Skin-protective biological activities of bio-fermented Aframomum angustifolium extract by a consortium of microorganisms.

Background: Aframomum sp. is a genus of plants in the Zingiberaceae family. It includes several species, some of which are used in cosmetics for their various properties, making them useful in skincare products, particularly for anti-aging, moisturizing, and brightening the skin. However, to date, there is no experimental evidence on its natural extracts obtained or modified using microorganisms (bio-fermentation) as an anti-aging agent. Objective: The present study aimed to evaluate the antiaging effect of a Bio-fermented Aframomum angustifolium (BAA) extract on 3D bioprinted skin equivalent. Methods: The consortium of microorganisms contained Komagataeibacter, Gluconobacter, Acetobacter, Saccharomyces, Torulaspora, Brettanomyces, Hanseniaspora, Leuconostoc, Lactobacillus, Schizosaccharomyces. It was developed on a media containing water, sugar, and infused black tea leaves. The seeds of Aframomum angustifolium previously grounded were mixed with the culture medium, and the ferments in growth; this fermentation step lasted 10 days. Then, the medium was collected and filtered (0.22 µm) to obtain the BAA extract. To enhance our comprehension of the impact of BAA extract on skin aging, we developed skin equivalents using bio-printing methods with the presence or absence of keratinocyte stem cells (KSC). These skin equivalents were derived from keratinocytes obtained from both a middle-aged donor, with and without KSC. Moreover, we examined the effects of treating the KSC-depleted skin equivalents with Bio-fermented Aframomum angustifolium (BAA) extract for 5 days. Skin equivalents containing KSC-depleted keratinocytes exhibited histological characteristics typical of aged skin and were compared to skin equivalents derived from young donors. Results: The BAA extract contained specific organic acids such as lactic, gluconic, succinic acid and polyphenols. KSC-depleted skin equivalents that were treated with BAA extract exhibited higher specular reflection, indicating better hydration of the stratum corneum, higher mitotic activity in the epidermis basal layer, improved dermal-epidermal connectivity, and increased rigidity of the dermal-epidermal junction compared to non-treated KSC-depleted equivalents. BAA extract treatments also resulted in changes at the dermis level, with an increase in total collagen and a decrease in global laxity, suggesting that this extract could help maintain youthful-looking skin. Conclusion: In summary, our findings indicated that BAA extract treatments have pleiotropic beneficial effects on skin equivalents and that the bio-fermentation provides new biological activities to this plant.

A Preliminary Study for an Intraoperative 3D Bioprinting Treatment of Severe Burn Injuries.

Intraoperative three-dimensional fabrication of living tissues could be the next biomedical revolution in patient treatment. APPROACH: We developed a surgery-ready robotic three-dimensional bioprinter and demonstrated that a bioprinting procedure using medical grade hydrogel could be performed using a 6-axis robotic arm in vivo for treating burn injuries. RESULTS: We conducted a pilot swine animal study on a deep third-degree severe burn model. We observed that the use of cell-laden bioink as treatment substantially affects skin regeneration, producing in situ fibroblast growth factor and vascular endothelial growth factor, necessary for tissue regeneration and re-epidermalization of the wound. CONCLUSIONS: We described an animal study of intraoperative three-dimensional bioprinting living tissue. This emerging technology brings the first proof of in vivo skin printing feasibility using a surgery-ready robotic arm-based bioprinter. Our positive outcome in skin regeneration, joined with this procedure's feasibility, allow us to envision the possibility of using this innovative approach in a human clinical trial in the near future.

The Whitening Properties of the Mixture Composed of Pomegranate, Osmanthus and Olive and the Protective Effects Against Ultraviolet Deleterious Effects.

BACKGROUND: Ultraviolet (UV) rays are the major environmental factor that damage skin physiology causing deleterious effects such as oxidation, photoaging and pigmentation. There has been considerable interest in using botanicals to prevent skin damages caused by UV irradiation. AIM: In this study, three plant extracts were tested either individually or combined together (mixture) as well as their corresponding main active compound: pomegranate/punicalagin, osmanthus/verbascoside and olive/hydroxytyrosol. We evaluated the whitening and anti-photoaging properties of the nutritional mixture using 2D human culture model and a 3D full-thickness pigmented skin model exposed to UVB and UVA. METHODS: For exploring skin pigmentation, oxidation and aging, we performed cell viability, tyrosinase activity and melanin content assays as well as histology analysis (Whartin-Starry staining), immunodetection (PMEL, MDA, collagen type I and elastin) and carbonylated proteins analysis by electrophoresis separation. RESULTS: Results showed that the pomegranate extract and the active molecule punicalagin could reduce the tyrosinase activity and melanin content in melanocytes (P < 0.05). The mixture, pomegranate extract and punicalagin inhibited the melanin production and pre-melanosomal protein (PMEL) expression in the 3D skin pigmented model (P < 0.001). Furthermore, the mixture treatment repaired the expressions of collagen I and elastin decrease by UV exposure (P < 0.01). The mixture also significantly decreased lipid peroxidation (P < 0.001) and carbonylated proteins (P < 0.05) in the skin model compared to the UV-exposed condition. CONCLUSION: To conclude, the mixture composed of pomegranate, osmanthus and olive extracts protects human skin from UV rays deleterious effects and exhibits antioxidative, anti-aging and skin whitening properties. Our data suggested pomegranate contributed to the whitening properties of the mixture notably through its main active compound, punicalagin. The mixture might be a good candidates for further development as natural antioxidant and skin care product.

