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BACKGROUND Wound infection can impair postoperative healing. Topical antibiotics have potential to treat wound infection and inflammation and minimize the adverse effects associated with systemic antibiotics. METHODS Full-thickness porcine wounds were infected with Staphylococcus aureus. Using polyurethane wound enclosure devices, wounds were treated with topical 100 μg/ml minocycline, topical 1000 μg/ml minocycline, topical saline control, or 4 mg/kg intravenous minocycline. Bacteria were quantified in wound tissue and fluid obtained over 9 hours. Immunosorbent assays were used to analyze inflammatory marker concentrations. Minocycline's effect on in vitro migration and proliferation of human keratinocytes and fibroblasts was tested using scratch assays and metabolic assays, respectively. RESULTS After 6 hours, 100 and 1000 μg/ml topical minocycline decreased bacteria in wound tissue to 3.5 ± 0.87 and 2.9 ± 2.3 log colony-forming units/g respectively, compared to 8.3 ± 0.9 log colony-forming units/g in control wounds (p < 0.001) and 6.9 ± 0.2 log colony-forming units/g in wounds treated with 4 mg/kg intravenous minocycline (p < 0.01). After 2 hours, topical minocycline reduced concentrations of the inflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α (p < 0.01), and inflammatory cell counts in wound tissue (p < 0.05). In noninfected wounds, topical minocycline significantly reduced interleukin-1β, interleukin-6, and inflammatory cell counts after 4 hours (p < 0.01). Matrix metalloproteinase-9 concentrations decreased after 1-hour treatment (p < 0.05). Keratinocyte and fibroblast in vitro functions were not adversely affected by 10 μg/ml minocycline or less. CONCLUSIONS Topical minocycline significantly reduces bacterial burden and inflammation in infected wounds compared with wounds treated with intravenous minocycline or control wounds. Minocycline also decreases local inflammation independently of its antimicrobial effect.

Cell migration requires spatiotemporal integration of signals that regulate cytoskeletal dynamics. In response to a migration-promoting agent, cells begin to polarise and extend protrusions in the direction of migration. These cytoskeletal rearrangements are orchestrated by a variety of proteins, including focal adhesion kinase (FAK) and the Rho family of GTPases. CCN2, also known as connective tissue growth factor, has emerged as a regulator of cell migration but the mechanism by which CCN2 regulates keratinocyte function is not well understood. In this article, we sought to elucidate the basic mechanism of CCN2-induced cell migration in human keratinocytes. Immunohistochemical staining was used to demonstrate that treatment with CCN2 induces a migratory phenotype through actin disassembly, spreading of lamellipodia and re-orientation of the Golgi. In vitro assays were used to show that CCN2-induced cell migration is dependent on FAK, RhoA and Cdc42, but independent of Rac1. CCN2-treated keratinocytes displayed increased Cdc42 activity and decreased RhoA activity up to 12 hours post-treatment, with upregulation of p190RhoGAP. An improved understanding of how CCN2 regulates cell migration may establish the foundation for future therapeutics in fibrotic and neoplastic diseases.

Transplantation of skin micrografts in a 1:100 ratio regenerate the epidermis of full-thickness wounds in pigs within 14 days in a wet environment. The aim of the current study was to combine micrografts and commercially available moist dressings. We hypothesized that micrografts regenerate the epidermis when covered with a moist dressing. 5cm×5cm and 10cm×10cm full-thickness wounds were created on the backs of pigs. Wounds were transplanted with 0.8mm×0.8mm micrografts created from a split-thickness skin graft in a 1:100 ratio. 5cm×5cm wounds were treated with wound chambers, moist dressings or dry gauze (non-transplanted control group). 10cm×10cm wounds were compared to non-transplanted wounds, both covered with moist dressings. Reepithelialization was assessed in biopsies from day 10, 14 and 18 post-transplantation. 5cm×5cm transplanted wounds covered with moist dressings showed 69.5±20.6% reepithelialization by day 14 and 90.5±10.4% by day 18, similar to wounds covered with a wound chamber (63.9±16.7 and 86.2±11.9%, respectively). 18 days post-transplantation, 10cm×10cm transplanted wounds covered with moist dressings showed 66.1±10.3% reepithelialization, whereas nontransplanted wounds covered with moist dressings were 40.6±6.6% reepithelialized. We conclude that micrografts combined with clinically available moist dressings regenerate the epidermis of full-thickness wounds.

