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Notes: We propose that the canine subglottic stenosis model can be used to investigate the pathophysiologic characteristics of excessive fibrosis. Submucosal and cartilaginous subglottic laser injuries in 22 dogs were created to compare normal wound healing with canine subglottic stenosis formation, human subglottic stenosis, and human hypertrophic scars. Tissues were evaluated for the content of proliferating cell nuclear antigen, collagen, and elastic fibers. Compared with normal mucosal healing, subglottic stenosis formation showed intensified and prolonged inflammation, delayed epithelialization, more mitotic activity, and greater collagen deposition. The model has several useful features. Stenoses can be produced predictably in dogs, with each animal serving as its own control. Endoscopic harvesting of subglottic scar can be performed repeatedly because restenosis occurs. Also, canine subglottic stenosis shares histologic characteristics with human subglottic stenosis and, perhaps, human hypertrophic scars. Future experimentation with this model should facilitate the understanding of excessive fibrosis.

Notes: Wound healing is impaired in the diabetic state because of, at least in part, low expression of growth factors. Individual growth factors can partially activate healing, yet the actual wound environment presents a dynamic continuum of multiple cellular signals. Complex interactions among growth factors and target cells can have synergistic effects, and several examples of combinatorial, in vivo activity are evident in the literature. In this study, the implantation of a combination of basic fibroblast growth factor and transforming growth factor-β in rats induced fivefold to sevenfold increases in granulation tissue formation in comparison with implantation of each growth factor alone. Diabetes was induced in adult, male Sprague-Dawley rats with streptozotocin. Incisional wounds and sponge granulation tissue were produced in separate groups and then treated with an injection of 2 µg transforming growth factor-β1 combined with 10 µg basic fibroblast growth factor on day 3. DNA, collagen, and protein were analyzed in granulation tissue on days 7 and 9, and biomechanical properties of incisions were tested on days 7, 14, and 21. The combination of transforming growth factor-β1 and basic fibroblast growth factor had marked, positive effects on biochemical parameters of wound healing and reversed the tensile strength deficit of diabetic wounds. Nonradioactive in situ hybridization showed increased expression of messenger RNA for type I and III procollagen and transforming growth factor-β1 in normal and diabetic wounds, whereas ultrastructural examination showed a marked reorganization of collagen fibrils. These findings reinforce the concept that appropriate mixtures of cytokines rather than individual cytokines may more adequately stimulate tissues in cases of impaired wound healing.

Notes: Fibroblast growth factor – binding protein (FGF-BP) is a secreted protein that appears to function as a low affinity heparin –binding protein. FGF-BP binds to FGF-1 and -2 in a non-covalent, reversible manner to mobilize and solubilize these growth factors from their storage sites in the extracellular matrix. FGF-BP is involved in both developmental and adult tissue homeostasis as well as in angiogenesis and tumorogenesis involving FGF-1/2. FGF-BP is overexpressed in several tumor types: head and neck, skin, cervical, and lung cancer, squamous cell carcinoma, and colon and breast adenocarcinoma. To establish the effect of FGF-BP on wound healing, several forms of FGF-BP cDNA were administered by particle-mediated gene transfer into various animal wound models using the gene gun (Bio-Rad). In a rat incisional wound model, gene gun cDNA delivery of full length FGF-BP at the time of surgery produced a 117% increase of wound strength in diabetic rats at 10d, although the relative increase did not reach statistical significance (P 〈 0.08). Two truncated variants of FGF-BP (pFGFbp10 and 17) were also administered in the rat incisional wound model by gene gun technique. pFGFbp17 increased the wound strength in diabetic rat 129%(p 〈 0.03), and the relative increase reach statistical significance (P 〈 0.008). In the rabbit ear ulcer model, particle-mediated transduction of full length FGF-BP increased collagen content by 195% and wound closure rate 38% at 10d post-surgery. These findings show that FGF-BP gene overexpression has a greater relative effect on wound healing in the diabetic rat model. The cDNA also had a significant effect in a rabbit excisional wound model that depended on granulation tissue formation. Truncated forms of the molecule may have higher therapeutic potency. FGF-BP has an important role in FGF-1/2 mobilization and macrophage functions, and FGF-BP gene therapy for wound healing can improve the process by stimulating angiogenesis, epithelization and collagen synthesis in target tissue.

