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Insulin-like growth factor-I (IGF-I) and keratinocyte growth factor (KGF) cDNA gene transfer individually improves dermal and epidermal regeneration. The aim of the present study was to determine whether the combination of IGF-I plus KGF cDNA further improves wound healing and by which mechanisms these changes occur. Rats received an acute wound and were divided into four groups to receive weekly subcutaneous injections of liposomes plus Lac Z cDNA, liposomes plus IGF-I cDNA, liposomes plus KGF cDNA, or liposomes plus IGF-I/KGF cDNA. Planimetry, immunological assays, histological and immunohistochemical techniques were used to determine IGF-I, KGF, platelet-derived growth factor, fibroblast growth factor (FGF), transforming growth factor-beta and vascular endothelial growth factor (VEGF) expression and different types of collagen (I, III and IV). IGF-I, KGF and their combination cDNA treatment significantly (P<0.05) accelerated re-epithelization, increased IGF-I, KGF, FGF, VEGF and collagen type IV expression, while it had no effect on collagen type I and III expression. The combination of IGF-I plus KGF cDNA increased (P<0.05) neovascularization and VEGF expression when compared to IGF-I cDNA, KGF cDNA groups and controls. In conclusion, exogenous administration of liposomal IGF-I plus KGF cDNA enhanced dermal and epidermal regeneration which is due to increased neovascularization.

Gene therapy was traditionally considered a treatment modality for patients with congenital defects of key metabolic functions or late-stage malignancies. The realization that gene therapy applications were much vaster has opened up endless opportunities for therapeutic genetic manipulations, especially in the skin and external wounds. Cutaneous wound healing is a complicated, multistep process with numerous mediators that act in a network of activation and inhibition processes. Gene delivery in this environment poses a particular challenge. Numerous models of gene delivery have been developed, including naked DNA application, viral transfection, high-pressure injection, liposomal delivery, and more. Of the various methods for gene transfer, cationic cholesterol-containing liposomal constructs are emerging as a method with great potential for non-viral gene transfer in the wound. This article aims to review the research on gene therapy in wound healing and possible future directions in this exciting field.

Liposomal gene transfer is an effective therapeutic approach to improve dermal and epidermal regeneration. The purpose of the present study was to define whether the biological or chemical structure of a liposome influences cellular and biological regeneration in the skin, and to determine by which mechanisms possible changes occur. Rats were inflicted a full-excision acute wound and divided into three groups to receive weekly subcutaneous injections of DMRIE liposomes plus the Lac Z gene, or DOTAP/Chol liposomes plus the Lac Z gene, or saline. Planimetry, immunological assays, histological and immunohistochemical techniques were used to determine cellular responses after gene transfer, protein expression, dermal and epidermal regeneration. DOTAP/Chol increased IGF-I and KGF protein concentration and caused concomitant cellular responses, for example, by increasing IGFBP-3, P<0.05. DOTAP/Chol liposomes improved epidermal regeneration by exhibiting the most rapid area and linear wound re-epithelization compared to DMRIE or control, P<0.001. DOTAP/Chol and DMRIE exerted promitogenic and antiapoptotic effects on basal keratinocytes, P<0.05. Dermal regeneration was improved in DOTAP/Chol-treated animals by an increased collagen deposition and morphology, P<0.001. DOTAP/Chol liposomes increased vascular endothelial growth factor concentrations and thus neovascularization when compared with DMRIE and saline, P<0.001. In the present study, we showed that different liposomes have different effects on intracellular and biological responses based on its chemical and molecular structure. For gene transfer in acute wounds, the administration of DOTAP/Chol liposomes appears to be beneficial.

