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Notes: The healing process in acute wounds has been extensively studied and the knowledge derived from these studies has often been extrapolated to the care of chronic wounds, on the assumption that nonhealing chronic wounds were simply aberrations of the normal tissue repair process. However, this approach is less than satisfactory, as the chronic wound healing process differs in many important respects from that seen in acute wounds. In chronic wounds, the orderly sequence of events seen in acute wounds becomes disrupted or “stuck” at one or more of the different stages of wound healing. For the normal repair process to resume, the barrier to healing must be identified and removed through application of the correct techniques. It is important, therefore, to understand the molecular events that are involved in the wound healing process in order to select the most appropriate intervention. Wound bed preparation is the management of a wound in order to accelerate endogenous healing or to facilitate the effectiveness of other therapeutic measures. Experts in wound management consider that wound bed preparation is an important concept with significant potential as an educational tool in wound management.This article was developed after a meeting of wound healing experts in June 2002 and is intended to provide an overview of the current status, role, and key elements of wound bed preparation. Readers will be able to examine the following issues;• the current status of wound bed preparation;• an analysis of the acute and chronic wound environments;• how wound healing can take place in these environments;• the role of wound bed preparation in the clinic;• the clinical and cellular components of the wound bed preparation concept;• a detailed analysis of the components of wound bed preparation. (WOUND REP REG 2003;11:1–28)

Notes: The perforated rat mesentery model was used to study the effect of transforming growth factor-β (TGF-β) on connective tissue repair and influx of macrophages into the peritoneal cavity during such repair. Sprague-Dawley rats were laparotomized, and mesenteric wounds were made with a scalpel. A daily intraperitoneal injection of 0.5 µg TGF-β was given for either 2 or 4 days. After 1 to 10 days, the animals received an intravenous injection of tritium-labeled thymidine before decapitation. Macrophages were collected by peritoneal washing, and the number of closed perforations was counted. Peritoneal cells were quantitated and a labeling index was determined by autoradiography. TGF-β given for either 2 (p 〈 0.001) or 4 (p 〈 0.004) days accelerated closure of perforations on days 3 to 7 after injury. Laparotomy as such significantly increased leukocyte influx (p 〈 0.004), as well as macrophage-labeling index (p 〈 0.02). However, TGF-β did not significantly influence either leukocyte influx or macrophage-labeling index. We concluded that TGF-β significantly enhances connective tissue repair in this perforated rat mesentery model and that TGF-β—induced stimulation of repair is not caused by an increased influx of macrophages into the peritoneal cavity.

Notes: Transforming growth factor-β1 appears to play important roles in normal wound healing by increasing synthesis of extracellular matrix components. However, the role of transforming growth factor-β1 in the production of excessive scar tissue by fibroblasts from stenotic lesions of the larynx has not been evaluated. We examined the effect of transforming growth factor-β1 on the steady-state messenger RNA levels of elastin, α2(l) procollagen, and lysyl oxidase (the enzyme that cross-links both of these structural proteins) in cell cultures of diploid human fibroblasts established from fetal skin, newborn foreskin, and an adult laryngeal stenotic lesion. Time-course and dose-response experiments demonstrated that treatment with 500 pmol/L transforming growth factor-β1 for 20 hours induced maximal levels of mRNA for elastin (7- to 59-fold) and α2(l) procollagen (1.7- to 2.4-fold) in all three cultures of fibroblasts. Transforming growth factor-β1 also increased levels of lysyl oxidase mRNA in fibroblasts cultured from newborn foreskin (2.4-fold) and a stenotic lesion (10-fold) but had minimal effects on the fibroblasts cultured from fetal skin (1.1-fold), which constitutively expressed high levels of lysyl oxidase mRNA. Furthermore, the fibroblast culture established from a laryngeal stenotic lesion responded with the highest fold-induction for all three mRNAs. Inhibition of mRNA synthesis by acti-nomycin D showed that transcription was required for transforming growth factor-β1 induction of elastin, α2(l) procollagen, and lysyl oxidase mRNA in all three cultures of fibroblasts. Inhibition of protein synthesis by cycloheximide showed that translation was required for maximal induction by transforming growth factor-β1 of elastin mRNA but had no observable effect on α2(l) procollagen mRNA in all three cultures of fibroblasts. In addition, translation was required for maximal induction of the lysyl oxidase mRNA by transforming growth factor-β1 in the fibroblasts cultured from a stenotic lesion but not for fibroblast cultures established from fetal and adult skin. These results show that transforming growth factor-β1 coordinately increases mRNA levels for the structural extracellular matrix proteins collagen and elastin, as well as for the cross-linking enzyme, lysyl oxidase. These data also support the hypothesis that transforming growth factor-β1 may contribute to the formation of laryngeal stenotic lesions.

