Biocompatibility and biosafety analysis of chitosan hydrogels using organotypic epidermal models

Background: Frequent dog consultations are due to skin wounds (Holland, 2019), with different trauma causes, like bites, burns and others. The majority of these wounds are not fatal, but cause long-lasting discomfort, demanding frequent medical care which may affect owners financially (Fahie & Shettko, 2007). Despite several pharmaceuticals available for dog wound treatment, new antibiotic-free efficient options, also more ecological are needed. Chitosan hydrogels offer a promising solution. Besides, being very ecological and biodegradable, chitosan hydrogels allow the maintenance of a moist environment that assists the exchange of fluids, essential for wound healing, and can also incorporate agents to avoid the development of infectious agents (Stashak, Farstvedt, & Othic, 2004). The aim of this study was to screen chitosan-based hydrogels for veterinary applications, supplied by the company BrInova Biochemistry as part of a collaborative project (NAQUIBIO DPSA). Screening was based on toxicity, biosafety, and efficacy “in vitro” tests, using organotypic epidermal models. Methods: Epidermal canine keratinocytes and human fibroblasts primary cells (supplied by CELLnTEC) were seeded in flasks and incubated to reach a desired level of growth, as indicated by the supplier. For toxicity evaluation, both cell lines were used. Viability tests were performed in 96 wells cell-culture plaques. Culture media and Triton X-100 was used as negative and positive controls, respectively. Firstly, hydrogels and potentially useful additives were tested individually, in several concentrations. Nontoxic components and doses were used in the production of several hydrogel-based composites, subsequentially tested. Viability was accessed by dehydrogenase activity (CCK-8, Sigma-Aldrich). For the efficacy of the hydrogel-based composites a wound scratch (WS) cellular assay was used, applied to both cell lines. For biosafety tests, a 3D keratinocyte culture was prepared, and irritation, corrosion, and sensitization protocols (adapted of EPISKINTM) were used. Results: Toxicity testes allowed the selection of 4 hydrogels, 2 nanoparticles and 2 plant essential oils to use in hydrogel-based composites, as well as the suitable concentrations range for each. Based on that information, 16 composites were prepared by other members of the NAQUIBIO team, and their toxicity was also evaluated, allowing the selection of 6 hydrogel-based composites. Those composites were evaluated for efficacy by a WS assay and the best compounds were those that could induce a fast closure of the scratch gap. The intersection of the toxicity and efficacy results allowed the selection of one hydrogel-based composite as the most promising one, whose biosafety was later evaluated. This proved to be non-corrosive, non-toxic and non-sensitizing. This compound was selected to proceed to the animal testing phase, in accordance with the goals of the NAQUIBIO project. Conclusions: As initially planned, we were able to select a mixture of hydrogels with essential oils and nanoparticles, to be further in vivo evaluated in dogs, or more specifically we have in advanced development a new wound dressing for veterinary use, capable of accelerating and creating better conditions for wound healing.