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Notes: Open wounds in the fetal rabbit do not heal by contraction and actually expand between 60% and 90% over a period of 5 days. Experiments were carried out to determine whether transforming growth factor-β1 can reduce expansion of open wounds in the fetal rabbit. This study was based on the concept that transforming growth factor-β1 causes differentiation of fibroblasts into contractile fibroblasts or “myofibroblasts.” To test this hypothesis, pregnant New Zealand White rabbits underwent laparotomy and hysterotomy on day 24 of gestation. A circular full-thickness cutaneous wound was made on the back of each fetus. After wounding, either vehicle alone or vehicle with transforming growth factor-β1 was applied topically to the wound site, and each fetus was then returned to the uterus. The hysterotomy and laparotomy were closed in standard fashion. On postoperative day 5, fetuses were harvested by repeat Cesarean section. Wound areas were determined from photographs, calculated as percentage of original wound size, and expressed in square millimeters. In addition, a portion of each wound was fixed and processed for histologic and immunohistochemical analysis. At harvest, the control wounds had expanded by an average of 87% of the original area. In marked contrast, the transforming growth factor-β1-treated wounds had only expanded an average of 16%. Thus, transforming growth factor-β1 significantly decreased the area of the open fetal wounds compared with control (p 〈 0.001). By histologic examination, no significant difference was found between the test group and the control group with regards to inflammation, neovascularization, collagen deposition, elastin content, glycosaminoglycan content, or hyaluronic acid content. Most notably, however, there was an increased density of fibroblasts in the transforming growth factor-β1-treated group. In addition, immunohistochemical staining with an anti-α-smooth muscle actin antibody showed the presence of contractile fibroblasts in the wound margins in the transforming growth factor-β1-treated group but failed to show any positive-staining fibroblasts in the matrices of the control group. These results indicate that open wounds in the fetal rabbit treated in vivo with transforming growth factor-β1 were significantly smaller than control wounds. This process appears to result from the recruitment and differentiation of normal dermal fibroblasts into contractile fibroblasts containing α-smooth muscle actin.

Notes: Extracellular matrix degradation during dermal wound healing involves multiple levels of regulation by several enzymes of the matrix metalloproteinase family, their activators, and their inhibitors. This study tested the hypothesis that a temporal pattern of interstitial collagenase appearance occurs during normal dermal wound healing, with matrix metalloproteinase-8 originating from neutrophils appearing earlier than the fibroblast-derived matrix metalloproteinase-1. Open (6 mm) full-thickness dermal wounds, which were covered by transparent occlusive dressings, were made in healthy human volunteers (n = 20). Wound fluids from under the dressings were collected daily through day 8, and wound tissue biopsies were obtained on days 0, 2, 4, 14, and 28. Collagenases were extracted from homogenized tissue biopsies for analysis. Samples were analyzed for the presence of matrix metalloproteinase-1 and matrix metalloproteinase-8 by enzyme-linked immunosorbent assays and by collagenase activity assays using purified types I and III collagen as substrates. In addition, tissue inhibitor of metalloproteinases-1 and matrix metalloproteinase-1/tissue inhibitor of metalloproteinases-1 complexes in wound fluids were measured. Results showed a differential temporal pattern of matrix metalloproteinase-1 and matrix metalloproteinase-8 in wound exudates with peak levels of matrix metalloproteinase-8 occurring on day 4 and matrix metalloproteinase-1 peak levels on day 7. Maximal levels in tissue for both enzymes occurred on day 2. At all time points examined, levels of matrix metalloproteinase-8 were statistically higher than matrix metalloproteinase-1 (100-fold to 200-fold). Tissue inhibitor of metalloproteinases-1 levels declined over time, whereas levels of matrix metalloproteinase-1/tissue inhibitor of metalloproteinase-1 complexes increased to a plateau on day 7. This study provides new evidence implicating matrix metalloproteinase-8 as a major collagenase in healing human dermal wounds. It also shows a temporal pattern in the appearance of the matrix metalloproteinases, tissue inhibitor of metalloproteinase-1, and matrix metalloproteinase-1/tissue inhibitor of metalloproteinases-1 complexes, suggesting that a tightly regulated pattern of expression of matrix metalloproteinases and their inhibitors is essential for normal wound healing in humans.