CCN2 (previously known as connective tissue growth factor) is a multifunctional matricellular protein that has numerous effects on cell life and cell interactions with the connective tissue. Although the importance of CCN2 for the fibrotic process in wound healing has been well studied, the involvement of CCN2 in keratinocyte function has not yet been explored. Therefore, the aim of the present study was to investigate the role of CCN2 in the epidermis during wound healing.Immunohistochemistry was done on sections from full-thickness porcine wounds. The effect of CCN2 on the migration of cultured human keratinocytes exposed to scratch wounds, the effect on phosphorylation of extracellular signal-related kinases (ERK), and the effect of adding inhibitors to the ERK/mitogen-activated protein kinase pathway to human keratinocytes were studied.The CCN2 protein was transiently expressed in vivo at the leading keratinocyte edge during re-epithelialization of full-thickness porcine wounds. In vitro, exogenous addition of CCN2 to human keratinocyte cultures regulated keratinocyte migration and resulted in phosphorylation of ERK. The addition of inhibitors of ERK/mitogen-activated protein kinase counteracted the effect of CCN2 on migration.CCN2 was transiently expressed at the leading keratinocyte edge in vivo. The biologic importance of this was supported in vitro, because CCN2 regulated human keratinocyte migration through activation of the Ras-mitogen-activated protein kinase kinase-ERK signal transduction pathway.

Background Transplantation of skin micrografts (MGs), split-thickness skin grafts (STSGs), or cultured autologous keratinocytes (CKs) enhances the healing of large full-thickness wounds. This study compares these methods in a porcine wound model, investigating the utility of micrograft transplantation in skin restoration. Study Design Full-thickness wounds were created on Yorkshire pigs and assigned to one of the following treatment groups: MGs, STSGs, CKs, wet nontransplanted, or dry nontransplanted. Dry wounds were covered with gauze and the other groups' wounds were enclosed in a polyurethane chamber containing saline. Biopsies were collected 6, 12, and 18 days after wounding. Quantitative and qualitative wound healing parameters including macroscopic scar appearance, wound contraction, neoepidermal maturation, rete ridge formation, granulation tissue thickness and width, and scar tissue formation were studied. Results Transplanted wounds scored lower on the Vancouver Scar Scale compared with nontransplanted wounds, indicating a better healing outcome. All transplanted wounds exhibited significantly lower contraction compared with nontransplanted wounds. Wounds transplanted with either MGs, STSGs, or CKs showed a significant increase in re-epithelialization compared with nontransplanted wounds. Wounds transplanted with MGs or STSGs exhibited improved epidermal healing compared with nongrafted wounds. Furthermore, transplantation with STSGs or MGs led to less scar tissue formation compared with the nontransplanted wounds. No significant impact on scar formation was observed after transplantation of CKs. Conclusions Qualitative and quantitative measurements collected from full-thickness porcine wounds show that transplantation of MGs improve wound healing parameters and is comparable to treatment with STSGs.

Transplantation of genetically modified keratinocytes has been shown to accelerate wound healing. However, this method is labor-intensive and time-consuming. We have developed a new technique of intraoperative gene delivery to wounds that involves transplantation of transfected minced skin particles (MSPs) derived from harvested partial-thickness skin.MSPs measuring 0.8 × 0.8 × 0.35 mm were created from a split-thickness skin graft of a pig. In vitro transfection was carried out with adenoviral LacZ (Ad-LacZ) for qualitative and adenoviral vascular endothelial growth factor (Ad-VEGF) for quantitative analysis. Transfected MSPs were transplanted to each of 2.5 × 2.5 cm full-thickness wounds on the dorsum of the pig. Nontransfected MSPs served as controls. Wound chambers were applied and injected with saline to create a wet environment.LacZ expression was detected in migrating cells originating from MSPs both in vitro and in vivo. VEGF expression in the wound fluid of Ad-VEGF-MSP-transplanted wounds on each of days 2 to 4 (mean ± SEM 6.74 ± 1.89 ng/mL, day 2; 9.88 ± 2.27 ng/mL, day 3; 9.87 ± 1.28 ng/mL, day 4) was significantly higher (p0.0001) compared with wounds transplanted with either untransfected MSPs, Ad-LacZ-MSPs, or untransplanted controls. In vitro VEGF expression was significantly higher (p0.0001) in Ad-VEGF 1 × 10(10) transfected MSPs compared with either Ad-VEGF 1 × 10(9) transfected MSPs or untransfected MSPs. Wounds transplanted with Ad-VEGF-MSPs showed significantly higher (p0.0001) numbers of newly formed blood vessels (12.6 ± 0.9 vessels/high power field [HPF]) compared with wounds transplanted with either Ad-LacZ-MSPs (4.4 ± 0.5 vessels/HPF) or untransfected MSPs (5.2 ± 0.7 vessels/HPF). All MSP-transplanted wounds (Ad-VEGF-MSPs, untransfected MSPs, Ad-LacZ-MSPs) showed significantly higher re-epithelialization compared with untransplanted wounds on days 10 and 14 (p0.0001).We demonstrated successful transfection of MSPs that can be transplanted to wounds as a source of gene-expressing cells. This technique can be used to deliver growth-modulating genes in wound healing.

