In Vitro Characterization of Variable Porosity Wound Dressing With Anti-Scar Properties

Abstract

Introduction: New options are needed to improve wound healing while preventing excessive scar formation. Temporary primary dressings are important options in topical wound management that allow the natural healing process. Methods: We evaluated a novel primary dressing consisting of a biosynthetic, variable porosity, matrix-containing gelatin and Aloe Vera extract and a derivative dressing coated with the anti-scarring agent salinomycin for their ability to promote cell growth, reduce myofibroblast formation, and regulate cytokine production. In addition, salinomycin-coated primary dressings were tested for antimicrobial activity. Results: Both primary wound dressings permitted cell growth and attenuated TGFβ-induced scar-forming myofibroblast formation. The primary wound dressings also reduced IL-6 production by 50%, IL-8 by 20%, MCP-1 by 75%, and GRO by 60% in human mesenchymal stem cells treated with TGFβ. Salinomycin coating of the dressing showed antimicrobial activity by preventing Staphylococcus aureus growth. Conclusions: Both primary wound dressings support the growth of human fibroblasts and stem cells, as well as reduce inflammatory cytokine production, demonstrating their potential to serve as temporary wound dressings.

Keywords: antimicrobial; excessive scarring; mesenchymal stem cell; myofibroblast; salinomycin.

Figure 1

The novel primary wound dressings reduce myofibroblast formation in human dermal fibroblasts. Human dermal fibroblasts were cultured with or without temporary wound dressings in the presence of TGFβ (5 ng/mL) for 5 days. Cells were then fixed and stained with AlexaFluor 594–conjugated phalloidin (yellow, binds and stains filamentous actin) and DAPI (blue, binds nucleic acids and stains cell nuclei) for fluorescence imaging. (a) Vehicle-treated cells; (b) TGFβ-treated cells; (c) TGFβ with dressing C; and (d) TGFβ with dressing A. Original magnification ×200.

Figure 2

The novel primary wound dressings reduce myofibroblast formation in human mesenchymal stem cells. Human adipose-derived mesenchymal stem cells were cultured with or without temporary wound dressings in the presence of TGFβ (5 ng/mL) for 5 days. Cells were then fixed and stained with AlexaFluor 594–conjugated phalloidin (yellow, binds and stains filamentous actin) and DAPI (blue, binds nucleic acids and stains cell nuclei) for fluorescence imaging. (a) Vehicle-treated cells; (b) TGFβ-treated cells; (c) TGFβ with dressing C; and (d) TGFβ with dressing A. Original magnification ×200.

Figure 3

Human mesenchymal stem cells express lower levels of proinflammatory cytokines when cultured with the novel primary wound dressings. Human mesenchymal stem cells were cultured in the presence or absence of dressing C or A for 4 days in the presence or absence of TGFβ before supernatants were collected and analyzed by Luminex for production of the inflammatory cytokines, IL-6 (a), MCP-1 (b), IL-8 (c), and GRO, also called CXCL1 (d). The assay was performed in triplicate, and data are presented as mean ± SEM (^*P < .05, ^**P < .01, ^***P < .001 compared with no membrane).

Figure 4

Human mesenchymal stem cells grow and are viable in the presence of the temporary wound dressings. Human adipose-derived mesenchymal stem cells were treated with vehicle (DMSO), IL-1β (100 pg/mL), or TGFβ (5 ng/mL) with Alamar blue reagent as described in the “Methods” section, and fluorescence was measured to quantify viability and cell growth. The assay was performed in triplicate, and data are presented as mean ± SEM. RFU indicates relative fluorescence unit.

Figure 5

Salinomycin-coated dressings have antimicrobial activity against S aureus. (a) Salinomycin (120 ng to 7.5 μg) was spotted onto 6-mm diameter filter discs. Vancomycin (30 μg) was used as a positive control. Discs were added to agar plates containing S aureus strain UAMS-1 and incubated at 37°C overnight. Zones of inhibition are labeled. (b, c) 12-mm diameter sections of dressing C were soaked with various concentrations of salinomycin (30 μg/mL to 7.5 mg/mL). Dressing C was then placed on an agar plates containing S aureus and incubated as previously. Dressing C without salinomycin showed no zone of inhibition, whereas a zone of inhibition was observed with dressing C coated with 230 μg/mL of salinomycin.