This chapter summarizes the current state of the literature regarding gender-specific differences in outcome for patients with burn injuries. The main focus of this chapter will be a consideration of the reasons for the sexual dimorphism in survival from burns. Despite increasing research directed at women's health, the literature is limited and quite inconsistent. The lack of gender-specific research in burn patients becomes even more obvious when seeking explanations for the underlying mechanisms that cause the vulnerability of women to burn injury. Ultimately, if female gender influenced burn mortality it would be imperative to consider gender-related differences in the development of novel therapeutic interventions in burn patients.

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.

Burn and blast injuries are frequently complicated by invasive infections, which lead to poor wound healing, delay in treatment, disability, or death. Traditional approach centers on early debridement, fluid resuscitation, and adjunct intravenous antibiotics. These modalities often prove inadequate in burns, where compromised local vasculature limits the tissue penetration of systemic antibiotics. Here, we demonstrate the treatment of infected burns with topical delivery of ultrahigh concentrations of antibiotics. Standardized burns were inoculated with Staphylococcus aureus or Pseudomonas aeruginosa. After debridement, burns were treated with either gentamicin (2 mg/mL) or minocycline (1 mg/mL) at concentrations greater than 1,000 times the minimum inhibitory concentration. Amount of bacteria was quantified in tissue biopsies and wound fluid following treatment. After six days of gentamicin or minocycline treatment, S. aureus counts decreased from 4.2 to 0.31 and 0.72 log CFU/g in tissue, respectively. Similarly, P. aeruginosa counts decreased from 2.5 to 0.0 and 1.5 log CFU/g in tissue, respectively. Counts of both S. aureus and P. aeruginosa remained at a baseline of 0.0 log CFU/mL in wound fluid for both treatment groups. The findings here demonstrate that super-therapeutic concentrations of antibiotics delivered topically can rapidly reduce bacterial counts in infected full-thickness porcine burns. This treatment approach may aid wound bed preparation and accelerate time to grafting.

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.

The process of wound healing is dynamic and takes place over months to years, during which there is a resolution of angiogenesis, continued wound contraction, and connective tissue remodeling. The outcome of this process is most commonly the formation of a scar, defined as a fibrous tissue replacing normal tissues destroyed by injury or disease. Scars often have a lowered or total loss of vital skin functions and imbue a large burden on both the patient and the health care system as a whole. Scar treatments are plentiful but are often unsatisfactory or inconsistent. No single treatment method has been universally adopted. To evaluate the clinical treatment as well as research focused on developing novel methods for scar management, objective studies of the progression of scar formation and the properties of mature scars are needed. Several parameters, including barrier function as well as mechanical and physiological properties, need to be taken into account when both categorizing and treating healing wounds and scars. To date, there is no available methodology that provides a comprehensive evaluation of a scar's properties. This review aims at presenting an overview of available scar assessment methods and devices, ranging from analysis of collagen properties in tissue biopsies to noninvasive methods for studies of mechanical parameters such as breaking strength and skin elasticity. In the cases where conclusive studies have been performed, the differences between normal skin and scar with respect to the above parameters are presented. Furthermore, this review highlights areas where the development of additional modalities are needed.

OBJECTIVE:In the United States, around 50% of all musculoskeletal injuries are soft tissue injuries including ligaments and tendons. The objective of this study is to assess the role of amnion-derived cellular cytokine solution (ACCS) in carboxy-methyl cellulose (CMC) gel in the healing of Achilles tendon in a rat model, and to examine its effects on mechanical properties and collagen content.METHODS:Achilles tendons of Sprague-Dawley rats were exposed and transected. The distal and proximal ends were injected with either saline or ACCS in CMC, in a standardized fashion, and then sutured using a Kessler technique. Tendons from both groups were collected at 1, 2, 4, 6, and 8 weeks postoperatively and assessed for material properties. Collagen studies were performed, including collagen content, collagen cross-linking, tendon hydration, and immunohistochemistry. Tendons were also evaluated histologically for cross-sectional area.RESULTS:Mechanical testing demonstrated that treatment with ACCS in CMC significantly enhances breaking strength, ultimate tensile strength, yield strength, and Young's modulus in the tendon repair at early time points. In context, collagen content, as well as collagen cross-linking, was also significantly affected by the treatment.CONCLUSION:The application of ACCS in CMC has a positive effect on healing tendons by improving mechanical properties at early time points. Previous studies on onetime application of ACCS (not in CMC) did not show significant improvement on tendon healing at any time point. Therefore, the delivery in a slow release media like CMC seems to be essential for the effects of ACCS demonstrated in this study.