Injury to the skin can predispose individuals to invasive infection. The standard of care for infected wounds is treatment with intravenous antibiotics. However, antibiotics delivered intravenously may have poor tissue penetration and be dose limited by systemic side effects. Topical delivery of antibiotics reduces systemic complications and delivers increased drug concentrations directly to the wound.Porcine full-thickness wounds infected with Staphylococcus aureus were treated with ultrahigh concentrations (over 1000 times the minimum inhibitory concentration) of gentamicin using an incubator-like wound healing platform. The aim of the present study was to evaluate clearance of infection and reduction in inflammation following treatment. Gentamicin cytotoxicity was evaluated by in vitro assays.Application of 2000 μg/ml gentamicin decreased bacterial counts in wound tissue from 7.2 ± 0.3 log colony-forming units/g to 2.6 ± 0.6 log colony-forming units/g in 6 hours, with no reduction observed in saline controls (p0.005). Bacterial counts in wound fluid decreased from 5.7 ± 0.9 log colony-forming units/ml to 0.0 ± 0 log colony-forming units/ml in 1 hour, with no reduction observed in saline controls (p0.005). Levels of interleukin-1β were significantly reduced in gentamicin-treated wounds compared with saline controls (p0.005). In vitro, keratinocyte migration and proliferation were reduced at gentamicin concentrations between 100 and 1000 μg/ml.Topical delivery of ultrahigh concentrations of gentamicin rapidly decontaminates acutely infected wounds and maintains safe systemic levels. Treatment of infected wounds using the proposed methodology protects the wound and establishes a favorable baseline for subsequent treatment.

Background Major loss of skin following burns or trauma requires skin grafting for repair. In addition, chronic wounds frequently require skin grafts. Current treatments are either cumbersome, limited in possible expansion ratio, costly, or require extensive time for treatment. This study investigates a new way of regenerating skin after major burns and other trauma, providing 100-fold expansion of a split-thickness skin graft. Methods Submillimeter micrografts were created by controlled mincing of a split-thickness skin graft and transplanted to porcine full-thickness wounds. By creating an incubator-like microenvironment using wound chambers, the micrografts provide reepithelialization whether placed dermal side up or dermal side down. Results Transplantation of micrografts in a 1:100 expansion ratio results in complete epithelialization of both healthy and diabetic wounds within 14 days. In comparison, nontransplanted wounds showed 62 percent reepithelialization in healthy pigs and 49 percent in diabetic pigs at the corresponding time point. Conclusions Minced skin micrografts are very effective in wound repair and can provide 100-fold expansion of a skin graft. Early clinical results confirm the utility of this technique.

Decreased inflammatory response seen in wet wound healing may be correlated with diminished scarring. This study seeks to test this hypothesis and to validate a model of scarring in the Yorkshire pig. Four Yorkshire pigs were used to create 36 dorsal wounds per pig (144 wounds total) in the following groups: full-thickness excisional, partial thickness, meshed split-thickness skin grafts, sheet split-thickness skin grafts, minced skin, and incisional wounds. Wounds were randomized into wet and dry groups. Wet wounds were enclosed in polyurethane chambers with 2 mL of normal saline. Dry wounds were covered with regular gauze. Terminal biopsies were performed at 72 hours and day 28. Histology demonstrated significantly less inflammatory infiltrate, thicker neoepidermis, more pronounced rete ridge formation, and decreased scar tissue thickness in wet wounds. The mean macroscopic scar surface area was significantly decreased in full-thickness excisional wet wounds compared with dry wounds (61.2 mm(2) vs. 150.8 mm(2), p

INTRODUCTION: This study presents the new finding that connective tissue growth factor (CTGF/CCN2) is expressed by hyperproliferating keratinocytes during reepithelialization. CTGF/CCN2 is a marker and mediator of fibrosis, regulating important cellular events during wound healing. There has been contradicting data regarding whether CTGF/CCN2 is expressed by epidermis or not. This study uses a well-established porcine wound model to investigare the expression of CTGF/CCN2 during epithelialization of full-thickness wounds.

INTRODUCTION: Transplantation of split-thickness skin grafts, culturedautologouskeratinocytesandskinmicrograftsresultinhealing of full-thickness wounds. This study compares these methods in a porcine wound model, investigating the utility of micrograft transplantation in skin restoration. METHODS: Full-thickness wounds were created on Yorkshire pigs and assigned to one of the following treatment groups: meshed splitthickness skin graft (STSG), cultured autologous keratinocytes (CK), skin micrografts (MG), wet environment (W) and dry environment (D). Dry wounds were covered with gauze and the other groups were enclosed in a polyurethane chamber containing 2 mL of saline. Samples were taken 6, 12 and 18 days post wounding. Macroscopic wound healing was quantified using the Vancouver Scar Scale (VSS). Wound healing parameters such as contraction, reepithelialization, epidermal characteristics and scar formation was determined by quantitative morphometric or morphologic analysis. RESULTS: Grafted wounds had a lower VSS score compared to non-grafted wounds (STSG:4, CK:6, MG:3, W:8.5, D:10, unwounded skin:0). Wounds grafted with STSG and MG showed significantly reduced contraction compared to non-grafted wounds (STSG:25%, CK:35%, MG:28%, W:43%, D:44%). All grafted wounds showed increased epidermal healing compared to nongrafted wounds. Furthermore, transplantation with STSG or MG led to less scar tissue formation compared to the non-grafted wounds andnosignificantimpactonscarformationwasobservedaftertransplantation of CK.