Background: The process by which functional elastic fibers are produced, namely elastogenesis, is complex and difficult to assess in vitro. Identifying efficient elasticity-boosting ingredients thus represents a challenge.Aims: The elasticity-boosting properties of a novel extract of Murraya koenigii leafy stems were assessed in vitro in 3D culture models before being evaluated in human female volunteers.Methods: Synthesis of elastic fiber related proteins was evaluated in a skin-equivalent model. Using multiphoton microscopy, the structural organization of elastin deposits was studied within a scaffold-free dermal microtissue. Biomechanical properties of the 3D microtissue were also measured by atomic force microscopy. In vivo, fringe-projection and image analysis were used to evaluate nasogenian fold severity in a panel of Caucasian female volunteers. The impact of gravity on visible signs of facial aging was assessed by clinical scoring carried out alternatively in the supine and sitting positions.Results: We showed the Murraya koenigii extract increased protein expressions of elastin and fibrillin-1 in a 3D skin equivalent model. Using scaffold-free dermal microtissue, we confirmed that Murraya koenigii extract allowed a proper and ordered network of elastin deposits and consequently improved tissue elasticity. Clinical data showed that a twice-daily application for 98 days of the extract formulated at 1% allowed to visibly reduce nasogenian fold severity, jowl severity and to mitigate the impact of gravity on the facial signs of aging.Conclusion: The newly discovered extract of Murraya koenigii leafy stems represents an innovative antiaging ingredient suited for elasticity-boosting and antisagging claims.

The loss of elasticity is a hallmark of systemic aging or genetic syndromes (e.g. cutis laxa, Williams-Beuren and supravalvular aortic stenosis) with direct consequences on tissue functions, and particularly deleterious when associated to the cardiovascular system. Tissue elasticity is mainly provided by large elastic fibers composed of supramolecular complexes of elastin and microfibrils. In arteries, the mature elastic fibers are located in the media compartment and form concentric elastic lamellar units together with the smooth muscle cells (SMCs). The main function of vascular elastic fibers is to allow extension and recoil of the vessel walls in response to the intraluminal pressure generated by the blood flow following cardiac systole. The synthesis of elastic fibers (elastogenesis) mainly occurs during the last third of fetal life with a peak in the perinatal period and then slowly decreases until the end of growth; as a result, elastic fiber repair is almost non-existent in adults. To date, no treatment exists to restore or repair deficient or degraded elastic fibers. A few pharmacological compounds have been proposed, but their efficacy/side effects balance remains very unfavorable. As an alternative strategy, we developed a synthetic elastic protein (SEP) inspired by the human tropoelastin, the elastin soluble precursor, to provide an elastic molecular prosthesis capable of integrating and reinforcing endogenous elastic fibers. The SEP was easily produced in E. coli and purified by inversed transition cycling method. The resulting 55 kDa protein recapitulates the main physicochemical properties of the tropoelastin as thermal responsiveness, intrinsically disordered structures, and spherical self-assembly. The cross-linked SEP displays linear elastic mechanical properties under uniaxial tension loads. Using a co-culture in vitro model of the endothelial barrier, our results show that SEP is able to cross the cohesive endothelial monolayer to reach underlying SMCs. Moreover, SEP is processed by SMCs through a lysyl oxidase-dependent mechanism to form fibrillar structures that colocalize with fibrillin-rich microfibrils. The SEP was further characterized in vivo through the zebrafish model. The results indicate a global innocuity on zebrafish embryos and an absence of neutrophil recruitment following injection into the yolk sac of zebrafish. Finally, intravenous injection of a fluorescent SEP highlights its deposition in the wall of tortuous vessels which persists for several days after injection of the larvae. Taken together, our results demonstrate for the first time the incorporation of a naked tropoelastin-bioinspired polypeptide in endogenous elastic fibrillar deposits from SMCs, and its recognition by the lysyl- oxidase enzymatic machinery. In absence of toxicity and proinflammatory signal combined to a long-lasting accumulation in vessels in vivo , the SEP fulfills the first prerequisites for the development of an original biotherapeutic compound addressing the repair of elastic fibers.

