Céline Viennet

Contractile forces generated by striae distensae fibroblasts embedded in collagen lattices

Striae distensae are characterized by linear, smooth bands of atrophic-appearing skin that are reddish at first and finally white. They are due to stretching of the skin, as in rapid weight gain, or mechanical stress, as in weight lifting. The pathogenesis of striae distensae is unknown but probably relates to changes in the fibroblast phenotype. In order to characterize striae distensae fibroblasts, alpha-smooth muscle actin expression and contractile forces were studied. Five healthy women with early erythematous striae and five healthy women with older striae were selected. Paired biopsies were taken from the center of lesional striae and adjacent normal skin. Fibroblasts were obtained by an explant technique and expanded in vitro in Dulbecco’s modified Eagle‘s medium. Contractile forces generated by fibroblasts in collagen lattices were measured with the Glasbox device developed in our laboratory. Alpha-smooth muscle actin expression was studied by immunofluorescence labeling of cells and by flow cytometry. Fibroblasts from early striae distensae were the richest cells in alpha-smooth muscle actin filaments and generated the highest contractile forces. Their peak contractile force was 26% greater than normal fibroblasts. There was a 150% higher level of alpha-smooth muscle actin content in fibroblasts from early striae distensae compared with fibroblasts from normal skin. In contrast, there was no significant difference in force generation between old striae fibroblasts and normal fibroblasts with cells expressing no alpha-smooth muscle actin. The contractile properties of fibroblasts from striae distensae varies depending on the stage of the disease. In early striae distensae, fibroblasts acquire a more contractile phenotype, corresponding to that of myofibroblasts.

Indication of fibroblast apoptosis during the maturation of disc-shaped mechanically stressed collagen lattices

Human dermal fibroblasts seeded in tense disc-shaped collagen gels organized gradually into a tissue composed of three distinct superimposed layers of cells: a basal layer of aligned myofibroblasts, a middle layer of unoriented fibroblasts, and an upper layer of myofibroblasts oriented orthogonally to the basal myofibroblasts. Confocal observation of α-smooth muscle actin (α-SMA) immunolabelling as a marker of myofibroblasts showed that the upper myofibroblasts disappeared during maturation of the lattices. Observation of morphological modifications of cells, typical chromatin condensation identified by Hoechst 33258 staining, and detection of ssDNA after formamide-induced denaturation suggested the involvement of apoptosis in myofibroblast disappearance. It is suggested that the model of disc-shaped stressed collagen lattice is thus able to mimic conditions in wound repair: on the one hand, wound contraction during which fibroblasts undergo mechanical stress and, on the other, apoptotic disappearance of cells at the end of tissue retraction.

Effects of topical corticosteroids on cell proliferation, cell cycle progression and apoptosis: In vitro comparison on HaCaT

Topical-corticosteroids are mainly used for the treatment of inflammatory or hyperproliferative skin diseases. The in vivo assay to rank topical-corticosteroids potency, based on the skin blanching, is not adapted to compare their anti-proliferative efficacy. We have compared the antiproliferative effect of six topical-corticosteroids on a model of hyperproliferant keratinocytes (HaCaT). Betamethasone-dipropionate; clobetasol-propionate; betamethasone-valerate; desonide; hydrocortisone-butyrate and hydrocortisone-base, at different concentrations (10(-8)-10(-4)M) have been compared. HaCaT proliferation has been evaluated by MTT-assay and the mechanism of the death was evaluated by annexin V/propidium iodide staining and cell cycle phases analysis. Topical corticosteroids reduced cell growth in a dose-dependent manner. At 10(-4)M, betamethasone dipropionate was the most antiproliferative compound while hydrocortisone-butyrate was the less. Hydrocortisone-base which is usually considered as the less potent topical-corticosteroids showed a clear cytotoxic effect. Betamethasone-dipropionate and betamethasone-valerate induced more apoptosis than necrosis whereas the reverse has been observed for other topical-corticosteroids. All topical-corticosteroids, except clobetasol-propionate, arrested cell cycle mainly in G2-phase. Clobetasol-propionate arrested cell cycle in S-phase population. At 10(-8)M, topical-corticosteroids induced HaCaT proliferation. In terms of antiproliferative effect at 10(-4)M, we propose to rank topical corticosteroids as follow: betamethasone-dipropionate>desonide≥betamethasone-valerate=hydrocortisone-base=clobetasol-propionate>hydrocortisone-butyrate. This classification differs from the current ranking, based on the vasoconstrictive effect, but is more adapted for hyperproliferative disease treatment.

