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 beta 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 nonviral methods. Levels of hBD-3 mRNA and protein in three-dimensional skin tissue produced from NIKS(hBD) (-3) were determined using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Tissue architecture was characterized by hematoxylin and eosin staining and by indirect immunofluorescence 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 fivefold 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 Staphylococcus aureus multiple peptide resistance factor (mprF) compared with 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.

OBJECTIVE:The goal of this study was to assess the immunogenicity and antigenicity of StrataGraft skin tissue in a randomized phase I/II clinical trial for the temporary management of full-thickness skin loss.BACKGROUND:StrataGraft skin tissue consists of a dermal equivalent containing human dermal fibroblasts and a fully stratified, biologically active epidermis derived from Near-diploid Immortalized Keratinocyte S (NIKS) cells, a pathogen-free, long-lived, consistent, human keratinocyte progenitor.METHODS:Traumatic skin wounds often require temporary allograft coverage to stabilize the wound bed until autografting is possible. StrataGraft and cadaveric allograft were placed side by side on 15 patients with full-thickness skin defects for 1 week before autografting. Allografts were removed from the wound bed and examined for allogeneic immune responses. Immunohistochemistry and indirect immunofluorescence were used to assess tissue structure and cellular composition of allografts. In vitro lymphocyte proliferation assays, chromium-release assays, and development of antibodies were used to examine allogeneic responses.RESULTS:One week after patient exposure to allografts, there were no differences in the numbers of T or B lymphocytes or Langerhans cells present in StrataGraft skin substitute compared to cadaver allograft, the standard of care. Importantly, exposure to StrataGraft skin substitute did not induce the proliferation of patient peripheral blood mononuclear cells to NIKS keratinocytes or enhance cell-mediated lysis of NIKS keratinocytes in vitro. Similarly, no evidence of antibody generation targeted to the NIKS keratinocytes was seen.CONCLUSIONS:These findings indicate that StrataGraft tissue is well-tolerated and not acutely immunogenic in patients with traumatic skin wounds. Notably, exposure to StrataGraft did not increase patient sensitivity toward or elicit immune responses against the NIKS keratinocytes. We envision that this novel skin tissue technology will be widely used to facilitate the healing of traumatic cutaneous wounds.This study was registered at www.clinicaltrials.gov (NCT00618839).

When skin is compromised, a cascade of signals initiates the rapid repair of the epidermis to prevent fluid loss and provide defense against invading microbes. During this response, keratinocytes produce host defense peptides (HDPs) that have antimicrobial activity against a diverse set of pathogens. Using nonviral vectors we have genetically modified the novel, nontumorigenic, pathogenfree human keratinocyte progenitor cell line (NIKS) to express the human cathelicidin HDP in a tissue-specific manner. NIKS skin tissue that expresses elevated levels of cathelicidin possesses key histological features of normal epidermis and displays enhanced antimicrobial activity against bacteria in vitro. Moreover, in an in vivo infected burn wound model, this tissue results in a two log reduction in a clinical isolate of multidrug-resistant Acinetobacter baumannii. Taken together, these results suggest that this genetically engineered human tissue could be applied to burns and ulcers to counteract bacterial contamination and prevent infection.

Background: Large wounds often require temporary allograft placement to optimize the wound bed and prevent infection until permanent closure is feasible. We developed and clinically tested a second-generation living human skin substitute (StrataGraft). StrataGraft provides both a dermis and a fully-stratified, biologically-functional epidermis generated from a pathogen-free, long-lived human keratinocyte progenitor cell line, Neonatal Immortalized Keratinocytes (NIKS).Methods: Histology, electron microscopy, quantitative polymerase chain reaction, and bacterial growth in vitro were used to analyze-human skin substitutes generated from primary human keratinocytes or NIKS cells. A phase I/II, National Institute of Health-funded, randomized, safety, and dose escalation trial was performed to assess autograft take in 15 patients 2 weeks after coverage with StrataGraft skin substitute or cryopreserved cadaver allograft.Results: StrataGraft skin substitute exhibited a fully stratified epidermis with multilamellar lipid sheets and barrier function as well as robust human beta defensin-3 mRNA levels. Analysis of the primary endpoint in the clinical study revealed no differences in autograft take between wound sites pretreated with StrataGraft skin substitute or cadaver allograft. No StrataGraft-related adverse events or serious adverse events were observed.Conclusions: The major finding of this phase I/II clinical study is that performance of StrataGraft skin substitute was comparable to cadaver allograft for the temporary management of complex skin defects. StrataGraft skin substitute may also eliminate the risk for disease transmission associated with allograft tissue and offer additional protection to the wound bed through inherent antimicrobial properties. StrataGraft is a pathogen-free human skin substitute that is ideal for the management of severe skin wounds before autografting.