23 Chlorhexidine Delays Wound Healing in Human Skin

Abstract

Introduction Chlorhexidine (CHG) is ubiquitous in surgical perioperative care. In vivo studies of CHG cytotoxicity on human skin are lacking. Given the use of CHG for daily wound cares and as a presurgical scrub, including donor site preparation, we sought to identify if CHG cytotoxicity would persist in a clinically relevant in vivo human skin xenograft model. Methods Human skin tissues were obtained from elective surgeries. Partial thickness wounds were created ex vivo in human skin using a 4 mm punch biopsy. 2% CHG (treatment) or PBS (control) was applied to the wounds for 30 minutes followed by rinsing the tissue +/- mechanical disruptive irrigation. Tissues were cultured at the air-liquid interface for 24 hours in culture media after treatment and tissue viability was performed using an MTT assay. For in vivo studies, athymic mice (n=4) were grafted on bilateral flanks with human skin. Eight weeks after engraftment and normalization of skin architecture, 4 mm partial thickness wounds were created on each xenograft (2 per mouse – treatment and control). 2% CHG was applied daily for 2 minutes followed by irrigation with PBS in the treatment wound. The control wound received PBS application and irrigation. The xenografts received treatment daily for 14 days to mimic daily wound cares, and digital images were obtained to document presence of infection and gross wound healing. On day 14, the xenografts were harvested and stained for lactate dehydrogenase and H&E to assess cell viability and wound re-epithelization, respectively. Results An MTT assay on ex vivo human skin wounds showed that CHG treated groups (irrigation or non-irrigation) had lower cell viability compared to the PBS treated group, however irrigation mitigates the cytotoxicity of CHG on human skin. In the in vivo xenograft study, no signs of infection were identified in either PBS or CHG treated wounds throughout the study. The wound size appeared larger on gross inspection in the CHG treated group compared to the PBS group as early as day 2.Microscopically, the PBS treated wounds were fully re-epithelialized (n=2) or had significantly more re-epithelialization (n=2) than the CHG treated wounds (n=4) after 14 days of treatment. The PBS-treated wounds were viable throughout the tissue, indicating the irrigation procedure was not harmful to the cells. In the CHG-treated wounds, nonviable cells were observed in the dermis beneath the wound that was directly in contact with CHG suggesting penetration of CHG contributes to cytotoxicity in acute wounds. Conclusions Daily CHG use is cytotoxic to human skin and impedes wound healing.

In Vitro Evaluation of Mangostanin as an Antimicrobial and Biocompatible Topical Antiseptic for Skin and Oral Tissues

“…These results are consistent with our previous study, which demonstrated the lack of the cytotoxicity effect of modified HA combined with MGTN on the 3D model of the gingival tissue equivalent (Munar-Bestard et al; Mangostanin hyaluronic acid hydrogel as an effective biocompatible alternative to chlorhexidine, 2023; manuscript submitted for publication). In addition, in the commercial 3D model of oral mucosa epithelium, the treatment with HA 0.05% MGTN gel showed low IL-1α levels, contrary to the positive control or to the gels containing CHX, thus showing excellent biocompatibility with tissues, as observed for the Betadine and Prontosan gels, whereas Crystalmina gel that contains 1% CHX exhibited a higher cytotoxicity, in concordance with the literature. − The lack of effect in cell viability induced by the positive control on the 3D model of human epidermis can be explained by the keratinized external layer of this tissue and/or to the relatively low SDS concentration used in this assay, evidencing the low sensibility of the 3D model of human epidermis tissue for MTT . In contrast, comparing the effects of HA with 0.05% MGTN with the commercial periodontal gels, we observed that the commercial periodontal gels containing 0.2% CHX exhibited a higher cytotoxicity in the 3D oral mucosa model, in line with CHX effects .…”