Non-viral human beta defensin-3 expression in a bioengineered human skin tissue: a therapeutic alternative for infected wounds

The innate immune system differentially regulates the expression of host defense peptides to combat infection during wound healing. We enhanced the expression of a host defense peptide, human β defensin-3 (hBD-3), in keratinocytes to generate a three-dimensional biologic dressing to improve healing of infected wounds. The NIKS human keratinocyte cell line was stably transfected ex vivo with a construct containing an epidermis-specific promoter driving hBD-3(NIKS hBD-3 ) using non-viral methods. Levels of hBD-3 mRNA and protein in three-dimensional skin tissue produced from NIKS hBD-3 were determined using quantitative PCR and ELISA, respectively. Tissue architecture was characterized by hematoxylin and eosin staining, and by indirect immunofluorence using proliferation and keratinocyte differentiation markers. Antimicrobial activity was assessed using an in vitro bacterial growth assay and in vivo using a murine burn infection model. Three-dimensional full thickness skin tissues containing epidermal NIKS hBD-3 or control NIKS possessed histologic features of interfollicular epidermis and exhibited normal tissue growth and differentiation. NIKS hBD-3 tissue contained approximately 5-fold more hBD-3 protein than tissue containing unmodified control NIKS. In vitro studies showed that NIKS hBD-3 tissue produced a significant reduction in the growth of Staphylococcusaureus ( S. aureus) mprF compared to control tissue. In an in vivo infected murine burn model, NIKS hBD-3 tissue resulted in a 90% reduction in bacterial growth. These results demonstrate that sustained delivery of hBD-3 by a bioengineered skin tissue results in a therapeutically relevant reduction in growth of a S. aureus strain in an animal model of infected third degree burn wounds.