StrataGraft skin substitute is well-tolerated and is not acutely immunogenic in patients with traumatic wounds: results from a prospective, randomized, controlled dose escalation trial

Abstract

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).

Figures

Figure 1

In vitro-generated tissues have characteristics comparable to those of native skin. (A) H&E stained sections from NIKS and NHEK skin substitutes, and human neonatal foreskin tissue, exhibit typical epidermal morphology, containing a single layer of basal cells, beneath stratified spinous, granular, and cornified layers. Scale bar = 50μm. (B) Barrier function was measured using a Nova DermaPhase meter (DPM) which measures skin surface electrical impedance in arbitrary units. DPM values reflect electrical impedence and are inversely related to surface hydration, therefore, lower DPM values indicate a dryer surface. Tissues made from NIKS (StrataGraft) or NHEK exhibit barrier function comparable to that of native human skin. The values obtained for intact skin and skin where the barrier function has been disrupted by tape stripping are presented as controls. Data represent mean ± SEM. (C) Antibodies specific to type I transglutaminase (TG), filaggrin, and collagen IV (Coll IV) were used to examine the differentiation pattern present in NIKS and NHEK tissues and human neonatal foreskin. Grey scale images for each protein examined are shown. Color-merged images display sections counterstained with Hoechst 33258 to visualize nuclei (blue). Scale bars = 50μm. (D) Expression of MHC antigens was also assessed (grey scale), with color-merged images depicting HLA-ABC in red, HLA-DR in green, and Hoechst 33258 in blue. Scale bars = 50 μm.

Figure 2

Overview of clinical trial design. Fifteen patients with full-thickness skin defects of ≥5% total body surface area were surgically debrided and wound beds were covered with StrataGraft and cadaver allografts placed adjacent to each other. Allografts were removed after one week and processed for histologic analysis. Patients were autografted when the wound beds were deemed by the clinician to be suitable for autografting. The primary efficacy endpoint was the percent of autograft take two weeks after autograft placement. A panel of secondary safety and efficacy analyses were performed at baseline, one week after allograft placement, and after three months.

Figure 3

StrataGraft skin substitute remains intact and viable after placement in the wound bed. (A) Representative paraffin-embedded H&E-stained sections of StrataGraft and cadaver allografts after removal from the wound bed of two different patients. Scale bar = 50 μm. (B). Representative images of StrataGraft and cadaver allograft samples stained for Ki67 expression after removal from the wound beds of two different patients reveal Ki67 staining of viable cells present in basal and suprabasal layers (brown). Scale bar = 50 μm. (C) Sections were assessed by two blinded observers and basal keratinocytes were scored as positive or negative for Ki67 staining. At least 400 cells from each sample were counted and the proliferation index (PI) (ratio of positive cells to the total cells counted) was determined. Shown are paired StrataGraft and cadaver allograft sample PI values from eight individual patients. The bar indicates the median value. McNemar’s test, P = 0.13.

Figure 4

StrataGraft skin substitute does not exhibit acute inflammatory infiltrates. Representative images of StrataGraft and cadaver allografts removed from the wound beds of two different patients, stained (brown) for CD3 to indicate T cell infiltrates, CD20 as a marker of B lymphocytes, and CD1α to identify Langerhans cells. Scale bars = 100 μm.

Figure 5

HLA-ABC, but not HLA-DR, is expressed in the basal and suprabasal epidermal keratinocytes of both StrataGraft and cadaver allografts after placement in the wound bed. The expression and localization of HLA-ABC and HLA-DR was assessed by immunofluorescence. Shown are representative grey scale and color images of post-placement StrataGraft and cadaver skin allografts stained for HLA-ABC (A; shown in red) and HLA-DR (B; shown in green) after removal from the wound beds of 2 different patients. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar = 100 μm.