypan blue (p = 0.02), and the viability of endothelial cells, as shown by an increased release of tritiated adenine (p = 0.03). We conclude that the wound fluid obtained from beneath occlusive dressings applied to chronic wounds inhibits cell proliferation.

Fluid from acute surgical wounds and from nonhealing pressure ulcers was examined for the presence of several matrix metalloproteinases. Gelatin zymography demonstrated the presence of two major gelatinases with apparent molecular masses of 72 kDa and 92 kDa and two minor gelatinases with apparent mobilities of 68 kDa and 125 kDa. Antigen-specific sera identified the 72-kDa protein as matrix melloproteinase-2. The same sera also reacted with the 68-kDa protein, which is consistent with it being an activated form of matrix metalloproteinase-2. Antigen-specific sera identified the 92-kDa and 125-kDa proteins as matrix metalloproteinase-9. Levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 were elevated more than 10-fold and 25-fold, respectively, in fluids from pressure ulcers compared with fluids from healing wounds. Examination of total potential and actual collagenolytic activity revealed that fluid from pressure ulcers contained significantly greater levels of both total and active collagenase compared with that of acute surgical wounds. In addition, an enzyme-linked immunosorbent assay demonstrated that fluids from pressure ulcers contained significantly more collagenase complexed with the inhibitor, tissue inhibitor of metalloproteinases. Together, these observations suggest that an imbalance exists between levels of matrix metalloproteinases and their inhibitors in the fluids of pressure ulcers and that this is primarily the result of elevated levels of the matrix metalloproteinases. The presence of excessive levels of activated forms of matrix-degrading enzymes at the wound surface of pressure ulcers may impede the healing of these wounds and may be relevant to the development of new rationales for treatment.