Homologous fibronectin enhances healing of excised wounds in rats.

In order to evaluate the effects of a topical application of homologous fibronectin on the healing of skin wounds, we made 2 excisional wounds on the back skin of each rat, applied ointment with or without fibronectin purified from citrated homologous plasma, and evaluated the effect according to wound size and microscopic findings. Excised lesions treated with carrier alone, but the difference was significant only in the early phase of wound healing, 2 and 3 days, according to wound size and microscopic changes. A significant decrease in wound size could be found in both groups, treated with ointment containing and not containing fibronectin, between day 4 and 9 when wound contraction was a major contributor to wound closure. Therefore it can be concluded that topical application of fibronectin has a beneficial effect on wound healing during its early phase, but no significant influence on wound contraction.     

Effects of systemic and topical estrogen application on the healing of full-thickness skin wounds in diabetic rats

In this research, the possible effects of systemic and topical estrogen were investigated on wound healing in normal and diabetic male rats. One hundred and ten male Wistar rats were divided into two groups (normal and diabetic). After induction of diabetes by streptozotocin in rats, each group was divided into three subgroups (control, sham, and test). A round full-thickness skin excision with 1.5-cm diameter was performed on the dorsum of each rat. In systemic use, 10?μG/SC of estradiol benzoate was administered daily to test subgroups for 28?days. In topical use, the wounds in the test subgroup rats were treated with a daily topical dose of 0.5?mg estrogen. Sham subgroup was injected with placebo and the control subgroup received nothing. The area of the wounds was measured by using scion image software at 3, 5, 7, 14, 21, 28?days. Histopathologic evaluation was assessed semi-quantitatively for different parameters including re-epithelization, neoangiogenesis, and granulation tissue formation. In macroscopic study, estradiol subgroups (systemic and topical application) wound healing had considerable changes in day?7 (p?<?0.05). Histopathologic evaluation was revealed increased vascularization, re-epithelialization in estradiol subgroups. So, this research expressed that systemic and topical estrogen can improve the impaired healing of diabetic wounds.     

Accelerated wound closure of pressure ulcers in aged mice by chitosan scaffolds with and without bFGF

Pressure ulcers are a significant healthcare concern, especially for elderly populations. Our work served to ameliorate the chronicity of these ulcers by addressing ischemia–reperfusion injury mediated by neutrophils and the concomitant loss of vasculature in these wounds. To this end, chitosan scaffolds loaded with basic fibroblast growth factor (bFGF) contained in gelatin microparticles were developed and tested for clinical relevance in an aged mouse model. Pressure ulcers were induced in aged mice, and efficacy of treatment was assessed. On days 3 and 7, both chitosan and chitosan–bFGF scaffolds significantly accelerated wound closure compared to gauze control. By day 10, all wounds achieved similar closure. Delivery and angiogenic function of bFGF was verified through ELISA and histology. Elevated neutrophil levels were observed in chitosan and chitosan–bFGF groups. Since neutrophil elastase contributes to the proteolytic environments of pressure ulcers, the effect of chitosan on elastase was assessed. In vitro, chitosan inhibited elastase activity. In vivo, elastase protein levels in wounds were reduced with chitosan–bFGF scaffolds by day 10. These results suggest that chitosan is an effective material for growth factor delivery and can help to heal chronic ulcers. Collectively, our data show that chitosan–bFGF scaffolds are effective in accelerating wound closure of pressure ulcers in aged animals.     

