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Notizen: 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.

Notizen: 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.

Notizen: 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.

Notizen: The stability of peptide growth factors exposed to fluids from healing surgical wounds and from nonhealing chronic wounds was examined in vitro. 125I-Labeled transforming growth factor-β1 or platelet-derived growth factor-BB was incubated with fluids from healing surgical wounds and fluids from venous stasis or pressure ulcers. Fluids from healing surgical wounds had no appreciable effect on the level of 125I corresponding to intact growth factor. In contrast, incubation with fluids from several venous stasis or pressure ulcers resulted in significant degradation of these growth factors. Degradation was blocked by broad-spectrum serine proteinase inhibitors and by specific inhibitors of neutrophil elastase. Levels of elastase activity in wound fluids correlated with the ability to degrade peptide growth factors. Further comparisons showed qualitative and quantitative differences in the endogenous proteinase inhibitors, α2-macroglobulin and α1-antiproteinase. These results could explain, in part, the variable growth factor levels which have been found in chronic wounds. More importantly, the ability of some chronic nonhealing wounds to rapidly degrade exogenously added growth factors has important implications with regard to past and future clinical attempts to use peptide growth factors to treat these types of problem wounds.

Notizen: A modification of the Sirius red and fast green dye staining technique which binds selectively to collagen and noncollagenous proteins, respectively, has been used to quantify the amount of collagen deposition occurring in wounded fetal mouse limbs. Wounded day 14 and 18 fetal mouse limbs were grown in serum-free organ culture for 1 to 7 days, fixed in formalin, embedded in paraffin, and sectioned. The sections were stained with Sirius red and fast green dyes, and those sections obtained from either wounded or unwounded tissue were identified microscopically. The sections were then scraped off microscopic slides. Dye bound to the tissue sections was then eluted with a mixture of sodium hydroxide and methanol, and the optical densities were determined spectrophotometrically. There was a 98.5% correlation between the absorbance of Sirius red dye (collagen) and fast green dye (noncollagenous proteins) of eluted stain and hydroxyproline and leucine content, respectively, as determined by high-performance liquid chromatography. In addition, there was a greater collagen/protein ratio in wounded compared with unwounded sections of day 18 limbs at 7 days after wounding (p = 0.005). However, no difference in the collagen/protein ratio was detected between wounded and unwounded regions of day 14 limbs at either day 1 or 7 after wounding. These results are consistent with previous histologic observations indicating greater collagen deposition in wounded regions of day 18 compared with day 14 limbs at 7 days after wounding. With the use of this technique, it is now possible to quantify the effects of putative fibrogenic agents on collagen deposition in wounded embryonic tissue.

Notizen: Chitosan is a large molecular weight, positively charged polysaccharide extracted and purified from the chitin of crab shells. This compound has been shown to have hemostatic activity and has been suggested for use as a topical agent in tissue repair. The objective of this study was to analyze the effect of chitosan on the wound healing response to a standardized injury in the rat. The polyvinyl alcohol sponge implant model was used as a means to deliver either chitosan or its vehicle to a standardized subcutaneous wound on the backs of Sprague-Dawley rats. On days 8 and 14, the chitosan-treated implants contained primarily polymorphonuclear leukocytes compared with the vehicle controls which contained mainly macrophages, fibroblasts, collagen, and new blood vessels. High-performance liquid chromatography analysis for hydroxy-l-proline deposited in the sponge implants showed significantly lower amounts on both days 8 and 14 in the chitosan treatment group. These histologic and biochemical studies suggest that chitosan modulates wound healing by first reducing the influx of activated tissue macrophages, which in turn reduces the subsequent events of angiogenesis, fibroplasia, and connective tissue deposition.

