A versatile, bioengineered skin reconstruction device designed for use in austere environments

Abstract

Austere environments in which access to medical facilities, medical personnel, or even water and electricity is limited or unavailable pose unique challenges for medical device product design. Currently existing skin substitutes are severely inadequate for the treatment of severe burns, chronic wounds, battlefield injuries, or work-related injuries in resource-limited settings. For such settings, an ideal device should be biocompatible, bioresorbable, promote tissue healing, not require trained medical personnel for deployment and use, and should enable topical drug delivery. As proof of concept for such a device, silk fibroin and an antioxidant hyaluronic acid derivative were chosen as primary constituents. The final formulation was selected to optimize tensile strength while retaining mechanical compliance and protection from reactive oxygen species (ROS). The ultimate tensile strength of the device was 438.0 KPa. Viability of dermal fibroblasts challenged with ROS-generating menadione decreased to 49.7% of control, which was rescued by pre-treatment with the hyaluronic acid derivative to 85.0% of control. The final device formulation was also tested in a standardized, validated, in vitro skin irritation test which revealed no tissue damage or statistical difference from control. Improved topical drug delivery was achieved via an integrated silk fibroin microneedle array and selective device processing to generate crosslinked/through pores. The final device including these features showed a 223% increase in small molecule epidermal permeation relative to the control. Scaffold porosity and microneedle integrity before and after application were confirmed by electron microscopy. Next, the device was designed to be self-adherent to enable deployment without the need of traditional fixation methods. Device tissue adhesive strength (12.0 MPa) was evaluated and shown to be comparable to a commercial adhesive surgical drape (12.9 MPa) and superior to an over-the-counter liquid bandage (4.1 MPa). Finally, the device's wound healing potential was assessed in an in vitro full-thickness skin wound model which showed promising device integration into the tissue and cellular migration into and above the device. Overall, these results suggest that this prototype, specifically designed for use in austere environments, is mechanically robust, is cytocompatible, protects from ROS damage, is self-adherent without traditional fixation methods, and promotes tissue repair.

Keywords: austere environment; biomaterial; hyaluronic acid; microneedles; silk fibroin; skin reconstruction device; wound healing.

FIGURE 1

Infographic of the device assembly procedure. MN array template was manufactured with photopolymer resin on a 2PP 3D printer. A PDMS negative mold of this template was cast and used cast SF/PVA MN arrays. Separately, a SF/HAM solution is frozen and lyophilized to form a 1 mm thick scaffold. The scaffold and MN array are bonded together, then this assembly is rendered water insoluble by inducing β-sheets in SF. Finally, SF adhesive is aerosol coated onto the surface of the MNs.

FIGURE 2

(A) SEM micrograph of SF/PVA MN array. (B) SEM micrograph of SF/HAM scaffold surface and partial cross section. (C) Photograph of hydrated device showing MN surface and (D) demonstrating the devices’ translucent property allowing it to inherit the underlying colors.

FIGURE 3

Mechanical properties of scaffold and device. (A) UTS of scaffold made from solutions containing varying concentrations of SF with 0.2% HAM. (B) UTS of scaffold from solutions containing 12% SF and varying concentrations of HAM. (C) UTS of the final formations of the scaffold only (black) compared to the complete device (pink). (D) Representative stress-strain curve for scaffold only (black) compared to the complete device (pink). (A,B) n = 8; one-way ANOVA with Dunnett’s correction for multiple comparisons; *p < 0.05, ***p < 0.001, ****p < 0.0001. (C) n = 8; 2-sided t-test; ****p < 0.0001. NS, not significant.

FIGURE 4

(A) ROS protection of fibroblasts by treatment with 2 mg mL-1 HAM or equivalent concentrations of CMHA alone, Dmet alone, or a blend of CMHA + Dmet. Followed by treatment with ROS-generating menadione (indicated by grey fill). n = 4. (B) Skin irritation test of NC (vehicle), PC (5% SDS), and final device formulation (25 mg, powdered) performed in 3D in vitro human epidermis. Dotted line shows cutoff threshold indicating whether test compound is considered a possible irritant. n = 3. (A,B) Mean +SD; one-way ANOVA with Tukey’s correction for multiple comparisons vs. NC (blue) or PC (red); **p < 0.01, ***p < 0.001, ****p < 0.0001. NC, negative control; NS, not significant; PC, positive control; SDS, sodium dodecyl sulfate.

FIGURE 5

Fibroblast migration in SF/HAM scaffold. Representative immunofluorescent confocal micrographs of SF/HAM scaffolds cultured for 2 (top row) or 7 (bottom row) days following initial seeding with a 5 µL drop of primary fibroblasts directly onto the top center surface of the scaffold. Red circle shows approximate initial seeding area to illustrate extent of migration. Scale bar applies to all images. DAPI and auto-fluorescent SF (blue); vimentin staining identifies fibroblasts (green).

FIGURE 6

Device adhesive strength. Adhesive tensile strength of SF adhesive-coated complete device compared to a commercial surgical isolation drape and liquid bandage bonded to chamois leather (as skin analog) and pulled apart perpendicularly. n = 6; Mean ± SD; one-way ANOVA with Dunnett’s correction for multiple comparisons; **p < 0.01.

FIGURE 7

Microneedle skin penetration, drug permeability, and porosity. (A) Cross-sectional micrograph of RHE penetrated by SF/PVA MNs. (B) SEM micrograph mosaic of RHE surface following MN insertion and removal. Red circles highlight perforations caused by microneedle array; yellow inlay zooms in on a single perforation. SEM micrograph of SF/PVA MN film surface before (C) and after (D) being inserted in RHE for 24 h. (E) Cumulative permeated FDS at each timepoint and (F) percent of total applied FDS permeated over 24 h after application to the surface of each sample consisting of scaffold only (S; blue), scaffold with MN (S + MN; red), or scaffold with MN and SF adhesive coating (S + MN + A; green), which had been applied to the surface of an RHE. n = 4; one-way ANOVA with Tukey’s correction for multiple comparisons; *p < 0.05; ***p < 0.001. FDS, fluorescein disodium; NS, not significant.

FIGURE 8

Device wound healing in vitro skin model. (A) Photograph of wounded human skin equivalents (3 mm full-thickness wound) treated with reconstruction devices at the start of the wound healing experiment (day 0). Micrographs of sectioned and hematoxylin and eosin stained full-thickness human skin equivalent which was wounded with a 3 mm biopsy punch, then left untreated as a control (B) or treated with a skin reconstruction device (C), and allowed to heal for 20 days. (D) Immunofluorescent micrograph of wounded human skin equivalent treated with skin reconstruction device. DAPI and auto-fluorescent SF (blue); vimentin staining identifies fibroblasts (green); keratin 14 staining identifies keratinocytes (red).