Human neutrophil elastase and collagenase sequestration with phosphorylated cotton wound dressings

The design and preparation of wound dressings that redress the protease imbalance in chronic wounds is an important goal of wound healing and medical materials science. Chronic wounds contain high levels of tissue and cytokine-destroying proteases including matrix metalloprotease and neutrophil elastase. Thus, the lowering of excessive protease levels in the wound environment by wound dressing sequestration prevents the breakdown of extracellular matrix proteins and growth factors necessary for wound healing. Phosphorylated cotton wound dressings were prepared to target sequestration of proteases from chronic wound exudate through a cationic uptake binding mechanism involving salt bridge formation of the positively charged amino acid side chains of proteases with the phosphate counterions of the wound dressing fiber. Dressings were prepared by applying sodium hexametaphosphate and diammonium phosphate in separate formulations to cotton gauze by pad/dry/cure methods. Phosphorylated cotton dressings were assessed for their ability to lower elastase and collagenase activity. The phosphorylated cotton dressings lowered elastase and collagenase activity 40-80% more effectively than the untreated cotton wound dressings under conditions that mimic chronic wound exudate. Efficacy of the phosphorylated cotton was found to be related to the level of phosphorylation and a lower pH due to protonated phosphate at the surface of the dressing. The capacity of the modified gauze to sequester continued elastase secretions similar to that found in a chronic wound over a 24-h period was retained within a 80% retention of elastase sequestration and was dose-dependent.     

17β-Estradiol Inhibits Wound Healing in Male Mice via Estrogen Receptor-α

Although estrogens have long been known to accelerate healing in females, their roles in males remain to be established. To address this, we have investigated the influence of 17β-estradiol on acute wound repair in castrated male mice. We report that sustained exposure to estrogen markedly delays wound re-epithelialization. Our use of hairless mice revealed this response to be largely independent of hair follicle cycling, whereas other studies demonstrated that estrogen minimally influences wound inflammation in males. Additionally, we report reduced collagen accumulation and increased gelatinase activities in the wounds of estrogen-treated mice. Increased wound matrix metalloproteinase (MMP)-2 activity in these animals may i) contribute to their inability to heal skin wounds optimally and ii) stem, at least in part, from effects on the overall levels and spatial distribution of membrane-type 1-MMP and tissue inhibitor of MMP (TIMP)-3, which respectively facilitate and prevent MMP-2 activation. Using mice rendered null for either the α or β isoform of the estrogen receptor, we identified estrogen receptor-α as the likely effector of estrogen’s inhibitory effects on healing.Although anecdotal evidence has long suggested that differences exist in the abilities of females and males (particularly the elderly) to heal acute wounds, only recently have they been substantiated by published research. Indeed, it was observed sex differences in key parameters such as restoration of the basement membrane¹ and elastin regeneration² that previously encouraged us to make detailed comparisons of healing in males and females. We discovered that, although repair is broadly similar in intact (young) male and female mice, castrated males heal acute skin wounds far better than do their ovariectomized female counterparts.³ Furthermore, males and females differed in their responsiveness to macrophage migration inhibitory factor (MIF): a potent inhibitor of repair in females, in males it has minimal influence.These studies encouraged us to conclude that sex differences in the responses to cutaneous injury do exist but that they are masked in young individuals by the combined actions of gonadal sex steroids. In males, testosterone and its more potent metabolite 5α-dihydrotestosterone inhibit repair^1,4; in females, estrogens such as 17β-estradiol accelerate healing.^5,6Although the effects of estrogens on female cutaneous physiology are well characterized, their roles in males are poorly understood. A handful of studies have sought to address this. In a group of aged males, locally administered 17β-estradiol was shown to reduce macroscopically determined day 7 wound areas in an excisional wounding model.⁶ It was recently shown that an overwhelming majority of genes displaying different wound expression between young and elderly human males are subject to estrogenic control.⁷ In a separate study, thrice-weekly application of 17β-estradiol to sun-protected skin in aged males induced the synthesis of collagen I; increased dermal collagen bundle thickness and density; and stimulated keratinocyte proliferation.⁸ Although these and other studies have provided useful insights, little is yet known about the healing properties of i) systemic and ii) prolonged estrogen treatment.Having previously reported preliminary evidence that systemic 17β-estradiol treatment may impair cutaneous wound healing in castrated male mice,³ we aimed with the present study to fully characterize the effects of 17β-estradiol on the healing of acute wounds in males and to delineate the mechanisms underpinning any identified responses. Because estrogens are well-known to influence the cycling of hair follicles,⁹ which themselves were recently shown to be beneficial to repair,¹⁰ the contribution of hair to estrogen-impaired healing provided our initial focus. We report that estrogen treatment of castrated mice significantly retards wound re-epithelialization in both hairless (hr/hr) mice and strain-matched controls, confirming that systemic estrogen treatment does indeed inhibit repair and suggesting that the presence of cycling hair follicles is not critical to this response. Subsequent studies identified estrogen receptor (ER)-α as the likely effector of estrogenic inhibition and highlighted the potential involvement of increased matrix metalloproteinase (MMP)-2 activity in the reduced wound accumulation of collagen that we observed in estrogen-treated mice.     

