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

Abstract

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.

Figures

Fig. 1

Human β-defensin-3 (hBD-3) expression is elevated at the mRNA level in NIKS skin substitute. RNA was isolated from skin substitutes generated using GS-1-Ep, BC-1-Ep, and NIKS keratinocytes and relative hBD-3 mRNA levels were determined by qPCR. Equal amounts of input RNA were used and cyclophilin RNA levels served as internal reaction controls. hBD-3 expression was normalized to endogenous cyclophilin mRNA levels and then to that of NHEK cultures. Data represent three independent experiments. Statistical significance was determined using one-way ANOVA with Dunnett’s post-hoc test for multiple comparisons against a single reference (NIKS).

Fig. 2

NIKS tissue inhibits bacterial growth. Shown are the relative colony forming units (CFU)/mL of Staphylococcus carnosus incubated with submerged skin substitute tissues made from GS-1-Ep, BC-1-Ep, and NIKS keratinocytes. The average number of viable bacteria from three independent experiments is presented as the percentage of CFU/mL relative to the GS-1-Ep tissue. Statistical significance was determined using one-way ANOVA with Dunnett’s post-hoc test for multiple comparisons against a single reference (NIKS).

Fig. 3

StrataGraft skin tissue. Panel A shows an intact StrataGraft skin substitute which has a surface area of 44 cm2. Meshed StrataGraft skin substitute (1:1 ratio) is shown in Panel B.

Fig. 4

Tissue morphology and lipid lamellae in StrataGraft skin substitute. (A) Tissue sections were fixed, embedded, and stained with hematoxylin and eosin. StrataGraft substitute consists of an epidermal layer attached to a dermal equivalent containing fibro-blasts. Within the epidermis, a single layer of highly ordered basal keratinocytes lies below several layers of spinous cells. Between the compact stratum corneum and the spinous cells is a layer of distinct granular cells, with prominent intracellular keratohyalin granules. Magnification = ×200, scale bar = 200 μm. (B) Stratum corneum lipid lamellae from StrataGraft skin substitute were visualized by electron microscopy using ruthenium tetroxide. Lamellar structures with an alternating electron-dense and lucent banding pattern were observed. Magnification = ×420,000, scale bar = 100 nm.

Fig. 5

Visual appearance of StrataGraft skin substitute in the wound bed. One week after placement in the wound bed, the StrataGraft skin substitute (upper portion of the wound bed) appears viable and adherent to the wound in comparison to the cadaver allograft (lower portion of the wound bed) in the same wound bed. Note that the translucent nature of StrataGraft skin substitute allows for visualization of the wound bed, in contrast to the opaque appearance of the cadaver allograft.