Keratinocyte growth factor (KGF) stimulates epithelial cell differentiation and proliferation, which are of major importance for wound healing. Local protein administration, however, has been shown to be ineffective due to enzymes and proteases in the wound fluid. We hypothesized that delivering KGF as a non-viral liposomal cDNA gene complex is a new approach that would effectively enhance dermal and epidermal regeneration. Twenty-two rats were given an acute wound and divided into two groups to receive weekly subcutaneous injections of liposomes plus the LacZ gene (0.2 microg, vehicle), or liposomes plus the KGF cDNA (2.2 microg) and LacZ cDNA (0.2 microg). Transfection was confirmed by histochemical assays for beta-galactosidase. Planimetry, histological and immunohistochemical techniques were used to determine protein expression, dermal and epidermal regeneration. Transfection and subsequent KGF expression was found in diving cells in the granulation tissue. Epidermal regeneration was improved by 170% in rats receiving the KGF cDNA constructs by exhibiting the most rapid area and linear wound re-epithelialization, P < 0.0001. KGF improved epidermal cell net balance by increasing skin cell proliferation and decreasing skin cell apoptosis, P < 0.0001. Dermal regeneration was further improved in KGF cDNA treated animals by an increased collagen deposition and morphology, P < 0.0001. KGF cDNA increased neo-vascularization and concomitant VEGF concentrations when compared with vehicle, P < 0.01. KGF cDNA did not only stimulate epithelial cells, but also mesenchymal cells through increases in IGF-I concentration, P < 0.005. Liposomes containing the KGF cDNA gene constructs were effective in improving epidermal and dermal regeneration. KGF gene transfer to acute wounds may represent a new therapeutic strategy to enhance wound healing.

Enhancement of dermal and epidermal regeneration represents a crucial goal for the treatment of acute, e.g. burn and trauma wounds, and chronic wounds, e.g. diabetic, autoimmune, arterial and venous wounds. Studies defining molecular mechanisms of the complex cascade of wound healing have shown that growth factors represent a new therapeutic strategy. The clinical application of growth factors in the form of proteins has been shown to be of little benefit. Therefore new delivery systems and therapeutic strategies needed to be developed to improve dermal and epidermal regeneration, one of which is gene therapy. For successful gene delivery the selection of an appropriate vector has been shown to be paramount. Because Retroviruses, Adenoviruses and Adeno-Associated Viruses can cause immunologic reactions and mutations, non-viral delivery systems for gene therapy, such as liposomal gene transfer appear advantageous over viral gene therapy. This review discusses the success, potential and limitations of non-viral gene transfer to improve regeneration of dermal and epidermal structures.

Najath Abdul Kareem,1 Ayesha Aijaz,1 Marc G Jeschke1– 4 1Sunnybrook Research Institute, Toronto, ON, Canada; 2Department of Surgery, Division of Plastic Surgery, University of Toronto, Toronto, ON, Canada; 3Department of Immunology, University of Toronto, Toronto, ON, Canada; 4Ross Tilley Burn Centre, Sunnybrook Health Sciences Centre, Toronto, ON, CanadaCorrespondence: Marc G JeschkeRoss Tilley Burn Centre, Sunnybrook Health Sciences Centre, 2075 Bayview Ave, Rm D704, Toronto, ON, M4N 3M5, CanadaTel +1 416-480-6703Fax +1 416-480-6763Email marc.jeschke@sunnybrook.caAbstract: Burn injuries affect approximately 11 million people annually, with fatalities amounting up to 180,000. Burn injuries constitute a global health issue associated with high morbidity and mortality. Recent years have seen advancements in regenerative medicine for burn wound healing encompassing stem cells and stem cell-derived products such as exosomes and conditioned media with promising results compared to current treatment approaches. Sources of stem cells used for treatment vary ranging from hair follicle stem cells, embryonic stem cells, umbilical cord stem cells, to mesenchymal stem cells, such as adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and even stem cells harvested from discarded burn tissue. Stem cells utilize various pathways for wound healing, such as PI3/AKT pathway, WNT-β catenin pathway, TGF-β pathway, Notch and Hedgehog signaling pathway. Due to the paracrine signaling mechanism of stem cells, exosomes and conditioned media derived from stem cells have also been utilized in burn wound therapy. As exosomes and conditioned media are cell-free therapy and contain various biomolecules that facilitate wound healing, they are gaining popularity as an alternative treatment strategy with significant improvement in outcomes. The treatment is provided either as direct injections or embedded in a natural/artificial scaffold. This paper reviews in detail the different sources of stem cells, stem cell-derived products, their efficacy in burn wound repair, associated signaling pathways and modes of delivery for wound healing.Keywords: stem cells, burn injury, exosomes, conditioned media, wound healing