Dark circles are a cosmetic concern worldwide, often associated with tiredness or aging. Owing to its thinness, the highly vascularized eye contour area easily shows blood and lymphatic circulation disorders. Environmental stress alters skin microcirculation, endothelial barrier function and increases oxidation in the subocular area. The multifactorial nature of dark circles and puffiness represents a real challenge for in vitro efficacy testing of active ingredients. Therefore, to select an active ingredient with both anti-puffiness and anti-dark circle potentials, we have implemented a screening strategy that combined different biological models addressing relevant targets of skin microcirculation and endothelial barrier function. Using this screening approach on 22 plant extracts, we identified a particularly interesting plant extract which significantly reduced adhesion protein VCAM-1 synthesis in dermal microvascular endothelial cell cultures, significantly decreased leukocyte adhesion to endothelial cell membranes, while significantly increasing trans-endothelial electrical resistance. This ability to improve skin microcirculation was illustrated in a vascularized 3D dermis model stimulated with TNF-α, in which the plant extract was shown to restore the basement membrane of capillary-like tubular structures, as shown by laminin expression. Additionally, the extract also favored hemoglobin degradation by stimulating HMOX-1 mRNA expression in dermal fibroblast cultures. It also increased chelation of ferrous ions, hemoglobin by-products that increase skin oxidation. The stepwise selection model we used allowed us to identify a unique plant extract with promising anti-puffiness and anti-dark circle potential, based on combined proteomic, genomic and biochemical methods in acellular assays, 2D and 3D cell models. Dedicated clinical study will be used in the near future to demonstrate the in vivo benefits of the ingredient.

The loss of elasticity is a characteristic feature of skin aging. Indeed, elastic fibers are poorly renewed and are degraded much quicker than synthesized. To compensate for this imbalance, a unique extract from the traditional ayurvedic plant Murraya koenigii was developed. First, a proteomic study combined with bioinformatics, revealed that monolayer cultures of human dermal fibroblasts (HDF) treated with M. koenigii extract expressed increased levels of dermal proteins involved in the maintenance and integrity of the extracellular matrix. More specifically, we demonstrated that M. koenigii was able to significantly increase the synthesis and in situ deposition of elastin (+43%), fibrillin-1 (+22%) and fibulin-5 (+62%) in monolayer cultures of HDF. As both epidermis and dermis are required for proper elastic fiber formation, these results were further confirmed in a full-thickness 3D reconstructed skin model, (+103% elastin, +35% fibrillin-1 with M. koenigii extract). The plant extract also significantly reduced elastic fiber degradation by inhibiting MMP-12 elastase activity (-27%). To control both structural and functional quality of elastic fibers neo-synthesized in the presence of M. koenigii extract, we developed a 3D scaffold-free spheroid microtissue, in which the elastic fiber network was characterized using two-photon autofluorescence (2PAF) imaging. Based on 2PAF image analysis, the dermis microtissue treated with M. koenigii extract exhibited a denser and more mature elastic fiber network. Regarding the biomechanical properties, we showed using atomic force microscopy (AFM), a 3-fold decrease in the relative dermal stiffness after treatment with M. koenigii. The 2PAF and AFM combined analyses allowed to draw a correlation map of apparent elastic modulus and elastin deposit density, which revealed that elastin-rich areas were less stiff. To conclude, M. koenigii extract displays undeniable elasticity boosting properties, highlighted at molecular, cellular and tissue levels, and appears as a powerful anti-aging ingredient.

Adipose tissue plays a critical role in energy balance and lipid metabolism. Disorders of adipose function can lead, amongst others, to type II diabetes, obesity and unsightly conditions such as cellulite. Monolayer cell culture systems have proven useful to understand many aspects of fat biology, but these 2D in vitro systems do not reflect the complexity of human adipose tissue. To overcome this limitation, we took advantage of the ability of pre-adipocytes to form spheroids when cultured in ultra-low attachment systems. Therefore, the goal of our study was to develop adipose spheroids and validate their lipolysis responsiveness, when stimulated with reference molecules. Human pre-adipocytes were seeded in GravityTRAP ULA™ 96-well plates and cultured for 17 days using proliferation, differentiation and maturation media. We first showed that cells were able to generate spheroids with homogeneous size distribution (about 400 μm of diameter), to produce extracellular matrix and to secrete adiponectin in a culture plate format suitable for screening. Quantitation of spheroid volume demonstrated that spheroids expanded over time, mainly by accumulation of lipid droplets increasing in size within the spheroid. We showed that adipocyte maturation was inhibited (-30% of spheroid volume and strong decrease of lipid droplet size) when TNF-a was added in both differentiation and maturation media. We then characterized the lipolytic effects of caffeine (C) and evodiamine (E) by showing that both molecules decreased spheroid volume (C: -26%, E: -14%) in concomitance with fewer (C: -70%, E: -47%) and smaller-sized (C: -72%, E: -55%) lipid droplets. We finally showed that these lipolytic effects resulted in an increased release of free fatty acids (C: +25%, E: +26%) and a decreased secretion of adiponectin (C: -80%). In summary, we showed that adipose spheroids provide an interesting in vitro model of pre-adipocyte maturation and adipocyte expansion. They represent relevant in vitro assays to study adipogenesis, adipose tissue dysfunction and evaluate pharmacological agents.

