Construction of biomimetic human skin based on nanobiocomposite matrix containing human fibroblasts and keratinocytes
DOI:
https://doi.org/10.34019/2179-3700.2024.v24.46195Keywords:
Chitosan, Cellulose nanofibers, Nanotoxicology, Cell culture, MTTAbstract
The skin, as the body's primary barrier, represents a major route of exposure to potentially toxic substances. With the rapid expansion of nanotechnology, products containing nanomaterials are increasingly applied directly to the skin. Safety and toxicity testing of these products is commonly conducted in two-dimensional (2D) in vitro cultures or in animals. However, due to limitations in prediction, phylogenetic differences, and ethical-political pressures, the search for alternative methods has become essential. In this context, three-dimensional (3D) in vitro models emerge as a promising alternative, as they can overcome the barriers posed by other methods. The aim of this study was to construct a biomimetic skin composed of a nanocomposite matrix based on chitosan and cellulose nanofibers (NFC), containing human fibroblasts and keratinocytes. The matrices were synthesized by the casting method, dissolving 1% (w/v) low molecular weight chitosan in a 1% (v/v) glacial acetic acid solution. Subsequently, the chitosan matrices were combined with different concentrations of NFC (0, 100, and 1000 µg/mL). Regarding cell culture, both cell lines were grown on the matrices using DMEM medium supplemented with fetal bovine serum (10%) and antibiotics (1% penicillin/streptomycin), maintained in an incubator with 5% CO₂ at 37°C in a humidified atmosphere. Cell morphology was evaluated by light microscopy, and cellular metabolism by MTT assay. Microscopy results showed changes in cell morphology, suggesting better exploitation of 3D space by the cells. The MTT test revealed a reduction in fibroblast mitochondrial activity at 24 and 48 hours, and in keratinocytes after 24 hours, suggesting that cell-matrix interaction interferes with cellular metabolism. However, keratinocytes demonstrated a greater adaptive capacity, returning to metabolic levels similar to the control after 48 hours of exposure. The matrices synthesized by the casting method proved viable for cell culture, showing a significant influence on cellular metabolism. The results indicate that these matrices have great potential to support cell growth and modulate cellular responses. Therefore, new perspectives arise for their application in tissue engineering and regenerative medicine, as well as providing promising alternatives for in vitro toxicological testing, contributing to the development of more advanced and ethical methods.
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