Role of endothelial selectins in wound repair.

P- and E-selectins are adhesion molecules expressed on activated endothelium and platelets at sites of vascular injury and inflammation. The selectins are important for leukocyte recruitment. Because little is known about the role of selectins in wound healing, we studied cutaneous wound repair of full-thickness excisional skin wounds in mice lacking P-selectin, E-selectin, or both of these selectins. The absence of either selectin alone had no notable effect on healing, and the only deficit observed was a delay in early neutrophil extravasation in the P-selectin-deficient mice. Mice deficient in both P- and E-selectins had markedly reduced recruitment of inflammatory cells and impaired closure of the wounds. Wound sections, studied up to 3 days after wounding, showed significant impairment of neutrophil influx. Macrophage numbers were also reduced in the double mutants at 3 and 7 days after wounding as compared with wild-type mice. Additionally, a wider epithelial gap in the wounds of the P- and E-selectin-double-deficient mice 3 days after wounding indicated delayed keratinocyte migration. These results demonstrate an important combined role for P- and E-selectins in processes leading to wound healing.     

Recombinant hepatocyte growth factor accelerates cutaneous wound healing in a diabetic mouse model

We examined effects of recombinant hepatocyte growth factor (HGF) on cutaneous wound healing, using a full-thickness cutaneous excision model in diabetic mice. Topical administration of HGF, as well as basic fibroblast growth factor (bFGF), promoted the rate of wound closure and re-epithelialization. Both HGF and bFGF enhanced expansion of the granulation tissue and stimulated neovascularization on day 7 postwounding, wherein the increase in microvessel density in HGF-treated wounds was higher than that in bFGF-treated wounds. Matrix metalloproteinases (MMP-2 and MMP-9) activities involved in cell migration, angiogenesis, and extracellular matrix (ECM) remodeling, were enhanced by HGF-treatment on day 7. On day 28 postwounding (later stages of wound healing), granulation tissue in bFGF-treated wounds remained to a greater extent than that seen in saline- and HGF-treated wounds. Likewise, bFGF- but not HGF-treatment stimulated DNA synthesis of fibroblasts in granulation tissue, suggesting that HGF stimulates wound healing with lesser degree of susceptibility to cutaneous scarring. We propose that supplement of HGF may be a potential therapeutic approach for treatment of cutaneous ulcer.     

Recombinant Hepatocyte Growth Factor Accelerates Cutaneous Wound Healing in a Diabetic Mouse Model

