Attenuation of negative effects of senescence in human skin using an extract from Sphingomonas hydrophobicum: development of new skin care solution.

OBJECTIVE: Intrinsic skin ageing is mainly caused by cellular senescence. p16 and p21 are two important tumour suppressor proteins that play essential roles during cell proliferation and ageing through regulating the expression of several genes. Moreover, physical changes between the ages of 55 and 60 years affect one's physical and disrupt self-esteem. The cosmetics industry has focused on bioactive substances derived from natural products such as plants, mushrooms and marine algae to counteract the deleterious effect on skin senescence. Besides these products, compounds produced by bacteria may decelerate individual senescence. METHODS: In order to evaluate the potential benefits of bacteria extract over skin ageing, we investigated whether a Sphingomonas hydrophobicum (SH) extract is able to protect our skin against senescence mechanisms. We used an ageing full-thickness skin equivalent model, which was treated or not with the bacteria extract in a systemic way for 42 days. p21 and p16 and senescence-associated galactosidase activity were used to detect cellular senescence with immunohistology. Using a psychobiological approach, we evaluated in vivo the effect of SH extract on self-esteem, isotropy and suppleness. RESULTS: Sphingomonas extract significantly suppressed senescence associated with ß-galactosidase activation. It also significantly inhibited the expression of cell cycle inhibitors (p21 and p16). At the same time, the bacteria extract has a significant positive impact on the issue by increasing the expression of versican and fibrillin-1. Significant improvements of self-esteem were reported after 56 days of SH extract application. These psychological benefits were accompanied by a significant improvement in skin suppleness and isotropy. CONCLUSION: Sphingomonas extract delays intrinsic skin ageing process by inhibiting cellular senescence, and has also the capability to restructure the skin. These beneficial physiological effects induced by SH extract have a positive influence on self-esteem. Because skin ageing causes emotional distress, SH extract can serve as an anti-ageing cosmeceutical agent and help to build a better psychological health, and help individuals to assume ageing.

OBJECTIF: Le vieillissement intrinsèque de la peau est principalement causé par la sénescence cellulaire. p16 et p21 sont deux importantes protéines suppressives de tumeurs qui jouent un rôle essentiel dans la prolifération et le vieillissement cellulaire en régulant l'expression de plusieurs gènes. De plus, les changements physiques survenant entre 55 et 60 ans affectent le physique et perturbent l'estime de soi. L'industrie cosmétique s'est concentrée sur les substances bioactives dérivées de produits naturels tels que les plantes, les champignons et les algues marines pour contrer les effets délétères sur la sénescence de la peau. En plus de ces produits, les composés produits par les bactéries peuvent ralentir la sénescence individuelle. MÉTHODES: Afin d'évaluer les bénéfices potentiels de l'extrait de bactérie sur le vieillissement cutané, nous avons étudié si un extrait de Sphingomonas hydrophobicum (SH) est capable de protéger notre peau des mécanismes de sénescence. Nous avons utilisé un modèle équivalent de peau vieillissante de pleine épaisseur, qui a été traitée ou non avec l'extrait de bactérie de façon systémique pendant 42 jours. p21 et p16, et l'activité galactosidase associée à la sénescence ont été utilisés pour détecter la sénescence cellulaire par immunohistologie. En utilisant une approche psychobiologique, nous avons évalué in vivo l'effet de l'extrait de SH sur l'estime de soi, l'isotropie et la souplesse. RÉSULTATS: L'extrait de Sphingomonas a considérablement supprimé la sénescence associée à l'activation de ß-galactosidase. Il a également inhibé de manière significative l'expression des inhibiteurs du cycle cellulaire (p21 et p16). En même temps, l'extrait de bactérie a un impact positif significatif sur le problème en augmentant l'expression du versican et de la fibrilline-1. Des améliorations significatives de l'estime de soi ont été rapportées après 56 jours d'application de l'extrait de SH. Ces bienfaits psychologiques s'accompagnaient d'une amélioration significative de la souplesse et de l'isotropie de la peau. CONCLUSION: L'extrait de Sphingomonas retarde le processus de vieillissement intrinsèque de la peau en inhibant la sénescence cellulaire et a également la capacité de restructurer la peau. Ces effets physiologiques bénéfiques induits par l'extrait de SH ont une influence positive sur l'estime de soi. Parce que le vieillissement de la peau provoque une détresse émotionnelle, l'extrait de SH peut servir d'agent cosméceutique anti-âge et aider à construire une meilleure santé psychologique, ainsi qu'aider les individus à assumer le vieillissement.

A simple way to reconstruct a human 3-d hypodermis: a useful tool for pharmacological functionality.

