Elizabeth Kiwanuka

Harnessing growth factors to influence wound healing.

Cutaneous wound healing is a dynamic process with the ultimate goal of restoring skin integrity. On injury to the skin, inflammatory cells, endothelial cells, fibroblasts, and keratinocytes undergo changes in gene expression and phenotype, leading to cell proliferation, migration, and differentiation. Cytokines and growth factors play an essential role in initiating and directing the phases of wound healing. These signaling peptides are produced by a variety of cells and lead to a concerted effort to restore the skin barrier function.

Epidermal regeneration by micrograft transplantation with immediate 100-fold expansion.

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.

Assessing quality of healing in skin: review of available methods and devices.

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 scars 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.

Moist dressing coverage supports proliferation and migration of transplanted skin micrografts in full-thickness porcine wounds

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 external microenvironment of healing skin wounds.

The skin wound microenvironment can be divided into two main components that influence healing: the external wound microenvironment, which is outside the wound surface; and the internal wound microenvironment, underneath the surface, to which the cells within the wound are exposed. Treatment methods that directly alter the features of the external wound microenvironment indirectly affect the internal wound microenvironment due to the exchange between the two compartments. In this review, we focus on the effects of temperature, pressure (positive and negative), hydration, gases (oxygen and carbon dioxide), pH, and anti‐microbial treatment on the wound. These factors are well described in the literature and can be modified with treatment methods available in the clinic. Understanding the roles of these factors in wound pathophysiology is of central importance in wound treatment.

CCN2 promotes keratinocyte adhesion and migration via integrin α5β1

BACKGROUND CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration. MATERIALS AND METHODS Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine-glycine-aspartic acid (RGD), 500 μM arginine-glycine-glutamate-serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2. RESULTS CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration. CONCLUSIONS Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.

Topical delivery of ultrahigh concentrations of gentamicin is highly effective in reducing bacterial levels in infected porcine full-thickness wounds.

Background: 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. Methods: 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. Results: Application of 2000 &mgr;g/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 (p < 0.005). 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 (p < 0.005). Levels of interleukin-1&bgr; were significantly reduced in gentamicin-treated wounds compared with saline controls (p < 0.005). In vitro, keratinocyte migration and proliferation were reduced at gentamicin concentrations between 100 and 1000 &mgr;g/ml. Conclusions: 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.

Use of high-frequency ultrasound guidance for intraoperative zygomatic arch fracture reduction.

Abstract Zygomatic arch fractures are common facial fractures; the management depends on the extent of the injury, the displacement of the bone, and coronoid impingement. For fractures without a need for fixation, an intraoral approach, known as Keen, or a temporal hairline approach, known as Gillies, can be used. However, without direct visualization of the fracture line, there is a risk for inadequate reduction. We have therefore begun to use ultrasound assistance to confirm proper reduction. We believe that intraoperative ultrasound guidance can be used to guide the surgeon toward the most precise fracture reduction and present 3 examples from our practice. We recommend the use of ultrasound in the reduction of zygomatic arch fractures.

Full-thickness porcine burns infected with Staphylococcus aureus or Pseudomonas aeruginosa can be effectively treated with topical antibiotics

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.

Cdc42 and p190RhoGAP activation by CCN2 regulates cell spreading and polarity and induces actin disassembly in migrating keratinocytes

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.