We examined effects of recombinant hepatocyte growth factor (HGF) on cutaneous wound healing, using a full-thickness cutaneous excision model in diabetic mice. Topical administration of HGF, as well as basic fibroblast growth factor (bFGF), promoted the rate of wound closure and re-epithelialization. Both HGF and bFGF enhanced expansion of the granulation tissue and stimulated neovascularization on day 7 postwounding, wherein the increase in microvessel density in HGF-treated wounds was higher than that in bFGF-treated wounds. Matrix metalloproteinases (MMP-2 and MMP-9) activities involved in cell migration, angiogenesis, and extracellular matrix (ECM) remodeling, were enhanced by HGF-treatment on day 7. On day 28 postwounding (later stages of wound healing), granulation tissue in bFGF-treated wounds remained to a greater extent than that seen in saline- and HGF-treated wounds. Likewise, bFGF- but not HGF-treatment stimulated DNA synthesis of fibroblasts in granulation tissue, suggesting that HGF stimulates wound healing with lesser degree of susceptibility to cutaneous scarring. We propose that supplement of HGF may be a potential therapeutic approach for treatment of cutaneous ulcer.     

Androgens influence expression of matrix proteins and proteolytic factors during cutaneous wound healing

Excessive proteolytic activity is a feature of chronic wounds such as venous ulcers, in which resolution of the inflammatory response fails and restorative matrix accumulation is delayed as a consequence. The inflammatory actions of native androgens during the healing of acute skin wounds have lately been characterized. We have now investigated the hypothesis that such activities may impact upon the balance between anabolic and catabolic processes during wound healing. We report that wound deposition of both type I collagen and fibronectin is increased in castrated rats compared with control animals. This response is accompanied by early increases and later decreases in overall wound levels of the key collagenolytic enzymes, matrix metalloproteinase (MMP)-1 and MMP-13. Moreover, the activities of MMP-2 and MMP-9, two further enzymes that contribute to collagen digestion during venous ulceration, were significantly decreased in the wounds of castrated rats. Additional analyses provide evidence that androgens directly stimulate dermal fibroblast collagen production, which supports the suggestion that increased wound collagen deposition in androgen-deprived rats results from reduced matrix degradation (as opposed to enhanced matrix protein biosynthesis). Androgen-mediated dysregulation of the parallel processes of collagen deposition and turnover may underscore the delayed healing of cutaneous wounds in elderly male patients and further contribute to the increased incidence of non-healing wounds in this population.     

Topical pluronic F-127 gel application enhances cutaneous wound healing in rats

Pluronic F-127 gel is used as vehicle for various topical applications. In the present study, effects of topical application of pluronic F-127 gel were evaluated in cutaneous wound healing in Wistar rats. Normal saline solution and pluronic F-127 gel (25%) were applied topically on open excision wounds for 14 days. Photography, determination of percentage wound contraction, and collection of granulation tissue were done on days 3, 7, 11 and 14 post-wounding. Topical application of gel (once daily) significantly increased the wound closure on days 11 and 14. The gel application increased the expressions of vascular endothelial growth factor (VEGF) and transforming growth factor-beta1 (TGF-β₁) on days 3 and 7. Histopathologically, more leukocyte infiltration followed by well formed granulation tissue with marked fibroblast proliferation was evident in the gel-treated group, as compared to the saline-treated control group. Immunohistochemistry of CD31 on day 7 revealed significant higher microvessel density in gel-treated wounds. Picrosirius staining demonstrated higher collagen fraction in gel-treated wounds. Thus, from the results, it could be concluded that pluronic F-127 gel has a mild inflammatory nature and enhanced the healing by stimulating expression of VEGF and TGF-β₁.     

Age-related changes in the temporal and spatial distributions of fibrillin and elastin mRNAS and proteins in acute cutaneous wounds of healthy humans

