Development of 3D gingival in vitro models using primary gingival cells.

Since March 2013, animal testing for toxicity evaluation of cosmetic ingredients is banned in Europe. This directive applies to all personal care ingredients including oral ingredients. Gingival in vitro 3D models are commercially available. However, it is essential to develop "in house model" to modulate several parameters to study oral diseases, determine the toxicity of ingredients, test biocompatibility, and evaluate different formulations of cosmetic ingredients. Our expertise in tissue engineering allowed us to reconstruct human oral tissues from normal human gingival cells (fibroblasts and keratinocytes). Indeed, isolation from surgical leftover was performed to culture these gingival cells. These cells keep their endogenous capacity to proliferate allowing reconstruction of equivalent tissue close to in vivo tissue. Reconstruction of gingival epithelium, chorion equivalent, and the combination of these two tissues (full thickness) using primary gingival cells displayed all characteristics of an in vivo gingival model.

The impact of airborne ultrafine particulate matter on human keratinocyte stem cells.

OBJECTIVE: Air pollution is today fully acknowledged to be a significant public health problem. Rapid urbanization exposed us to a variety of unhealthy ambient air pollutants at high concentrations. The emergence of airborne ultrafine particles has added an additional dimension to this already complex problem of air pollution. The skin has different functions, one of them being the protection against the deleterious effect of external agents. The aim of this study is to evaluate the impact of airborne ultrafine particles (UFP) pollution on skin aging and on keratinocyte differentiation. METHODS: Ex vivo human skin biopsies and cultured keratinocytes stem cells (KSC) were submitted to diesel exhaust-derived UFP. Reactive oxygen species (ROS) production was assessed with the MitoSOX™ probe. Keratinocyte stemness potential was evaluated by the immunodetection of keratin 15 (K15) and p63 (∆N isoforms). Effect of UFP on the epithelial niche maintenance was evaluated by immunodetection of Sox9. Reconstructed epidermis model was used to assess the impact of UFP on keratinocyte differentiation and aging. RESULTS: UFP exposure induced ROS production and disturbed K15, ∆Np63 and Sox9 expression in KSC or ex vivo skin. Finally, investigations on reconstructed epidermis revealed a phenotype marked by impaired keratinocyte differentiation. CONCLUSION: These results indicate that UFP pollution is a potent extrinsic factor of skin aging, affecting the keratinocyte stem cell potential and the skin renewal process.

OBJECTIF: La pollution de l'air est désormais pleinement reconnue comme un problème de santé publique important. L'urbanisation rapide nous a exposés à une variété de polluants atmosphériques ambiants malsains à des concentrations élevées. L'émergence de particules ultrafines en suspension dans l'air a ajouté une dimension supplémentaire à ce problème déjà complexe de la pollution de l'air. La peau exerce différentes fonctions, l'une d'elles étant la protection contre l'effet délétère d'agents extérieurs. L'objectif de cette étude est d'évaluer l'impact de la pollution par les particules ultrafines (UFP) aéroportées sur le vieillissement cutané et sur la différenciation des kératinocytes. MÉTHODES: Des biopsies de peau humaine ex vivo et des kératinocytes souches (KSC) en culture ont été mis en présence d'UFP provenant d'échappement de véhicule diesel. La production d'espèces réactives de l'oxygène (ROS) a été évaluée avec la sonde MitoSOX™. Le potentiel de souche des kératinocytes a été évalué par immunodétection de la kératine 15 (K15) et p63 (isoformes ∆N). L'effet des UFP sur la niche épithéliale a été évalué par immunodétection de Sox9. Un modèle d'épiderme reconstruit a été utilisé pour évaluer l'impact des UFP sur la différenciation et le vieillissement des kératinocytes. RÉSULTATS: L'exposition aux UFP a induit la production de ROS, a perturbé l'expression de K15, ∆Np63 et de Sox9 dans les KSC et dans la peau ex vivo. Enfin, des investigations sur des épidermes reconstruits ont révélé un phénotype marqué par une différenciation altérée des kératinocytes. CONCLUSION: Ces résultats indiquent que la pollution par les UFP est un facteur extrinsèque puissant du vieillissement cutané, affectant le potentiel des cellules souches de kératinocytes et le processus de renouvellement cutané.

Reconstruction of functional human epidermis equivalent containing 5%IPS-derived keratinocytes treated with mitochondrial stimulating plant extracts.

Reconstructed human epidermis equivalents (RHE) have been developed as a clinical skin substitute and as the replacement for animal testing in both research and industry. KiPS, or keratinocytes derived from induced pluripotent stem cells (iPSCs) are frequently used to generate RHE. In this study, we focus on the mitochondrial performance of the KiPS derived from iPSCs obtained from two donors. We found that the KiPS derived from the older donor have more defective mitochondria. Treatment of these KiPS with a plant extract enriched in compounds known to protect mitochondria improved mitochondrial respiration and rendered them fully competent to derive high-quality RHE. Overall, our results suggest that improving mitochondrial function in KiPS is one of the key aspects to obtain a functional RHE and that our plant extracts can improve in this process.

Glyoxal Induces Senescence in Human Keratinocytes through Oxidative Stress and Activation of the Protein Kinase B/FOXO3a/p27KIP1 Pathway.