In the shadow of the wrinkle: theories

As time passes, wrinkles typically appear. These skin depressions that become deeper and deeper draw more and more coarser lines on almost all the visible parts of aging individual’s skin. They are indeed the most obvious and maybe disliked signs of skin aging, and thus, preventing and treating them are a major topic for dermo‐cosmetic laboratories. However, the cause and occurrence mechanism of these simplistic looking lines are not yet fully understood. Wrinkling is thought to be a complex biophysical process resulting from repeated strains on a progressively, structurally and biochemistry altered aging skin with impaired mechanical properties. Focus is made on the specific histological features of the wrinkle compared to the surrounding aging skin. The numerous age‐related changes in human skin that are supposed to be involved in wrinkling are briefly reviewed, and the current theories on wrinkle formation linked to these changes are also discussed.

Artocarpus altilis heartwood extract protects skin against UVB in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE Artocarpus altilis (Moreceae) has been widely used as a traditional folk medicine in Southeast Asia for the treatment of many diseases, including skin disorders, such as ulcers and dermatitis. AIM OF THE STUDY The present study aimed to investigate the ability of an artocarpin-enriched extract to prevent ultraviolet radiation B-induced photodamage. MATERIALS AND METHODS The content of artocarpin in the extract was determined by high performance liquid chromatography (HPLC). A DPPH assay was used to evaluate the free radical scavenging activity of the extract, which was compared with those of l-ascorbic acid and α-tocopherol. Cytotoxicity and proliferation of cells treated with the extract were determined using XTT and BrdU assays, respectively. Human skin fibroblasts and keratinocytes were pretreated with the extract for 24h and later irradiated with ultraviolet radiation B at 128 J/cm(2). The levels of TNF-α and IL-6 released from ultraviolet radiation B-irradiated keratinocytes and, MMP-1 and type-I procollagen produced by ultraviolet radiation B-irradiated fibroblasts were measured by ELISA and/or western blotting. The hairless skin of male mice (outbred ICR) was treated with the extract or l-ascorbic acid solution prior to exposure to ultraviolet radiation B irradiation. The dose of ultraviolet B irradiation was consecutively increased to 18, 36, 54, and 72 J/cm(2) at weeks 1-4, 4-7, 7-10, and 10-12, respectively. The epidermal thickness and collagen content in the skin of ultraviolet radiation B-irradiated mice were evaluated. RESULTS The extract concentration of 50 µg/mL was not toxic and did not inhibit the proliferation of fibroblasts. The pretreatment of fibroblasts with 50 µg/mL extract prior to ultraviolet radiation B irradiation attenuated MMP-1 production but did not affect type-I procollagen production. The extract also decreased the ultraviolet radiation B-induced production of TNF-α and IL-6 in keratinocytes. Moreover, the topical administration of the extract suppressed epidermal thickening and collagen loss in chronically ultraviolet radiation B-exposed skin in mice. CONCLUSIONS The experimental study revealed that A. altilis extract suppresses structural alterations in skin damaged by ultraviolet radiation B irradiation. This suppression was, at least partially, mediated by decrease in MMP-1 production in fibroblasts and TNF-α and IL-6 productions in keratinocytes.

In the shadow of the wrinkle: experimental models

Research on aging has run for decades, and knowledge on the biologic process of skin chronological and photoaging is still increasing thanks to read across results generated between human, animal, and in vitro studies. However, wrinkles should not be considered to result only from the aging process. There are few reports on specific wrinkle histological features compared to the surrounding skin, and there is thus a need in really wrinkling skin animal and in vitro models. UV‐irradiated Hr mouse is a good model because it develops wrinkles. Nevertheless, as mouse skin is somehow different from human skin, the innovative model of wrinkling human skin xenograft on SCID mice seems to be really promising. Concerning in vitro and ex vivo models, although there have been considerable advances in reconstructing realistic aged skins, there is still a lack of in vitro wrinkling skin model, and unfortunately, this gap will probably be difficult to fill.

Development and characterization of a human dermal equivalent with physiological mechanical properties.

Different models of reconstructed skin are available, either to provide skin wound healing when this process is deficient, or to be used as an in vitro model. Nevertheless, few studies have focused on the mechanical properties of skin equivalent. Indeed, human skin is naturally under tension. Taking into account these features, the purpose of this work was to obtain a cellularized dermal equivalent (CDE), composed of collagen and dermal fibroblasts.