Poly(ethylene glycol) (PEG) hydrogels have been extensively used as scaffolds for tissue engineering applications, owing to their biocompatibility, chemical versatility, and tunable mechanical properties. However, their bio-inert properties require them to be associated with additional functional moieties to interact with cells. To circumvent this need, we propose here to reticulate PEG molecules with poly(L-lysine) dendrigrafts (DGL) to provide intrinsic cell functionalities to PEG-based hydrogels. The physico-chemical characteristics of the resulting hydrogels were studied in regard of the concentration of each component. With increasing amounts of DGL, the cross-linking time and swelling ratio could be decreased, conversely to mechanical properties, which could be tailored from 7.7 ± 0.7 to 90 ± 28.8 kPa. Furthermore, fibroblasts adhesion, viability, and morphology on hydrogels were then assessed. While cell adhesion significantly increased with the concentration of DGL, cell viability was dependant of the ratio of DGL and PEG. Cell morphology and proliferation; however, appeared mainly related to the overall hydrogel rigidity. To allow cell infiltration and cell growth in 3D, the hydrogels were rendered porous. The biocompatibility of resulting hydrogels of different compositions and porosities was evaluated by 3 week subcutaneous implantations in mice. Hydrogels allowed an extensive cellular infiltration with a mild foreign body reaction, histological evidence of hydrogel degradation, and neovascularization.

Spheroids as microtissues are a powerful alternative to standard 2D cell culture for in vitro studies. 3D scaffold-free spheroids are formed within a few days from a cell suspension using hanging drop technology. The advantages of spheroids exclusively composed of fibroblasts rely on the physiological production of the extracellular matrix thanks to the aggregative capability of fibroblasts to self-assemble in a round tridimensional structure. This microdermis presents a complex tissue organization that closely mimics the architecture and composition of the human dermis in vivo. The aim of this study was to characterize structural and biomechanical properties of spheroids composed of normal human fibroblasts. The density and the structural organization of the elastic fiber network within spheroids were firstly characterized using second harmonic generation microscopy, a powerful technique for autofluorescent observation of dense and ordered fibrillary macrostructures. Biomechanical properties of spheroids were then evaluated using atomic force microscopy, a nanoindentation tool that can determine the Young's modulus and therefore elastic properties of the microdermis. The combination of these two analysis techniques on a same spheroid allowed us to establish a perfect correlation between the fluorescent signal indicating elastic fiber presence and the mechanical properties of the spheroid. Indeed, we demonstrated that the increase of elastin amount within a spheroid resulted in the decrease of the elastic modulus value. These results highlight the elastic properties of the microdermis which is mechanically softer thanks to the presence of elastic fibers. Therefore, a spheroid is a biomimetic model in which mechanical and structural elastic properties are related to each other. This correlative study outlines spheroids as a reliable microdermis model to test active ingredient efficiency on elastin synthesis and biomechanical properties of the microtissue.