OBJECTIVE:Tendon injuries produce considerable morbidity, long-lasting disability, and remain a considerable challenge for clinicians and patients. The objective of the study was to assess the effect of amnion-derived multipotent progenitor (AMP) cells and amnion-derived cell cytokine solution on Achilles tendon healing by using a rat model.METHODS:Achilles tendons of Sprague-Dawley rats were exposed and transected. The distal and proximal ends were injected with either saline, amnion-derived cell cytokine solution, or AMP cells in a standardized fashion and then sutured by using a Kessler technique. Tendons from each group (n = 6-13) were collected at weeks 1, 2, and 4 postoperatively and assessed for material properties (ultimate tensile strength, Young modulus, yield strength, and breaking strength). Tendons were also evaluated histologically for cross-sectional area by using hematoxylin-eosin and trichrome stains.RESULTS:Mechanical testing showed that the Young modulus was significantly higher in AMP cells-treated tendons at week 4 compared with both saline-treated and amnion-derived cell cytokine solution-treated tendons. Yield strength was significantly higher in the AMP cells-treated group compared with saline-treated controls at week 4. No significant differences were observed between the study groups at weeks 1 and 2.DISCUSSION:Amnion-derived multipotent progenitor cells have a positive effect on healing tendons by improving mechanical strength and elastic modulus during the healing process. The presented findings suggest the clinical utility of AMP cells in facilitating the healing of ruptured tendons. Both the Young modulus and yield strengths of tendons increased significantly following treatment with AMP cells.

Background: 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.Materials and methods: 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.Results: 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.Conclusions: 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. Published by Elsevier Inc.

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.

Background: Diabetic wounds result in significant morbidity, prolonged hospitalization, and enormous healthcare expenses. Among the complications, wound infection is a major contributing factor for amputations. Pigs have been shown to have wound healing resembling that in humans. The Problem: The presented studies aimed to establish a diabetic wound healing model in large animals and investigate the implication of human-beta defensin-3, a host defense peptide, on the healing process. Basic/Clinical Science: Diabetes was induced in pigs using streptozotocin injection followed by full-thickness wound creation. Nondiabetic pigs with or without high glucose wound fluid concentrations served as controls. Wound fluid was analyzed for insulin-like growth factor, platelet-derived growth factor, and transforming growth factor. In the second study diabetic wounds in pigs were infected with Staphylococcus aureus and microseeded with an adenoviral vector expressing human beta-defensin-3 (hBD-3). Wounds were analyzed for transgene expression, bacterial infection, and reepithelialization. Clinical Care Relevance: Diabetic wounds are difficult to treat because of their poor response to conventional treatment options. Gene delivery to wounds expressing antimicrobial peptides like hBD-3 may serve as a new treatment strategy. Conclusion: An experimental diabetic wound healing model in pigs could be established that showed significantly delayed healing compared to healthy pigs. Moreover, it could be shown that hBD-3 expression significantly promotes healing in S. aureus‐infected diabetic full-thickness wounds in a preclinical large animal model. BACKGROUND

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.

BACKGROUND: 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.STUDY DESIGN: MSPs measuring 0.8 X 0.8 X 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 X 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.RESULTS: 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 (p < 0.0001) compared with wounds transplanted with either untransfected MSPs, Ad-LacZ-MSPs, or untransplanted controls. In vitro VEGF expression was significantly higher (p < 0.0001) in Ad-VEGF 1 X 10(10) transfected MSPs compared with either Ad-VEGF 1 X 10(9) transfected MSPs or untransfected MSPs. Wounds transplanted with Ad-VEGF-MSPs showed significantly higher (p < 0.0001) 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 (p < 0.0001).CONCLUSIONS: 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. (J Am Coll Surg 2011;212:340-348. (c) 2011 by the American College of Surgeons)

Kiwanuka, Elizabeth MD; Hackl, Florian MD; Caterson, Edward J. MD, PhD; Junker, Johan P.E. PhD; Gerdin, Bengt MD, PhD; Nowinski, Daniel MD, PhD; Eriksson, Elof MD, PhD, FACS Author Information

Objective: The purpose of this study was to determine whether amnion-derived cellular cytokine solution (ACCS) could improve the quality of epithelialization and accelerate closure of dermatome-created partial-thickness wounds in normal and streptozotocin-induced diabetic pigs. Methods: Dermatome-created partial-thickness wounds were sealed with wound chambers in healthy and diabetic pigs and were injected with ACCS. Wound fluid was exchanged daily for total protein concentration, and biopsies were taken on days 6, 8, 10, and 12. Epithelialization, thickness of epidermis, number of epidermal cell layers, and rete ridges were evaluated. Results: The macroscopic appearance of the wounds and speed of healing was similar in all groups at each time point. All wounds were healed by day 6. The epidermis was thicker in the ACCS-treated diabetic wounds than in the controls (140.6 μm vs 82.7 μm on day 12 in diabetic pigs). There were more cell layers (13 vs 7.7) in ACCS-treated diabetic pigs on day 12. The number of rete ridges per 2.5 mm was greater on day 12 in the ACCS-treated diabetic wounds (13 vs 8). There was also a significant increase in the number of rete ridges in ACCS-treated nondiabetic pigs but no difference in epidermal thickness or number of cell layers. Conclusion: In diabetic pigs, we found a significantly thicker epidermis and more cell layers and rete ridges in the ACCS-treated wounds. Healthy pigs showed more rete ridges but no difference in thickness of epidermis or number of cell layers on day 12.

ABSTRACTDecreased 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 mm2 vs. 150.8 mm2, p<0.01). Hydroxyproline content was decreased in full‐thickness wet compared with dry groups (44.81 vs. 62.21 mg/g, p<0.01). Tensile strength was 90% greater in full‐thickness wet compared with dry groups (p<0.01). Healing in the liquid environment significantly reduced scar formation. This model will allow for future investigation of high‐concentration topical scar‐modulating agents in the liquid environment.