Notes: Rat mesenteric perforations heal by contraction within 5 to 7 days, whereas mouse mesenteric perforations seldom close within 3 weeks unless stimulated by transforming growth factor-β1. In this article, we quantified the expression of alpha-smooth muscle actin by quantitative-reverse transcription-polymerase chain reaction and the orientation of actin filaments at the wound margin by Fourier transformation image analysis after treatment with transforming growth factor-β1. The expression of transforming growth factor-β1 and its type II receptor was also assessed. Actin filaments were shown to increase with time at the wound margin in both species and the expression of alpha-smooth muscle actin mRNA increased simultaneously. Transforming growth factor-β1 enhanced the alpha-smooth muscle actin expression four to five times in rats and three to four times in mice on day 5, but the number of copies expressed per cell was 15-fold higher in rats than in mice. Transforming growth factor-β1 was down-regulated after wounding in free peritoneal cells of rats, but maintained until day 5 in transforming growth factor-β1-treated mice. The main finding of this study was that untreated, normal rats expressed substantially more alpha-smooth muscle actin than mice. After treatment with transforming growth factor-β1, this expression increased similarly in both species. It can be hypothesized that normal closure of mesenteric perforations requires a minimum level of actin expression. This level is not reached in normal mice, but is exceeded after stimulation. Perforations in the rat always close, because the alpha-smooth muscle actin expression is always above this level.

Notes: Previous analyses of fluids collected from chronic, nonhealing wounds found elevated levels of inflammatory cytokines, elevated levels of proteinases, and low levels of growth factor activity compared with fluids collected from acute, healing wounds. This led to the general hypothesis that chronic inflammation in acute wounds produces elevated levels of proteinases that destroy essential growth factors, receptors, and extracellular matrix proteins, which ultimately prevent wounds from healing. To test this hypothesis further, pro- and activated matrix metalloproteinases (MMP-2 and MMP-9), tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), and the ratios of MMPs/TIMPs were assayed in fluids and biopsies collected from 56 patients with chronic pressure ulcers. Specimens included ulcers treated for 0, 10, and 36 days with conventional therapy or with exogenous cytokine therapies. Quantitative assay data were correlated with the amount of healing. The average MMP-9/TIMP-1 ratio in fluids from 56 ulcers decreased significantly as the chronic pressure ulcers healed. Furthermore, the average MMP-9/TIMP-1 ratio was significantly lower for fluids collected on day 0 from wounds that ultimately healed well (≥85% reduction in initial wound volume) compared with wounds that healed poorly (〈50% wound volume reduction). These data show that the ratio of MMP-9/TIMP-1 levels is a predictor of healing in pressure ulcers and they provide additional support for the hypothesis that high levels of MMP activity and low levels of MMP inhibitor impair wound healing in chronic pressure ulcers.

Notes: A healing wound represents a complex series of interactions between cells, soluble mediators, and extracellular matrix. Within this multifaceted environment, there are multiple regulatory points which control the ordered series of events that lead to normal tissue repair. An alteration in this physiologic network can lead to the development of a chronic wound. This article presents an update on the numerous mediators that exist within the wound environment in both acute normal healing and chronic nonhealing wounds. We also present a hypothesis which may provide a conceptual pathophysiologic mechanism with which to understand all chronic wounds.

