Comparative assessment of commercially available wound gels in ex vivo human skin reveals major differences in immune response-modulatory effects

Abstract

Wound healing is a crucial process for maintaining the function of human skin as a protective barrier to pathogens and other external stress factors. Hydrogels-in combination with antimicrobials-are often used, as moist wound care has been widely accepted as standard therapy. Recently, we reported about immune response-modulatory effects of an octenidine-based hydrogel, however little is known about the mechanism of action of other hydrogels including antiseptic molecules or chlorine-based and chlorine-releasing agents, respectively. The aim of this study was the comparative assessment of commercially available wound gels (octenilin^®, Prontosan^®, Lavanid^®, Betadona^®, ActiMaris^®, Microdacyn₆₀^®, Veriforte^TMmed) with regard to their effects on the secretion of distinct cytokines (IL-6, IL-8, IL-10), matrix-metalloproteinases as well as their potential to cause alterations in skin structure and apoptosis. Hence, tape-stripped human ex vivo skin biopsies were treated topically with wound gels and cultured for 48 h. Enzyme-linked immunosorbent assays and an enzyme activity assay of culture supernatants revealed that octenilin^® demonstrates significantly broader anti-inflammatory and protease-inhibitory capacities than other wound gels. Further, haematoxylin & eosin as well as caspase-3 staining of treated biopsies showed that octenilin^® does not alter skin morphology and shows the least interfering effect on human epidermal cells compared to untreated controls. Overall, this study clearly demonstrates totally different effects for several commercially available hydrogels in our wound model, which gives also new insight into their tissue compatibility and mode of action.

Figure 1

Quantification of cytokines in superficially wounded ex vivo skin treated with commercially available wound gels. Shown are (a) IL-6, (b) IL-8, (c) IL-10, (d) MMP2 concentration levels in culture supernatants of 48 h cultured human tape-stripped (TS) skin biopsies topically treated with indicated wound gels as well as a TS untreated control. Samples were analysed in duplicates with an enzyme-linked immunosorbent assay (ELISA). Data is presented as a mean ± standard deviation. A Wilcoxon matched-pairs signed rank test was performed with GraphPad Prism 9.3.1. *p ≤ 0.05; n = 6, female donors, location: abdomen, age range: 20–45 years (panel 1).

Figure 2

Not all wound gels inactivate MMPs in wounded human ex vivo skin. Shown are activated (a) MMP1, MMP2, MMP8, MMP9, MMP12, MMP14 (3 h activation), (b) MMP3, MMP10 (24 h activation) concentration levels in culture supernatants of 48 h cultured human tape-stripped (TS) skin biopsies treated with indicated wound gels as well as a TS untreated control. Samples were analysed in duplicates with an MMP activity assay. Relative fluorescence units (RFU) are indicated. Data is presented as a mean ± standard deviation. A Wilcoxon matched-pairs signed rank test was performed with GraphPad Prism 9.3.1. *p ≤ 0.05; n = 6, female donors, location: abdomen, age range: 20–45 years (panel 1).

Figure 3

Assessment of the skin structure and apoptosis upon treatment with wound gels. Representative images and zoom-ins of the boxed areas of haematoxylin and eosin (H&E)-stained tape-stripped (TS) sections from biopsies (age 38 years) either left untreated or treated with indicated wound gels using a light microscope. The presence of caspase-3 expressing cells was evaluated using a caspase-3 (CASP3) antibody and visualisation with a secondary Alexa Fluor™ 546 AB (red). Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm.

Figure 4

Numbers of caspase-3⁺ cells in wounded human ex vivo tissue upon wound gel treatment. Shown is the number of caspase-3⁺ cells in treated skin biopsies counted within 4 fields of view (FOV) per biopsy (tape-stripped (TS), untreated control and treated with Oct, Pro, Lav, Bet, Act, Mic or Ver) of six donors. FOVs are assessed as technical replicates and data is presented as a mean ± standard deviation. Two-way analysis of variance (ANOVA) was performed using GraphPad Prism 9.3.1. ***p ≤ 0.001, ****p ≤ 0.0001.