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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Sierra Sánchez, Álvaro (Cell Production and Tissue Engineering Unit, Virgen de las Nieves University Hospital, 18014, Granada, Spain)


"Introduction: In recent years new therapies, such as skin cell lines injection and bioengineered autologous skin substitutes (BASS) technology, have emerged to promote re-epithelialization of damaged areas such as cutaneous wounds and ulcers or to treat patients with severe burns. Antiseptics are commonly used during wound healing to avoid serious infections, but some of them could have the opposite effect by delaying the healing process due to their apparent cytotoxicity against skin cells. Further studies on viability against standard procedures for wound healing and treatment protocol optimization are necessary for the improvement of BASS technology for clinical use. The aim of this study is to evaluate the effect of common antiseptics for clinical use in human fibroblasts and keratinocytes cell lines and in BASS, focusing on cell proliferation and viability, wound closure evaluation and inflammatory cytokine secretion as well as, epithelium and skin barrier integrity, in order to establish the least harmful treatment for wound care.

Methodology: The cytotoxicity of five antiseptics (ethanol, chlorhexidine digluconate, sodium hypochlorite, povidone iodine and polyhexanide) was evaluated in this in vitro study on human fibroblasts and keratinocytes and on BASS. Treatments were applied every 48 hours for 14 days to each cell type culture and for 16 days to BASS. To determine the cytotoxicity of the different antiseptics on skin cell lines, cell viability (Live/Dead®) and cell proliferation (AlamarBlue™) assays were performed on cell monolayers. The impact on cell migration capacity was also evaluated with a wound closure assay. For BASS, keratinocytes and dermal fibroblasts were isolated from skin samples and integrated into hyaluronic acid-based substitute. To determine the impact of treatments on BASS, cell viability (Live/Dead®), cytokine secretion (ELISA) and skin barrier integrity (Transepidermal Water Loss, TEWL) were evaluated.

Results: Chlorhexidine digluconate and ethanol significantly reduced the viability of keratinocytes and also inhibited cell migration compared to other treatments. For fibroblasts, povidone iodine followed by chlorhexidine digluconate significantly reduced cell viability. Wound closure assay showed that povidone iodine also inhibited cell migration. Likewise, sodium hypochlorite was the least detrimental to both cell types. Regarding BASS treatment, sodium hypochlorite was the only treatment that showed a high cell viability percentage throughout the evaluation time compared to other antiseptic treatments, as well as a similar cytokine secretion pattern as control BASS. No significant differences were found regarding epidermal barrier function.

Conclusions: This study revealed how antiseptics commonly used in clinical practice have high cytotoxicity on skin cell lines even at low concentrations, which can slow down the healing process by affecting cell viability and migration capacity. If the epithelial integrity is affected, the wound healing process can be altered, so the information gathered in this study may be useful in selecting the most suitable antiseptic after treatment with new emerging therapies to prevent infections. Interestingly, the findings of this research point towards sodium hypochlorite being the most suitable antiseptic treatment for BASS post-transplantation wound care."

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