Senescence is a well-characterized cellular state associated with specific markers such as permanent cell proliferation arrest and the secretion of messenger molecules by cells expressing the senescence-associated secretory phenotype. The senescence-associated secretory phenotype composition depends on many factors such as the cell type or the nature of the stress that induces senescence. Because the skin constitutes a barrier with the external environment, it is particularly subjected to different types of stresses and consequently prone to premature cellular aging. The dicarbonyl compounds glyoxal (GO) and methylglyoxal are precursors of advanced glycation end products, whose presence marks normal and pathological aging. In this study, we show that GO treatment provokes oxidative stress by increasing ROS and advanced glycation end-products levels and induces senescence in human keratinocytes. Furthermore, GO-induced senescence bears a unique molecular progression profile: an early-stage senescence when protein kinase B‒FOXO3a-p27KIP1 pathway mediates cell cycle arrest and a late-stage senescence maintained by the p16INK4/pRb pathway. Moreover, we characterized the resulting secretory phenotype during early-stage senescence by mass spectrometry. Our study provides evidence that GO can affect keratinocyte functions and act as a driver of human skin aging. Hence, senotherapeutics aimed at modulating GO-associated senescence phenotype hold promising potential.

Characterization of a New Full-Thickness In Vitro Skin Model.

Since 30 years, bioengineering allowed to reconstruct human tissues using normal human cells. Skin is one of the first organ to be reconstructed thanks to the development of specific cell culture media and supports favoring the culture of human skin cells, such as fibroblasts, keratinocytes, or melanocytes. Skin models have evolved from epidermis to complex models including a dermis. The purpose of the present study was to design a reconstructed full-thickness (FT) skin suitable to perform in vitro testing of both molecules and plant extracts. First, we reconstructed epidermis with normal human keratinocytes displaying the expected multilayered morphology and expressing specific epidermal proteins (e-cadherin, claudin-1, p63, Ki67, Keratin 10, filaggrin, and loricrin). Then, a dermal equivalent was developed using a collagen matrix allowing the growth of fibroblasts. The functionality of the dermis was demonstrated by the measurement of skin parameters such as rigidity or elasticity with Ballistometer® and other parameters such as the contraction over time and the expression of dermal proteins. The combination of these two compartments (dermis and epidermis) allowed to reconstruct an FT model. This study model allowed to study the communication between compartments and with the establishment of a dermoepidermal junction showing the expression of specific proteins (collagen XVII, laminin, and collagen IV). Impact statement The objective of our research project was to design a three-dimensional human full-thickness (FT) skin suitable to perform in vitro testing of molecules and plant ingredients. The combination of these two reconstructed compartments (dermis and epidermis) allowed to reconstruct an FT model. This study model allowed to study the communication between compartments and with the establishment of a dermoepidermal junction showing the expression of specific proteins (collagen XVII, laminin, and collagen IV). This in vitro model can be use by cosmetic and pharmaceutical industries to study the effect of chemical or natural compounds on the skin.

Establishment and performance assessment of an in-house 3D Reconstructed Human Cornea-Like Epithelium (RhCE) as a screening tool for the identification of liquid chemicals with potential eye hazard.

The objective of this project was to develop an in-house 3D Reconstructed Human Cornea-Like Epithelium (RhCE) to be used as a screening tool in early stages of product development. This study reports the establishment of the experimental procedure and the performance assessment of the model to discriminate liquid chemicals classified as causing serious eye damage/irritation (UN-GHS Category 1/2) from liquid chemicals not requiring such hazard classification (UN-GHS No Category). Histological examination of this ocular equivalent model, based on Normal Human Keratinocytes (NHK) cultured in a chemically defined medium, revealed a stratified and well-organized tissue construct. Moreover, barrier robustness and functionality were demonstrated by the effective time-50 (ET50) of Triton X-100 measurement. The prediction model is based on cytotoxicity assessment following a test chemical exposure. When the mean tissue viability was over 60%, the chemical was defined as No Category; otherwise, it was classified as Category 1/2. In accordance with the applicable OECD guidance document (ENV/JM/MONO (2015)23), the performance of the model for eye hazard identification was evaluated with the minimum list of reference chemicals. As a result, the method scored 84.4% Accuracy, 70.8% Specificity, 100% Sensitivity and 93.3% for Concordance, demonstrating prediction performances close to those of Validated Reference Methods that are commonly used for regulatory purposes. Finally, these results suggest that this in-house RhCE based test method is a qualitative and accurate screening tool for eye hazard identification of liquid chemicals.

Property characterization of reconstructed human epidermis equivalents, and performance as a skin irritation model.

In recent years, in vitro skin models combining cell biology and tissue engineering have been developed in order to replace animal models for toxicological studies and to serve as research support to better understand skin biology. This study reports the development and characterization of a epidermal tissue equivalent meant to be used to develop and to evaluate the effect of applied cosmetic ingredients, and for alternative toxicological testing. This epidermis equivalent model was characterized relative to the morphological characteristics of short- and long-term maintained tissues by performing histological studies. We also studied the integrity of the epidermal barrier. Finally, with the goal of validating its use as a skin irritation test, we studied the irritation potential of 20 chemical references listed in OECD Test Guideline N°439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method). In 2015, OECD officially published the updated version of the Validated Reference Method (VRM) that uses reconstructed human epidermis models for irritation testing, thus offering the possibility for proposed putative similar test methods to obtain a validation agreement through Performance Standards-based validation. In this study, we observed that the epidermal equivalent we developed showed similarities to human in vivo skin, based on the analyzed parameters. Moreover, its performances as a skin irritation test were similar to the ones described in the OECD Test Guideline N°439.