Notizen: In many species, open cutaneous fetal wounds do not heal in utero. Such open wounds have been shown to close only after their exclusion from amniotic fluid, thus leading to the hypothesis that amniotic fluid inhibits open wound healing. Therefore the effect of amniotic fluid exposure on the healing of open fetal skin wounds was studied. Fetuses of New Zealand White rabbits received a full-thickness circular 4 mm diameter skin punch biopsy wound. Wounds were left uncovered, covered with a latex patch, or covered with a latex patch with a central hole (doughnut). This third group provided for wound exposure to amniotic fluid while controlling for any wound splinting effect of the patch. Wounds were harvested after 5 days, the wound area was determined planimetrically, and wound edges were examined by means of light microscopy. Analysis of glycosaminoglycans in the wound extra-cellular matrix was performed on a separate group of wounds treated similarly. Uncovered wounds enlarged by an average of 60%, whereas wounds covered with the doughnut patch enlarged by an average of 24%. In contrast, the wounds in the patch-covered group decreased in size by an average of 84%. Histologically all groups contained proliferating fibroblasts and epithelial migration at the wound edge but also an absence of granulation tissue. The patch-covered wounds, which had decreased wound area, were significantly enriched in hyaluronic acid. These results suggest that the healing of the patch-covered wounds occurs without the formation of granulation tissue, presumably through a process of cellular migration and proliferation and that healing was inhibited by exposure to amniotic fluid. Hyaluronic acid has been shown to be permissive of cellular migration and to play a key role in tissue regeneration. Therefore, we speculate that direct exposure of open wounds to amniotic fluid during the late stages of fetal development in the rabbit prevents hyaluronic acid deposition, which in turn may alter wound closure.

Notizen: Our current understanding of the complex processes involved in wound healing is based mainly on studies of animal models. Although this information has been useful, it may not totally reflect the response found in human beings. For example, human beings have a tendency to either “overheal,” as seen in keloids and hypertrophic scar formation, or have deficient healing, as seen in chronic ulcer formation. No animal models are available to analyze these human clinical pathologic conditions. Therefore the objective of this study was to analyze the wound healing response in a large population (n = 40) of normal healthy human beings as a first step to begin studies of abnormal human wound healing. Simultaneously, a comparison was made between the polyvinyl alcohol implant and the expanded polytetrafluoroethylene implant model. Under sterile conditions with the use of local anesthesia, two preweighed polyvinyl alcohol implants and two standard 6 cm expanded polytetrafluoroethylene implants were placed subcutaneously in the upper arm of each subject. High-performance liquid chromatography was used to quantitate isoleucine and hydroxy-l-proline in acid hydrolysates of each implant. Isoleucine was used as an indicator of protein content in the tissue sample, whereas hydroxyproline reflected collagen content. No infectious or hemorrhagic complications were found in the 40 volunteers included in the study. No significant difference was found in isoleucine or hydroxy-l-proline content between postoperative day 7 polyvinyl alcohol implants and day 14 polyvinyl alcohol implants. In contrast, both isoleucine and hydroxy-l-proline content were significantly increased in day 14 expanded polytetrafluoroethylene implants compared with day 7 implants (p 〈 0.005 and p 〈 0.001, respectively). In addition, the ratio of hydroxy-l-proline to isoleucine was significantly increased in day 14 expanded polytetrafluoroethylene implants compared with day 7 expanded polytetrafluoroethylene and both day 7 and day 14 polyvinyl alcohol implants (p 〈 0.001). This observation suggests that by 14 days implantation of expanded polytetrafluoroethylene stimulated an increased deposition of collagen. No significant differences were found in the hydroxy-l-proline to isoleucine ratios among day 7 expanded polytetrafluoroethylene, day 7 polyvinyl alcohol, and day 14 polyvinyl alcohol implants. Histologic analyses correlated with the biochemical findings. These results suggest that expanded polytetrafluoroethylene may be the preferred implant for studies designed to examine pathologic processes associated with retarded wound healing. In contrast, the polyvinyl alcohol implant may be better suited for studies where a low background response is required. Moreover, the extreme variability in normal healthy volunteers seen in this study correlates clinically with the finding that, among the normal adult human population, there is a heterogeneous wound healing response.