Advanced human 3D-bioprinted skin constructs to model Inflamm’aging
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Presented by: Laure Vert
Throughout its life, human skin is constantly undergoing aggression from different origins, which cumulatively adds to its physiological aging. Exposed daily to these various physical or chemical stimuli, an inflammatory-like response can be triggered for the skin to efficiently resist and protect itself. However, the skin becomes more fragile, and several essential features are less efficient: the skin’s epidermal hydrolipid film becomes poorer, the intracellular cement does not properly perform its barrier function, opening the door to dehydration. The dermis becomes thinner, with elastic fiber degradation, fragmented collagen fibers, and dermal connective tissue damage. The term inflamm’aging reflects this close relationship between inflammation and aging. While we gained a lot of insights on the molecular mechanisms driving Inflamm’aging using 2D cellular models, a more integrated view is needed to grasp molecular and cellular consequences on human skin.
Here, we intended to mimic inflamm’aging on various predictive 3D skin models. We also tested a new advanced eco-extraction of Granville Rose (GR) with a process of magnetic waves and centrifugation, and assessed its effect on all skin layers. We set UV radiation (UVR) conditions to mimic inflamm’aging, as UVR is known to stimulate the release of inflammatory molecules.
We first used skin biopsies stressed with 5J/cm2 UVA followed by 200mJ/cm2 UVB treated or not with GR extract. One key structural component of the skin dermal elastic fiber network was visualized by immunostaining to assess the impact of inflammation on their organization. After 48 hours, UVR impacted the fibrillin-1 organization, with a marked decrease of their total length (-17%). In presence of GR extract, fibrillin fibers were protected from UVR (+13%). This result highlights the capacity of GR extract to prevent deleterious effects of UVR on skin dermal structure. They are also in accordance with the properties of GR extract to reduce the release of inflammatory molecules after different stress conditions, which we could observe ex-vivo.
Next, we established a reconstructed human skin model of inflamm’aging. First, we set the experimental conditions of irradiation on full thickness skin and measured the transepidermal water loss (TEWL) as a readout of skin proper barrier function. Morphological parameters of the skin were also analysed. Dermal equivalents were produced by using scaffold-free self-assembly method of human normal skin fibroblasts, on top of which human normal keratinocytes were manually seeded. Reconstructed skin samples were then irradiated with 12 or 16J/cm2 UVA for 3 or 6 days. Compared to unirradiated controls, UVA treatment strongly affected the epidermal barrier function, with a marked increase of the measured TEWL. This result correlated with a disorganized epidermal basal layer, and impaired dermal structure that appeared less dense after irradiation. A dose-dependent response was observed, as well as a stronger phenotype after 6 days compared to 3 days for the lower UVA treatment used.
To further mimic inflamm’aging, the dose of 12J/cm2 of UVA was therefore chosen and chronically applied on 3D bioprinted skin constructs. Epidermis bioprinting was performed using an inkjet bioprinter equipped with a piezoelectric nozzle. An optimized design bioprinting pattern was used to bring the best results in term of epidermis homogeneity and reproducibility. GR extract was added by systemic treatment from the dermal maturation until the bioprinting of the epidermal layers, as well as during the chronic UVA treatment up to 42 days. Several morphological parameters were analysed, such as the barrier function recovery, the thickness of the epidermal and dermal compartments. The release of cytokines was also determined in these conditions. Our results on these new predictive bioprinted model were aligned with those obtained with ex-vivo and conventional in-vitro model, further validating the protective effects of GR extract.
Altogether, our work identified GR extract as a key cosmetic ingredient to fight against skin inflamm’aging. We used state-of-the-art bioprinted skin models to highlight the protective effect of GR extract on all skin layers.
Here, we intended to mimic inflamm’aging on various predictive 3D skin models. We also tested a new advanced eco-extraction of Granville Rose (GR) with a process of magnetic waves and centrifugation, and assessed its effect on all skin layers. We set UV radiation (UVR) conditions to mimic inflamm’aging, as UVR is known to stimulate the release of inflammatory molecules.
We first used skin biopsies stressed with 5J/cm2 UVA followed by 200mJ/cm2 UVB treated or not with GR extract. One key structural component of the skin dermal elastic fiber network was visualized by immunostaining to assess the impact of inflammation on their organization. After 48 hours, UVR impacted the fibrillin-1 organization, with a marked decrease of their total length (-17%). In presence of GR extract, fibrillin fibers were protected from UVR (+13%). This result highlights the capacity of GR extract to prevent deleterious effects of UVR on skin dermal structure. They are also in accordance with the properties of GR extract to reduce the release of inflammatory molecules after different stress conditions, which we could observe ex-vivo.
Next, we established a reconstructed human skin model of inflamm’aging. First, we set the experimental conditions of irradiation on full thickness skin and measured the transepidermal water loss (TEWL) as a readout of skin proper barrier function. Morphological parameters of the skin were also analysed. Dermal equivalents were produced by using scaffold-free self-assembly method of human normal skin fibroblasts, on top of which human normal keratinocytes were manually seeded. Reconstructed skin samples were then irradiated with 12 or 16J/cm2 UVA for 3 or 6 days. Compared to unirradiated controls, UVA treatment strongly affected the epidermal barrier function, with a marked increase of the measured TEWL. This result correlated with a disorganized epidermal basal layer, and impaired dermal structure that appeared less dense after irradiation. A dose-dependent response was observed, as well as a stronger phenotype after 6 days compared to 3 days for the lower UVA treatment used.
To further mimic inflamm’aging, the dose of 12J/cm2 of UVA was therefore chosen and chronically applied on 3D bioprinted skin constructs. Epidermis bioprinting was performed using an inkjet bioprinter equipped with a piezoelectric nozzle. An optimized design bioprinting pattern was used to bring the best results in term of epidermis homogeneity and reproducibility. GR extract was added by systemic treatment from the dermal maturation until the bioprinting of the epidermal layers, as well as during the chronic UVA treatment up to 42 days. Several morphological parameters were analysed, such as the barrier function recovery, the thickness of the epidermal and dermal compartments. The release of cytokines was also determined in these conditions. Our results on these new predictive bioprinted model were aligned with those obtained with ex-vivo and conventional in-vitro model, further validating the protective effects of GR extract.
Altogether, our work identified GR extract as a key cosmetic ingredient to fight against skin inflamm’aging. We used state-of-the-art bioprinted skin models to highlight the protective effect of GR extract on all skin layers.