BACKGROUND: Adipose tissue engineering has been hampered by the inability to culture mature adipocytes. Adipose-derived stem cell (ASC) culture opens the way for the preparation of human 3-D hypodermis in large quantities. These models play a role in obesity-related active molecules and slimming agent screening. Moreover, they contribute to a better understanding of the mechanisms underpinning obesity. MATERIALS AND METHODS: Freshly extracted ASC from fat tissue were characterized by flow cytometry for CD73, CD90, CD105, HLA-ABC, CD14 and CD45 markers and by Western blot for pref-1. Their differentiation in mature adipocytes was followed by lipid and adiponectin secretion or by oil red O staining and radioimmunoassay. Neosynthesized extracellular matrix (ECM) of 3-D hypodermis was investigated by immunohistochemistry (collagen type I, V and VI) and transmission electron microscopy. RESULTS: Our results demonstrate that the culture of preadipocytes in proliferation medium for 15 days followed by 16 days of culture in differentiation medium allowed production of the thickest single-layer hypodermis in which preadipocytes and mature adipocytes coexist and synthesize adiponectin and ECM components. Functionality of our 3-D single-layer hypodermis was demonstrated both by a 3.5-fold glycerol production after its stimulation with norepinephrine (adrenergic agonist) and by its slimming after caffeine treatment versus the nontreated 3-D hypodermis. CONCLUSION: This economic 3-D model, easy to prepare and giving reproducible results after the treatment of actives, is useful for pharmacotoxicological trials as an alternative to animal experimentation.

Validation of the BacT/ALERT®3D automated culture system for the detection of microbial contamination of epithelial cell culture medium.

Living tissue engineering for regenerative therapy cannot withstand the usual pharmacopoeia methods of purification and terminal sterilization. Consequently, these products must be manufactured under aseptic conditions at microbiologically controlled environment facilities. This study was proposed to validate BacT/ALERT(®)3D automated culture system for microbiological control of epithelial cell culture medium (ECCM). Suspensions of the nine microorganisms recommended by the European Pharmacopoeia (Chap. 2.6.27: "Microbiological control of cellular products"), plus one species from oral mucosa and two negative controls with no microorganisms were prepared in ECCM. They were inoculated in FA (anaerobic) and SN (aerobic) culture bottles (Biomérieux, Lyon, France) and incubated in a BacT/ALERT(®)3D automated culture system. For each species, five sets of bottles were inoculated for reproducibility testing: one sample was incubated at the French Health Products Agency laboratory (reference) and the four others at Cell and Tissue Bank of Lyon, France. The specificity of the positive culture bottles was verified by Gram staining and then subcultured to identify the microorganism grown. The BacT/ALERT(®)3D system detected all the inoculated microorganisms in less than 2 days except Propionibacterium acnes which was detected in 3 days. In conclusion, this study demonstrates that the BacT/ALERT(®)3D system can detect both aerobic and anaerobic bacterial and fungal contamination of an epithelial cell culture medium consistent with the European Pharmacopoeia chapter 2.6.27 recommendations. It showed the specificity, sensitivity, and precision of the BacT/ALERT(®)3D method, since all the microorganisms seeded were detected in both sites and the uncontaminated medium ECCM remained negative at 7 days.

Assessment of transformed properties in vitro and of tumorigenicity in vivo in primary keratinocytes cultured for epidermal sheet transplantation.

Epidermal keratinocytes are used as a cell source for autologous and allogenic cell transplant therapy for skin burns. The question addressed here is to determine whether the culture process may induce cellular, molecular, or genetic alterations that might increase the risk of cellular transformation. Keratinocytes from four different human donors were investigated for molecular and cellular parameters indicative of transformation status, including (i) karyotype, (ii) telomere length, (iii) proliferation rate, (iv) epithelial-mesenchymal transition, (v) anchorage-independent growth potential, and (vi) tumorigenicity in nude mice. Results show that, despite increased cell survival in one keratinocyte strain, none of the cultures displayed characteristics of cell transformations, implying that the culture protocol does not generate artefacts leading to the selection of transformed cells. We conclude that the current protocol does not result in an increased risk of tumorigenicity of transplanted cells.

Evaluation of tumorigenic risk of tissue-engineered oral mucosal epithelial cells by using combinational examinations.

Recently, oral mucosal epithelial cells were proposed as a cell source of the autologous cell transplant therapy for corneal trauma or disease. The question addressed is to know if the biological conditions of grafting could induce certain cellular, molecular, and genetic alterations that might increase the risk of mutations and possibly of cellular transformation. Recent progress in cancer research enables us to depict the generation mechanisms and basic characteristics of human cancer cells from molecular, cytological, and biological aspects. The aim of this study is to evaluate the risk of tumorigenicity of the oral mucosal epithelial culture process in order to mitigate that risk, if any, before clinical application. Oral mucosal epithelial cells from three different human donors were investigated by combinational examinations to detect possible tumorigenic transformation. We investigated (i) clonogenic and karyology types, (ii) the validation of proliferation rate, (iii) the epithelial-mesenchymal transition, (iv) anchorage-independent growth potential, and (v) tumorigenicity on nude mice. Results show that the culture process used in this study presents no risk of tumorigenicity.