Elasticity and resilience of the skin are determined largely by the elastin framework, whose microfibrillar scaffold is composed of fibrillin. To date, the spatial and temporal patterns of expression of human elastin and fibrillin during wound healing have not been described. Ninety healthy human subjects underwent 4 mm cutaneous punch biopsy wounds from the upper inner arm, which were re-excised from day 3 to 3 months post-wounding. There were marked changes in the patterns of distribution and the amounts of elastin and fibrillin in sun-protected skin with ageing. However, there were no major age-related differences in the mRNA levels for elastin, fibrillin-1 and fibrillin-2 using in situ hybridization. Elastin and fibrillin appeared in greatest amounts in the wounds of the elderly, particularly in females. A regenerative pattern of elastin and fibrillin arcades at the dermo-epidermal junction was observed in the wounds of aged subjects. mRNA expression of elastin was greatest in the wounds of the aged (from day 3 to day 14 post-wounding) with a similar spatial and temporal pattern to fibrillin-1 expression; this suggests that fibrillin-1 is the major contributor to dermal elastic fibre construction during wound repair. Fibrillin-2 was expressed only in the wounds of the aged and expression was confined to areas proximal to dermal blood vessels. The clear-cut differences in the localization of the two members of the fibrillin family suggest that these have well-defined roles in normal skin and wound tissue. In summary, these data indicate that ageing is associated with increased expression of fibrillin and elastin during acute wound healing and concomitant restoration of the papillary dermal architecture with an improved quality of scarring. © 1997 by John Wiley & Sons, Ltd.     

Therapeutic Effects of Topical Application of Ozone on Acute Cutaneous Wound Healing

This study was undertaken to evaluate the therapeutic effects of topical ozonated olive oil on acute cutaneous wound healing in a guinea pig model and also to elucidate its therapeutic mechanism. After creating full-thickness skin wounds on the backs of guinea pigs by using a 6 mm punch biopsy, we examined the wound healing effect of topically applied ozonated olive oil (ozone group), as compared to the pure olive oil (oil group) and non-treatment (control group). The ozone group of guinea pig had a significantly smaller wound size and a residual wound area than the oil group, on days 5 (P<0.05) and 7 (P<0.01 and P<0.05) after wound surgery, respectively. Both hematoxylin-eosin staining and Masson-trichrome staining revealed an increased intensity of collagen fibers and a greater number of fibroblasts in the ozone group than that in the oil group on day 7. Immunohistochemical staining demonstrated upregulation of platelet derived growth factor (PDGF), transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF) expressions, but not fibroblast growth factor expression in the ozone group on day 7, as compared with the oil group. In conclusion, these results demonstrate that topical application of ozonated olive oil can accelerate acute cutaneous wound repair in a guinea pig in association with the increased expression of PDGF, TGF-β, and VEGF.     

Chemokines IL-8, GROα, MCP-1, IP-10, and Mig Are Sequentially and Differentially Expressed During Phase-Specific Infiltration of Leukocyte Subsets in Human Wound Healing

Healing of cutaneous wounds requires a complex integrated network of repair mechanisms, including the action of newly recruited leukocytes. Using a skin repair model in adult humans, we investigated the role chemokines play in sequential infiltration of leukocyte subsets during wound healing. At day 1 after injury, the C-X-C chemokines IL-8 and growth-related oncogene α are maximally expressed in the superficial wound bed and are spatially and temporally associated with neutrophil infiltration. IL-8 and growth-related oncogene α profiles also correlate with keratinocyte migration and subsequently subside after wound closure at day 4. Macrophage infiltration reaches the highest levels at day 2 and is paralleled by monocyte chemoattractant protein-1 mRNA expression in both the basal layer of the proliferative epidermis at the wound margins and mononuclear cells in the wound area. Other monocyte-attracting chemokines such as monocyte chemoattractant protein-3, macrophage inflammatory protein-1α and -1β, RANTES, and I309 are undetectable. At day 4, perivascular focal lymphocyte accumulation correlates with strong focal expression of the C-X-C chemokines Mig and IP-10. Our results suggest that a dynamic set of chemokines contributes to the spatially and temporally different infiltration of leukocyte subsets and thus integrates the inflammatory and reparative processes during wound repair.     