Submicron polycaprolactone particles as a carrier for imaging contrast agent for in vitro applications.

Fluorescent materials have recently attracted considerable attention due to their unique properties and high performance as imaging agent in biomedical fields. Different imaging agents have been encapsulated in order to restrict its delivery to a specific area. In this study, a fluorescent contrast agent was encapsulated for in vitro application by polycaprolactone (PCL) polymer. The encapsulation was performed using modified double emulsion solvent evaporation technique with sonication. Fluorescent nanoparticles (20 nm) were incorporated in the inner aqueous phase of double emulsion. A number of samples were fabricated using different concentrations of fluorescent contrast agent. The contrast agent-containing submicron particle was characterized by a zetasizer for average particle size, SEM and TEM for morphology observations and fluorescence spectrophotometer for encapsulation efficiency. Moreover, contrast agent distribution in the PCL matrix was determined by confocal microscopy. The incorporation of contrast agent in different concentrations did not affect the physicochemical properties of PCL particles and the average size of encapsulated particles was found to be in the submicron range.

Precise role of dermal fibroblasts on melanocyte pigmentation

Dermal fibroblasts are traditionally recognized as synthesizing, remodeling and depositing collagen and extracellular matrix, the structural framework for tissues, helping to bring thickness and firmness to the skin. However, the role of fibroblasts on skin pigmentation arouses concern recently. More is known about the interactions between epidermal melanocytes and keratinocytes. This review highlights the importance of fibroblast-derived melanogenic paracrine mediators in the regulation of melanocyte activities. Fibroblasts act on melanocytes directly and indirectly through neighboring cells by secreting a large number of cytokines (SCF), proteins (DKK1, sFRP, Sema7a, CCN, FAP-α) and growth factors (KGF, HGF, bFGF, NT-3, NRG-1, TGF-β) which bind to receptors and modulate intracellular signaling cascades (MAPK/ERK, cAMP/PKA, Wnt/β-catenin, PI3K/Akt) related to melanocyte functions. These factors influence the growth, the pigmentation of melanocytes via the expression of melanin-producing enzymes and melanosome transfer, as well as their dendricity, mobility and adhesive properties. Thus, fibroblasts are implicated in both skin physiological and pathological pigmentation. In order to investigate their contribution, various in vitro models have been developed, based on cellular senescence. UV exposure, a major factor implicated in pigmentary disorders, may affect the secretory crosstalk between dermal and epithelial cells. Therefore, identification of the interactions between fibroblasts and melanocytes could provide novel insights not only for the development of melanogenic agents in the clinical and cosmetic fields, but also for a better understanding of the melanocyte biology and melanogenesis regulation.

Polycaprolactone Based Nanoparticles Loaded with Indomethacin for Anti-Inflammatory Therapy: From Preparation to Ex Vivo Study

PurposeThis work focused on the preparation of polycaprolactone based nanoparticles containing indomethacin to provide topical analgesic and anti-inflammatory effect for symptomatic treatment of inflammatory diseases. Indomethacin loaded nanoparticles are prepared for topical application to decrease indomethacin side effects and administration frequency. Oppositely to already reported works, in this research non-invasive method has been used for the enhancement of indomethacin dermal drug penetration. Ex-vivo skin penetration study was carried out on fresh human skin.MethodsNanoprecipitation was used to prepare nanoparticles. Nanoparticles were characterized using numerous techniques; dynamic light scattering, SEM, TEM, DSC and FTIR. Regarding ex-vivo skin penetration of nanoparticles, confocal laser scanning microscopy has been used.ResultsThe results showed that NPs hydrodynamic size was between 220 to 245 nm and the zeta potential value ranges from −19 to −13 mV at pH 5 and 1 mM NaCl. The encapsulation efficiency was around 70% and the drug loading was about 14 to 17%. SEM and TEM images confirmed that the obtained nanoparticles were spherical with smooth surface. The prepared nanoparticles dispersions were stable for a period of 30 days under three temperatures of 4°C, 25°C and 40°C. In addition, CLSM images proved that obtained NPs can penetrate the skin as well.ConclusionThe prepared nanoparticles are submicron in nature, with good colloidal stability and penetrate the stratum corneum layer of the skin. This formulation potentiates IND skin penetration and as a promising strategy would be able to decline the side effects of IND.