cEpiderm, a Canine Skin Analog Suitable for In Vivo Testing Replacement

Skin is one of the organs most tested for toxicity and safety evaluation during the process of drug research and development and in the past has usually been performed in vivo using animals. Over the last few years, non-animal alternatives have been developed and validated epidermis models for human and rat skin are already available. Our goal was to develop a histotypical canine skin analog, suitable for non-animal biocompatibility and biosafety assessment. Canine keratinocytes were seeded in an air-lift culture using an adapted version of the CELLnTEC protocol. Corrosion and irritation protocols were adapted from human EpiSkinTM. For histological analysis, sample biopsies were fixed in neutral-buffered formalin, and paraffin slices were routinely processed and stained with hematoxylin and eosin. A canine multilayer and stratified epidermal-like tissue (cEpiderm), confirmed by histological analysis, was obtained. The cEpiderm tissue exhibited normal morphological and functional characteristics of epidermis, namely impermeability and an adequate response to stressors. The cEpiderm is a promising canine skin model for non-animal safety testing of veterinary pharmaceuticals and/or cosmetics, significantly contributing to reducing undesirable in vivo approaches. cEpiderm is therefore a valid canine skin model and may be made commercially available either as a service or as a product

Development of a canine skin analog

Background. Skin covers an extensive area of the body and comprises three major layers, subcutis, dermis, and epidermis. This organ is one of the most tested for toxicity and safety evaluation during the process of drug research and development, in past usually performed in vivo using animals. On the last years a concern of sustainable and eco-friendly approach has been developing, with the expansion of non-animal alternatives (Klicks et al.; Mathes et al.).A histotypical cell cultured-derived tissue may be used to replace animal testing and are imperative to avoid armful, drawn-out tests to assess chemicals for their capacity to erode, bother or sensitize the skin. There are validated epidermal models for human (EPISKINTM and EpiDermTM) and rat (TER) skin (Flaten et al.; NIEHS; Netzlaff et al.). The aim of our study was the development of a histotypical canine skin equivalent, that can be used for the assessment of corrosion, irritation and sensibilization, avoiding in vivo animal testing. Methods. Canine keratinocyte progenitor cells were seeded in inserts and were allowed to grow until differentiation was reached, using an adapted version of the CELLnTEC commercial protocol, specific for human cells (CELLnTEC). For histological analysis, samples were fixed in 10% neutral-buffered formalin. Three-micrometer paraffin sections were routinely processed for biopsies and stained with hematoxylin and eosin. Corrosion, irritation and sensibilization protocols were adapted from human equivalent validated tests. Results. A multilayer (3-4 cell layers thick) of canine keratinocytes was developed in air-lift culture, originating a stratified epidermal-like tissue, confirmed by histological analysis. This epidermal-like tissue exhibited functional characteristics of normal epidermis. It showed adequate impermeabilization, after 0.1% Triton X-100 exposure for 4h and responded adequately to the positive (5% SDS and glacial acetic acid) and negative (PBS) controls used in “in vitro” corrosion and irritation assessment. Conclusions. As predicted, a canine skin analog was developed. This is a promising skin model for non-animal safety tests of veterinary pharmaceuticals or cosmetics, reducing in vivo testing, and can be commercialized as a service or a product

Development of a canine epidermis equivalent model for evaluation of sensitization

Background Canine atopic dermatitis (cAD) is a genetic-predisposed allergic and pruritic inflammatory skin condition, associated with sensitization to environmental allergens. Keratinocytes can produce several inflammatory mediators, in response to several other host mediators, antigens, and pathogens by virtue of their wide range of surface receptors, some of them dysregulated in cAD individuals. A histotypical cell cultured-derived tissue may be used to replace animal testing and are imperative to avoid armful, drawn-out tests to assess chemicals for their capacity to disrupt skin or cause sensitization. The aim of our study was to develop a histotypical canine epidermis equivalent, that could be used for the assessment of skin irritation and sensitization, useful in studies of cAD pathogeny and pharmacology. Methods Canine keratinocytes progenitor cells were seeded in air-lift culture, using an adapted version of the CELLnTECTM protocol. Irritation and sensitization protocols were adapted from human equivalent validated tests. For histological analysis, samples fixed in neutral-buffered formalin and paraffin sections were routinely processed for biopsies and stained with hematoxylin and eosin. Results A multilayer (3-4 cell-layer thick) of canine keratinocytes was developed in air-lift culture, originating a stratified epidermal-like tissue, confirmed by histological analysis. This epidermal-like tissue exhibited functional characteristics of the normal epidermis, showing an adequate impermeabilization after 0.1% Triton X-100 exposure for 4h. Additionally, the epidermis model responded adequately to the positive [5% SDS, 20% salicylic acid in AOO (acetone and olive oil 4:1)] and negative (PBS and AOO) controls of the irritation and sensitization tests, assessed by the quantification of cellular viability and of the secreted IL-18. Conclusion As predicted, a canine epidermis analog was developed. This is a promising canine epidermis model that can be used for the study of skin-derived cAD triggering factors, and in the development and evaluation of new drugs for topical applications. Acknowledgements: Project co-financed by the European Union Fund – Portugal 2020, Alentejo 2020 – Ref. 03/SI/2017 (ALT20-03-247-FEDER-033578)

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

CENTR(AR): pulmões em andamento: manual de atividade física domiciliário

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