Accelerated wound closure in neutrophil-depleted mice

The infiltration of neutrophils into injured tissue is known to protect wounds from invading pathogens. However, more recent studies suggest that neutrophils might inhibit the wound repair process. To investigate the role of neutrophils in wounds, mice were neutrophil-depleted by injection with rabbit anti-mouse neutrophil serum. Remarkably, epidermal healing, measured by wound closure, proceeded significantly faster in neutropenic than control mice (77.7+14.2% vs. 41.2+0.9%, P<0.02 at day 2). Dermal healing was not affected by neutrophil depletion, as neither collagen deposition nor wound-breaking strength was significantly different between neutropenic and control mice. As the delayed repair of diabetic individuals exhibits robust inflammation, the effect of neutrophil depletion on diabetic wound healing was investigated. Similar to the observations in wild-type mice, wound closure was accelerated by nearly 50% in neutropenic, diabetic mice. The results suggest that although neutrophils may provide protection against infection, they may retard wound closure.     

Low molecular weight hyaluronic acid prevents oxygen free radical damage to granulation tissue during wound healing

Hyaluronic acid protects granulation tissue from oxygen free radical damage and stimulates wound healing, but its molecular weight prevents it from permeating the epidermal barrier A low molecular weight hyaluronic acid preparation is able to permeate the skin, but it is unknown whether or not it retains the scavenging effects of oxygen free radicals in granulation tissue. Our experiments were conducted in rats with excisional or incisional wounds. Wound contraction over 11 days and breaking strength on the fifth day were measured. Oxygen free radical production was induced by intraperitoneal administration of two different xenobiotics: phenazine methosulfate and zymosan. The wounds were treated topically with low molecular weight hyaluronic acid (0.2%) cream or placebo. In the incisional wound group, the effects of superoxide dismutase were also determined. Absolute controls received wounds and placebo but no xenobiotics. Wound healing was significantly slower in the xenobiotic group than in the control groups. These effects were strongly reduced by topical administration of low molecular weight hyaluronic acid (0.2%) cream and in incisional wounds by topically injected superoxide dismutase. Low molecular weight hyaluronic acid is effective as the native compound against oxygen free radicals. Its pharmacological effects through transdermal administration should be tested in appropriate models.     

The eosinophil as a cellular source of transforming growth factor alpha in healing cutaneous wounds.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are known to promote the healing of epithelial wounds. Eosinophils are present in healing wounds and have recently been shown to be capable of producing TGF-alpha. This investigation was done to determine if eosinophils infiltrated into healing wounds are capable of expressing this cytokine. Using the rabbit cutaneous open wound model, the study found that the eosinophil is one of the predominant cell types in the healing wound, beginning from the seventh day and thereafter. Most surprisingly, the majority of the eosinophils present in the healing wound were found to contain TGF-alpha mRNA and protein by in situ hybridization and immunohistochemistry. Thus it is proposed that the delivery of TGF-alpha by eosinophils to epithelial wound healing sites represents a normal body mechanism whereby this multifunctional cytokine can accelerate the wound healing process.     

Macroscopic aspects of wound healing (contraction and epithelialisation) after topical administration of allicin in dogs

Macroscopical aspects of second-intention healing of full-thickness excisional wounds were studied in five normal male mixed-breed dogs. Test wounds were treated topically with allicin 0.5% in methyl cellulose gel, and control wounds were treated with methyl cellulose gel only. Wound treatment started 24 h after wounding. The wounds were evaluated over a 4-week period. At days 0, 3, 7, 10, 14, 17, 21, 24 and 28, digital photographs were taken of all wounds. Rulers were held vertically and horizontally close to the wound as a reference. The area of epithelialisation and granulation tissue were measured for each wound using Scion Image software. Percentage wound contraction, epithelialisation and healing were calculated for each wound. Initially, all wound areas increased in size. After the initial enlargement, wound areas decreased rapidly in size between days 7 and 17 in both the test and control groups. Epithelialisation was first noticed at day 3 in control and day 5 in the test wounds. No significant differences were observed in the percentage of wound contraction, epithelialisation and healing between the test and control wounds (P> 0.05).