MicroRNA-23b-3p regulates human keratinocyte differentiation through repression of TGIF1 and activation of the TGF-ß-SMAD2 signalling pathway.

MicroRNAs (miRNAs) are a class of short non-coding RNAs capable of repressing gene expression at the post-transcriptional level. miRNAs participate in the control of numerous cellular mechanisms, including skin homeostasis and epidermal differentiation. However, few miRNAs involved in these processes have been identified so far in human skin, and the gene networks they control remain largely unknown. Here, we focused on miR-23b-3p, a miRNA that is expressed during the late step of human keratinocyte differentiation. We report that miR-23b-3p silencing modulates epidermal differentiation in human skin reconstructs. The SMAD transcriptional corepressor TGIF1 was identified on bioinformatic analysis as a potential target of miR-23b-3p. Expression analysis and reporter gene assays confirmed direct regulation of TGIF1 expression by miR-23b-3p. Finally, we showed that miR-23-3p was able to activate TGF-ß signalling in human keratinocytes by increasing SMAD2 phosphorylation through TGIF1 repression. Taken together, these data identify miR-23b-3p as a new regulator of human epidermal differentiation in line with TGF-ß signalling.

Perlecan expression influences the keratin 15-positive cell population fate in the epidermis of aging skin.

The epidermis is continuously renewed by stem cell proliferation and differentiation. Basal keratinocytes append the dermal-epidermal junction, a cell surface-associated, extracellular matrix that provides structural support and influences their behaviour. It consists of laminins, type IV collagen, nidogens, and perlecan, which are necessary for tissue organization and structural integrity. Perlecan is a heparan sulfate proteoglycan known to be involved in keratinocyte survival and differentiation. Aging affects the dermal epidermal junction resulting in decreased contact with keratinocytes, thus impacting epidermal renewal and homeostasis. We found that perlecan expression decreased during chronological skin aging. Our in vitro studies revealed reduced perlecan transcript levels in aged keratinocytes. The production of in vitro skin models revealed that aged keratinocytes formed a thin and poorly organized epidermis. Supplementing these models with purified perlecan reversed the phenomenon allowing restoration of a well-differentiated multi-layered epithelium. Perlecan down-regulation in cultured keratinocytes caused depletion of the cell population that expressed keratin 15. This phenomenon depended on the perlecan heparan sulphate moieties, which suggested the involvement of a growth factor. Finally, we found defects in keratin 15 expression in the epidermis of aging skin. This study highlighted a new role for perlecan in maintaining the self-renewal capacity of basal keratinocytes.

In vitro 3-D model based on extending time of culture for studying chronological epidermis aging.

Skin aging is a complex phenomenon in which several mechanisms operate simultaneously. Among them, intrinsic aging is a time-dependent process, which leads to gradual skin changes affecting its structure and function such as thinning down of both epidermal and dermal compartments and a flattening and fragility of the dermo-epidermal junction. Today, several approaches have been proposed for the generation of aged skin in vitro, including skin explants from aged donors and three-dimensional skin equivalent treated by aging-inducing chemical compounds or engineered with human cells isolated from aged donors. The aim of this study was to develop and validate a new in vitro model of aging based on skin equivalent demonstrating the same phenotypic changes that were observed in chronological aging. By using prolonged culture as a proxy for cellular aging, we extended to 120 days the culture time of a skin equivalent model based on collagen-glycosaminoglycan-chitosan porous polymer and engineered with human skin cells from photo-protected sites of young donors. Morphological, immunohistological and ultrastructural analysis at different time points of the culture allowed characterizing the phenotypic changes observed in our model in comparison to samples of non photo-exposed normal human skin from different ages. We firstly confirmed that long-term cultured skin equivalents are still morphologically consistent and functionally active even after 120 days of culture. However, similar to in vivo chronological skin aging a significant decrease of the epidermis thickness as well as the number of keratinocyte expressing proliferation marker Ki67 are observed in extended culture time skin equivalent. Epidermal differentiation markers loricrin, filaggrin, involucrin and transglutaminase, also strongly decreased. Ultrastructural analysis of basement membrane showed typical features of aged skin such as duplication of lamina densa and alterations of hemidesmosomes. Moreover, the expression of hyaluronan and its surface receptor CD44 drastically decreased as observed during chronological skin aging. Finally, we found that the level of p16INK4A expression significantly increased supporting cellular senescence process associated to our model. To conclude, the major morphological and ultrastructural epidermal modifications observed in both our extended culture skin equivalent model and skin biopsies from old donors validate the relevance of our model for studying chronological aging, understanding and elucidating age-related modifications of basic skin biological processes. In addition, our model provides a unique tool for identifying new targeted molecules intended at improving the appearance of aging skin.

Expression of estrogen-related receptor beta (ERRß) in human skin.

Skin is a non classical target tissue for estrogens, whose biological and mechanical properties are affected by the hormonal deprivation occurring at the menopause. Estrogen-related receptors (ERR), closely related to the estrogen receptors (ER), constitute a subfamily of orphan receptors, interfering with ER-mediated signalling pathways. The expression of ERRß has been detected in only a few adult tissues so far, such as the prostate and the inner ear. Here, we demonstrate the expression of ERRß in normal human skin. ERRß was detected in human epidermis, in both keratinocytes and Langerhans cells, by immunohistochemistry. These results were validated on freshly extracted epidermal cells and on monolayer-cultured keratinocytes by RT-PCR and western blotting (WB), suggesting the implication of ERRß in skin immunity and endocrine effects.