TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin.

The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial-stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin.

Nonviral 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 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.

Chimeric composite skin substitutes for delivery of autologous keratinocytes to promote tissue regeneration.

OBJECTIVE: We hypothesize that the pathogen-free NIKS human keratinocyte progenitor cell line cultured in a chimeric fashion with patient's primary keratinocytes would produce a fully stratified engineered skin substitute tissue and serve to deliver autologous keratinocytes to a cutaneous wound. SUMMARY OF BACKGROUND DATA: Chimeric autologous/allogeneic bioengineered skin substitutes offer an innovative regenerative medicine approach for providing wound coverage and restoring cutaneous barrier function while delivering autologous keratinocytes to the wound site. NIKS keratinocytes are an attractive allogeneic cell source for this application. METHODS: Mixed populations of green fluorescent protein (GFP)-labeled NIKS and unlabeled primary keratinocytes were used to model the allogeneic and autologous components in chimeric monolayer and organotypic cultures. RESULTS: In monolayer coculture, GFP-labeled NIKS had no effect on the growth rate of primary keratinocytes and cell-cell junction formation between labeled and unlabeled keratinocytes was observed. In organotypic culture employing dermal and epidermal compartments, chimeric composite skin substitutes generated using up to 90% GFP-labeled NIKS exhibited normal tissue architecture and possessed substantial regions attributable to the primary keratinocytes. Tissues expressed proteins essential for the structure and function of a contiguous, fully-stratified squamous epithelia and exhibited barrier function similar to that of native skin. Furthermore, chimeric human skin substitutes stably engrafted in an in vivo mouse model, with long-term retention of primary keratinocytes but loss of the GFP-labeled NIKS population by 28 days after surgical application. CONCLUSIONS: This study provides proof of concept for the use of NIKS keratinocytes as an allogeneic cell source for the formation of bioengineered chimeric skin substitute tissues, providing immediate formal wound coverage while simultaneously supplying autologous cells for tissue regeneration.

Phase I/II clinical evaluation of StrataGraft: a consistent, pathogen-free human skin substitute.

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.

Inhibition of multidrug-resistant Acinetobacter baumannii by nonviral expression of hCAP-18 in a bioengineered human skin tissue.

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, pathogen-free 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.

Comparison of therapeutic antibiotic treatments on tissue-engineered human skin substitutes.

For regenerative medicine to gain clinical acceptance, the effects of commonly used treatment regimens on bioengineered organs must be considered. The antibiotics mafenide acetate (mafenide) and neomycin plus polymyxin (neo/poly) are routinely used to irrigate postoperative skin grafts on contaminated wounds. The effects of these clinically used antibiotics were investigated using tissue-engineered human skin substitutes generated with primary human keratinocytes or the near-diploid human keratinocyte cell line, Near-diploid Immortal Keratinocytes. Following topical or dermal treatment, the skin substitutes were assayed for viability, tissue morphology, glycogen content, and the expression of active caspase 3. Mafenide, but not neo/poly, induced morphological and biochemical changes in tissue-engineered skin substitutes. Keratinocytes in all histological layers of mafenide-treated skin substitutes exhibited ballooning degeneration and glycogen depletion. Mafenide-treatment also triggered separation of basal keratinocytes from the underlying dermis. None of the antibiotic treatments induced apoptosis, as measured by active caspase 3 immunostaining. The results demonstrate that mafenide, but not neo/poly, is detrimental to the viability and structural integrity of tissue-engineered human skin substitutes. These findings highlight the need to identify treatment regimens that are compatible with and hence enable the therapeutic efficacy of first-generation bioengineered organs such as skin.