Notes: The process of wound healing involves a complex interaction between numerous cell types, extracellular matrix molecules, and soluble mediators including growth factors and cytokines. This complex milieu is under active investigation for the purposes of beginning to understand how this environment regulates tissue repair. Quantitation of growth factors, cytokines, and matrix metalloproteinases within surgical wound fluids may help to elucidate this regulatory network, not only in noncomplicated wound healing but also in pathologic lesions such as chronic ulcers.

Notes: The presence and cellular distribution of epidermal growth factor (EGF), TGF-α, and EGF-R were determined in the rat fascial and peritoneal tissue during healing of an incisional injury by means of immunohistochemistry and autoradiographic techniques. The immunostaining intensity for EGF in the regenerating wound area was substantially higher during the first 14 days, then decreased to near prewound levels during 14 to 35 days after surgery. Within the wound area, the most intense immunostaining occurred with inflammatory cells, followed by fascial striated muscle and arterioles, whereas fibroblasts in the regenerating area contained very low immunostaining intensity. The immunostaining pattern for TGF-α with the use of three separate polyclonal antibodies that were directed against the amino and carboxy termini of TGF-α precursor and a fragment of the mature 50-amino-acid form of TGF-α was similar to that seen with EGF and persisted until 28 days after injury. However, fibroblasts in the regenerating area immunostained intensely for TGF-α but not for EGF. Quantitative autoradiography of iodine 125—labeled EGF binding and immunohistochemical studies of the EGF-R with monoclonal antibodies that were directed against the extracellular binding domain of EGF-R demonstrated the presence of specific EGF-R in regenerating fascial and peritoneal tissue. Net grain density (100 µm2), representing specific binding of 125I-EGF, was calculated for different cell types in the wound. The grain density over fascial striated muscle, migratory fibroblasts and peritoneal fibroblasts increased by two and one half, three, and four times, respectively, at 7 days and decreased to the values in adjacent unwounded tissue by 21 days after injury (p 〈 0.05). Immunostaining for the EGF-R generated similar patterns, which persisted for 14 days after injury. The grain density and immunostaining for EGF-R over the arterioles in the wound did not change during the course of healing and was similar to that of the uninjured regions. In summary, these observations indicate that the local levels of EGF, TGF-α, and EGF-R increase during the early phases of healing in fascial and peritoneal injury, which suggests a role for these growth factors in the normal mechanism of fascial/peritoneal wound healing and fibrous adhesion formation.

Notes: The purpose of this study was to provide molecular and mechanistic evaluation of an ischemic wound model in rats to determine if it is a valid model for human chronic wounds. Compared to acute wounds, human chronic wounds contain markedly elevated levels of proinflammatory cytokines and matrix metalloproteinases, while matrix metalloproteinase inhibitors and growth factor activity are diminished. Accordingly, tissue from ischemic and normal rat wounds were analyzed for cytokine, proteases and growth factor levels. Dorsal full thickness punch wounds were created in rats using a reproducible template. The ischemic wound group (n = 10) had six uniformly placed wounds within a bipedicled dorsal flap. The control group (n = 10) had the same wounds created without elevation of a flap. On postwound days 3, 6 and 13 wounds were excised and analyzed. Protein levels for tumor necrosis factor-α were determined with a rat-specific enzyme-linked immunosorbent assay, while mRNA was determined by RNase protection assay. Matrix metalloproteinases and serine protease detection was done using gelatin and casein zymography, respectively. Significant delay in healing was achieved in the ischemic group: 50% healing for control wounds was at 7 days and 11 days for ischemic wounds (p 〈 0.001). No significant differences between wound groups were found for interleukin-1β, and mRNA for tumor necrosis factor-α and interleukin-1β. However, at day 13 ischemic wounds contained significantly more tumor necrosis factor-α than controls and normal skin (586 ± 106 pg/biopsy vs. 79 ± 7 pg/biopsy vs. 52 ± 2 pg/biopsy; p 〈 0.001). Zymography showed substantially greater quantities of matrix metalloproteinase-2, matrix metalloproteinase-9, and serine proteases in ischemic wounds. This model of delayed healing in rats shares many of the key biochemical, molecular and mechanistic characteristics found in human chronic wounds, namely elevated tumor necrosis factor-α and protease levels. As such, this model will likely prove to be useful in chronic wound research, particularly in developing novel therapeutics.