Notes: Cytomodulin (CM-1) is a synthetic heptapeptide. Human dermal fibroblasts cultured in the presence of CM-1 showed increased expression of transforming growth factor–ß(TGF-ß) and collagen I and decreased expression of MMP-1. TGF-β is involved in numerous vital processes in wound healing, and the rate of wound repair is influenced by its overexpression at the site of injury. To establish the effect of CM-1 in wound healing, we administered several concentrations of CM-1 to incisional wounds and to unwounded skin of collagen-luciferase transgenic mice. Luciferase bioluminescence served as a quantitative reporter of CO1A2 transcription. Imaging was performed to measure the luciferase activity on these mice at different time points after administration of 1, 10 and 100 ug CM-1. The highest amount (100 μg) showed the highest level of collagen expression both in unwounded and wounded skin. In another experiment, we used a scrambled peptide as a control and administered the peptides both topically and by injection. CM-1 was effective in stimulating luciferase expression both by topical and intradermal administration. To determine the effect of CM-1 on wound strength, we measured the tensile strength in incisional wounds of wild type C57B6 mice at 10 d. Histological analysis was also performed to assess the collagen content and organization of the tissue. The data showed a statistically significant increase in tensile strength and collagen content at 10 and 100 μg doses. These results illustrate the utility of CM-1 as a novel, low-molecular weight vulnerary agent.

Notes: Aging has been anecdotally reported to result in prolonged wound healing. Measurement of punch biopsy wound closure in young (4 month old) and old (36 month old) rats indicated there was a significant delay in wound closure by old rats during the early phase of repair, after which closure rates were equivalent. The delay in granulation tissue accumulation in older animals could involve premature programmed cell death (apoptosis); however, apoptotic fibroblasts in sponge granulation tissue and tissue culture were less abundant in samples from old rats relative to young rats. Myofibroblasts express α-smooth muscle actin, and they are believed to be important in wound contraction. There were no significant differences in overall abundance or distribution of α-smooth muscle actin containing myofibroblasts in granulation tissue and in cultured granulation tissue fibroblasts regardless of the age of the donor rat. The spatial distribution of myofibroblasts and apoptotic cells was distinct. Fibroblasts from granulation tissue and skin explants were placed in a collagen gel contraction assay prior to the 5th passage to determine their in vitro contractility. While granulation tissue fibroblasts from young and old rats showed similar collagen gel contractility, skin fibroblasts from old rats displayed greater collagen gel contractile behavior than young skin fibroblasts. Greater gel contractility of fibroblasts from old rats appeared to result, in large part, from the ability of those cells to cause generalized gel degradation. Gelatin zymography indicated a greater abundance of matrix metalloproteinase-2 in supernatants from gels containing skin fibroblasts from old rats. Taken together, these results suggest that the age-associated healing delay in the rat may not be related to the appearance or abundance of distinct myofibroblast or apoptotic cell populations. Proteolysis may have a significant role in delayed wound healing in aged animals.

Notes: Expression constructs encoding a full-length cDNA encoding the human epidermal growth factor receptor, or reporter gene for green fluorescent protein or luciferase were coated onto gold particles and driven into porcine skin using a gene gun delivery system. Strategies for epidermal growth factor receptor boosting were tested in two types of wounds. For grafted wounds, intact porcine skin was pretreated by the introduction of the epidermal growth factor receptor expression construct 24 hours before its harvesting as a split-thickness skin graft. Partial-thickness excisional wound beds (donor sites) were transfected at the time of their creation. Wound healing parameters were subsequently tested in the presence or absence of excess epidermal growth factor ligand. Initial distributions of gene gun delivered gold particles as well as luciferase expression levels suggested that optimal skin penetrations and expression levels were achieved at 500 psi for intact epidermis and 300 psi for exposed wound beds. At 2 days after gene delivery, visualization of green fluorescent protein by fluorescence microscopy showed focal expression of green fluorescent protein at the advancing epithelial outgrowths found at wound edges or surviving epithelial remnants. Green fluorescent protein expression appeared transient since no green fluorescent protein was noted in specimens removed at 4 days after injury. Northern blot analysis on mRNA isolated from wounds 2 days after introduction of epidermal growth factor receptor coated gold particles by gene gun confirmed the expression of the human epidermal growth factor receptor transgene in both skin grafts and excisional wounds. Skin grafts showed subsequent biological responses to the introduction of excessive epidermal growth factor receptor as well as expression of the human epidermal growth factor receptor construct within healing epidermis. While control autografts (reporter gene treated, epidermal growth factor alone, placebo formula, no treatment) showed few 5′-bromodeoxyuridine-labeled cells, epidermal growth factor receptor autografts showed 5′-bromodeoxyuridine labeling of nearly every basal cell. Favorable wound healing outcomes were also shown within excisional wounds following in vivo boosting of epidermal growth factor receptor. Four days after receiving epidermal growth factor receptor particle bombardment, resurfacing was significantly accelerated in those wounds receiving epidermal growth factor receptor transgene. Application of topical epidermal growth factor ligand resulted in the highest percentage of resurfacing. Maximal re-epithelialization was noted in wound beds receiving both receptor boosting and excessive daily epidermal growth factor ligand. A modest increase in the thickness of the granulation tissue followed gene therapy with epidermal growth factor receptor. In summary these in vivo data suggest that it is possible to boost in vivo expression of a tyrosine kinase receptor during wound repair. Increased epidermal growth factor receptor expression has an integral impact on cell proliferation, rates of resurfacing and dermal components and merits consideration as a possible therapeutic agent.