Les peptides élastiques (ELP, Elastin-like peptide) sont d'excellents exemples de polymères biomimétiques récemment proposés en médecine régénérative, en particulier dans le domaine de l'ingénierie tissulaire des tissus mous (peau, vaisseaux sanguins, poumons…) pour lesquels la modélisation est complexe car l'instruction correcte des cellules nécessite une élasticité fonctionnelle. L'ajustement précis de la structure primaire des ELP peut moduler voire améliorer les propriétés physico-chimiques, structurales et fonctionnelles de la protéine native. De plus, la capacité des ELP à ajuster leurs caractéristiques physico-chimiques en réponse à des stimuli externes (température, pH), les définit comme des polymères intelligents. Ces polymères bioactifs offrent ainsi une large gamme d'applications très prometteuses encore très peu explorées dans les technologies d'ingénierie tissulaire et les systèmes d'administration de médicaments. Dans ce travail de thèse, nous avons développé, caractérisé et évalué les potentiels thérapeutiques d'une protéine élastique synthétique, Elactiv', inspirée de la structure unique de la tropoélastine humaine, précurseur soluble de l'élastine. Elactiv' conserve les caractéristiques physico-chimiques (comportement thermosensible, propriétés d'autoassemblage) et les fonctions biologiques de la protéine native (prolifération, différenciation et survie des fibroblastes dermiques et kératinocytes humains, sensibilité à la dégradation enzymatique). De plus, Elactiv' possède la particularité in vitro de s'incorporer dans les fibres élastiques néo-synthétisées par des fibroblastes dermiques sains, et d'induire la synthèse de tropoélastine fibrillaire par des fibroblastes pathologiques, syndrome de Williams-Beuren, qui ne synthétisent pas ou très peu de fibres élastiques. Un hydrogel formé exclusivement d'Elactiv' a permis d'accéder aux propriétés mécaniques de l'ensemble et de vérifier sa biocompatibilité in vitro et son innocuité et sa résorption in vivo. Enfin, l'association de la protéine Elactiv' aux dendrigrafts de poly(L-lysine), polymères synthétiques hautement fonctionnalisables, a permis de faire évoluer l'architecture de l'hydrogel vers un biomatériau hybride dans le but d'augmenter ses propriétés mécaniques et biologiques. Ainsi, les potentiels biomimétiques et thérapeutiques de la protéine Elactiv' en font un candidat prometteur pour la régénération des tissus mous

Le microenvironnement est connu pour influencer le comportement cellulaire par des facteurs biochimiques et biomécaniques spécifiques pour chaque tissu. En ingénierie tissulaire, la modélisation des tissus mous est complexe dans la mesure où l’instruction correcte des cellules nécessite une élasticité fonctionnelle. Les recombinamères d’élastine (REL) sont des polymères versatiles se présentant comme de potentiels outils pour imiter le microenvironnement cellulaire élastique avec des propriétés thermo-mécaniques définies et les fonctions biologiques. Cette étude décrit le design, la synthèse et la caractérisation d’un REL particulier inspiré de la structure unique de la tropoélastine humaine. Ce REL de 50,6 kDa conserve un point isoélectrique très basique (10,31) et la capacité intrinsèque de coacervation de façon similaire à la tropoélastine, avec une température de transition de 35 °C. Cette propriété de coacervation permet notamment de purifier notre REL par le processus de cycle de transition inverse. Son repliement, déterminé par dichroïsme circulaire, est caractérisé par une hélice gauche de type polyproline II. La microscopie électronique révèle de petites sphères creuses individuelles à de faibles concentrations. Ces sphères tendent à fusionner et à se fondre en réseaux fibrillaires à des concentrations plus élevées, démontrant ainsi des propriétés d’auto-assemblage prometteuses. Enfin, en présence d’un agent réticulant, il est possible d’obtenir un hydrogel dont les propriétés mécaniques sont comparables à celles des tissus mous élastiques comme la peau.

Poly(L-lysine) (PLL) dendrigrafts (DGLs) are arborescent biosynthetic polymers of regular and controlled structures. They have specific properties such as biocompatibility and non-immunogenicity, and their surface density of NH2 functions can be easily modified and therefore appears as a powerful tool for the functionalization of hydrophobic polymers used in the context of tissue engineering. In this study, we evaluated several criteria of human skin fibroblasts when cultured with DGL of generations 2, 3 and 4, with linear PLL polymer as reference. In aqueous phase, DGLs and PLL displayed a similar cytotoxicity towards fibroblasts. Plastic culture plates grafted with DGLs were further characterized as homogeneous surfaces by atomic force microscopy and surface characterization by amino density estimation by colorimetric assay. Proliferation of fibroblasts was increased when cultured onto PLL and DGLs monolayers when compared with crude plates. Cellular adhesion was increased by 20% on DGLs in comparison to PLL. Integrin α5 subunit protein expression level was increased after 48 h of culture on DGLs, in comparison to control or PLL-coated surfaces. The presence of DGLs did not lead to overexpression or activation of matrix metalloproteinases 2 and 9. Finally, fibroblasts adhesion was increased by 40% on poly-(lactic-co-glycolic acid) matrices functionalized with DGLs when compared to PLL. Overall, these features make DGL promising candidates for the surface engineering of biomaterials in tissue engineering.