Effect of a DACC-coated dressing on keratinocytes and fibroblasts in wound healing using an in vitro scratch model

Abstract

Wound dressings that exert an antimicrobial effect in order to prevent and treat wound infections can be harmful to the wound healing process. Dressings with hydrophobic coatings, however, have been suggested to both reduce the microbial load and promote the healing process. Therefore, the potential effects of a dialkylcarbamoyl chloride (DACC)-coated dressing on fibroblasts and keratinocytes in wound healing were studied using mechanical scratch wounding of confluent cell layers as an in vitro model. Additionally, gene expression analysis by qRT-PCR was used to elucidate the longitudinal effects of the DACC-coated dressing on cell responses, specifically inflammation, growth factor induction and collagen synthesis. DACC promoted cell viability, did not stick to the cell layers, and supported normal wound healing progression in vitro. In contrast, cells became attached to the uncoated reference material, which inhibited scratch closure. Moreover, DACC slightly induced KGF, VEGF, and GM-CSF expression in HaCaT cells and NHDF. Physiological COL1A1 and COL3A1 gene expression by NHDF was observed under DACC treatment with no observable effect on S100A7 and RNASE7 levels in HaCaT cells. Overall, the DACC coating was found to be safe and may positively influence the wound healing outcome. Graphical abstract.

Graphical abstract

Fig. 1

Determination of the influence of the stress control, DACC-coated dressing, and uncoated RM on HaCaT cell viability (A) and NHDF viability (B) compared to the untreated (medium) control using the MTT test

Fig. 2

Comparison of NHDF adherence to the DACC-coated dressing samples and uncoated RM determined by measurement of the cellular ATP-content using the luminometric ATP assay. Asterisks indicate a significant deviation between DACC-coated dressing and uncoated RM (*p < 0.05; **p < 0.01; ***p < 0.001), while n.s. designates “not significant”

Fig. 3

Progression of HaCaT scratch healing (A) in the untreated (medium) control (B) and the stress control (C) as well as under treatment with DACC (D) and untreated RM (E) after 1, 8, 24, and 48 hours (h). Asterisks indicate a significant deviation at the respective time point from the untreated control (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 4

Progression of NHDF scratch healing (A) in the untreated (medium) control (B) and the stress control (C) as well as under treatment with DACC (D) and untreated RM (E) after 1, 8, 24, and 48 hours (h). Asterisks indicate a significant deviation at the respective time point from the untreated control (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 5

Treatment impact of DACC and uncoated RM on gene expression in keratinocytes (HaCaT cells) and fibroblasts (NHDF) during scratch wound healing compared to the untreated (medium) control and the stress control. Quantitative analyses of gene expression for the pro-inflammatory cytokine genes IL1A, IL6, CXCL8, and CXCL1 were performed at 4, 8, 24 and 48 hours (h). Relative gene expression was normalized to the housekeeping gene ACTB. Data are presented as—fold gene expression compared to the untreated (medium) control at the start of the experiment. Asterisks indicate a significant deviation at the respective time point from the untreated control (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 6

Influence of DACC and uncoated RM on gene expression in keratinocytes (HaCaT cells) and fibroblasts (NHDF) during scratch wound healing compared to the untreated (medium) control and the stress control. Quantitative analyses of gene expression for the growth factor genes KGF, TGFB1, FGF2, VEGF, GM-CSF, PDGFA, PDGFC, and EGF were performed at 4, 8, 24, and 48 hours (h). Relative gene expression was normalized to the housekeeping gene ACTB. Data are presented as—fold gene expression compared to the untreated (medium) control at the start of the experiment. n.d.—expression of EGF was not determined for HaCaT cells as keratinocytes do not produce this growth factor. Asterisks indicate a significant deviation at the respective time point from the untreated control (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 7

Gene expression of antimicrobial peptides under treatment with DACC and untreated RM during HaCaT scratch wound healing compared to the untreated (medium) control and the stress control. Quantitative analyses of gene expression for the antimicrobial peptide genes S100A7 and RNASE7 were performed at 4, 8, 24, and 48 hours (h). Relative gene expression was normalized to the housekeeping gene ACTB. Data are presented as—fold gene expression compared to the untreated (medium) control at the start of the experiment. Asterisks indicate a significant deviation at the respective time point from the untreated control (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 8

Collagen gene expression under treatment with DACC and uncoated RM during NHDF scratch wound healing compared to the untreated (medium) control and the stress control. Quantitative analyses of gene expression for collagen genes COL1A1 and COL3A1 were performed at 4, 8, 24, and 48 hours (h). Relative gene expression was normalized to the housekeeping gene ACTB. Data are presented as—fold gene expression compared to the untreated (medium) control at the start of the experiment. Asterisks indicate a significant deviation at the respective time point from the untreated control (*p < 0.05; **p < 0.01; ***p < 0.001)