Treatment of Skin Wounds with Cell Transplantation and Gene Transfer

Sammanfattning: Background: A large number of different wound coverings have been used in order to improve the wound microenvironment, and thus accelerate repair. Since almost two centuries, clinicians and researchers have developed techniques for skin transplantation. In the past several decades, growth factors have been used as tools for accelerating and modifying skin wound repair. The experimental work in this thesis has the goal of improving wound repair in these areas. Methods: In a porcine wound model, flexible transparent wound chambers containing saline or a growth medium with antibiotics were used in all experiments, either alone or compared to other dressings. Transplantation of suspensions of keratinocytes and fibroblasts was studied and compared to other transplantation methods. A tetracycline-inducible regulatory switch for controlling transgene expression was developed and tested in vitro and in vivo. Results: Wound healing in a liquid environment was faster as compared to dry and moist conditions. Transplantation of wounds with suspensions of keratinocytes or fibroblasts further accelerated repair. Transfection with beta-galactosidase confirmed that the transplanted cells could regenerate skin. Cotransplantation of keratinocytes and fibroblasts resulted in synergistic effects on epidermal repair. Transplantation of hEGF gene transfected fibroblasts resulted in significant hEGF expression in tissue and wound fluid. By transplanting cells that had been transfected with the combinations of the tetracycline-operator/hEGF genes and the tetracycline repressor gene, we showed that EGF expression in wounds may be precisely regulated by tetracycline. Conclusions: It is concluded that the use of the wound chamber allows for the addition of a liquid and transplanted cell suspensions, both of which accelerate healing. The feasibility of targeted regulated delivery of growth factors with ex vivo gene transfer incorporating a tetracycline switch was demonstrated. This provides a very precise tool for the delivery of therapeutic proteins.