Research of cellular physiological mechanisms

1. Dermatocosmetic ingredients testing on standardized cellular cultures Research of cellular physiological mechanisms Development of new concepts regarding anti-ageing active ingredients Development of cellular investigative techniques Comparative analysis and correlation with non-invasiveness tissue methods Innovation of dermatocosmetic products New solutions for antiageing topical product investigations Classical methods of diagnosis and testing Efficacy checking of intermediate or final formulations Innovation in dermatocosmetic, anti-ageing tests Innovation in clinical dermatology Technological process innovation / Rising performances for tested products CORRELATIVES AND COMPLEMENTARY EXPERIMENTAL MODELS FOR THE DEVELOPMENT OF ANTIAGEING DERMATOCOSMETIC PRODUCT Authors: Laura Olariu, Brindusa DumitriuBIOTEHNOS, Research & Development, Otopeni, Romania. Decreased cell function in aged andphotodamagedskin appears to be due to abnormalities in the signaling pathways that regulate matrix production. In human dermal fibroblasts, TGF-β not only functions as primary stimulus for collagen synthesis, but also reduced collagen degradation by inhibiting MMP-1 expression. INTRODUCTION: Despite the exponential develop of cosmetics with pharmaceutical activity, appropriates methods to quantify the effects are not yet established. The efficacy claim is a widespread effect for many skin care products, but usually a few test were done to scientific prove this action. IMPLEMENTATION OF “IN VITRO” AND “IN VIVO” METHODS FOR SCREENING TECHNIQUES AND PRODUCT DEVELOPMENT • DEFINITION: “in vitro” and “in vivo” screening techniques comprise an assembly of instrumental methods, comparative and correlative, oriented to prove: • the claimed effect and • the specific target action of an ingredient. “IN VITRO” METHODS TO PROVE THE “ANTIAGEING “ EFFECT OF DERMATOCOSMETIC INGREDIENTS: Cell cultures: • RECONSTITUTED HUMAN SKIN • STANDARDISED CELL LINES: dermal human fibroblasts (ex. HS27), normal human keratinocytes (ex. Immortalised cell line HaCat) Relevant cellular parameters: “IN VIVO” , NON-INVASIVE TECHNIQUES TO PROVE THE “ANTIAGEING “ EFFECT OF DERMATOCOSMETIC PRODUCTS: CITOTOXICITY • Studies on healthy volunteers using • NON-INVASIVE METHODS: Microscopic evaluation (selection) • MTT/MTS reduction Before treatment: R4= 0.217; R3= 0.642; Fo= 0.057 SKIN ELASTICITY CUTOMETER MTS is a tetrazolium salt which is transformed in a formazan type product by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan product as measured by 490nm absorbance is directly proportional to the number of living cells in culture After treatment: R4= 0.113; R3= 0.389; Fo= 0.040 LDH release COLAGEN DEPOSITS VISUALISATION The mechanical parameters R4 R3 and F0 (area) provided by Cutometer measurements are most indicative of human skin fatigue. Lactatdehidrogenase release in the extracellular medium is a potent indicator of citotoxic agent action Citotoxicity curve for a dermatocosmetic ingredient Riss, T. and Moravec, R.A. (2004) - Assay Drug Dev. Technol.2, 51–62. DERMASCAN • Collagen deposits visualization by • high frequency ultrasound scanners • (ex. DERMASCAN - Cortex Technology) The age-related decrease of skin elasticity results in larger fatigue of adult skin than young skin after applying multiple stress at one and the same anatomic region. The non-invasive method applied can be useful for objective and quantitative investigation of age-related changes in skin fatigue and evaluation of the effects of cosmetic and antiaging topical products. CELL PROLIFERATION CFSE (carboxyfluoresceindiacetatsuccinimidil ester ) is a cell permeantfluorescein-based dye which covalenty attaches to cytoplasmic components of cells, resulting in uniform bright fluorescence. Upon cell division, the dye is distributed equally between daughter cells, allowing the resolution of up to eight cycles of cell division by flow cytometry. Succesive generationproliferation for a dermatocosmetic ingredient ANTI-WRINKLE Before anti-ageing treatment ROUGHNESS SKIN VISIOMETER / VISIOSCAN SMOOTHNESS • D N A synthesis: Cell cycle progression visualized by flow cytometry After anti-ageing treatment PI staining of nuclear DNA show a histogram representation of G0/G1, S and G2/M cell cycle phases Cellcyclesequentiation for a dermatocosmetic ingredient BA Bach, WA Knappe, MG Edinger – Am. J.Pathol. 1991; 96:615-627 TGF-β ACTIVATION, SOLUBLE PROTEIN RESPONSIBLE FOR COLLAGEN HOMEOSTASIS • Application: • Compoundsdesigned in BiotehnosLaboratories: • Dermo – Oz – andDermo – O – phytocompoundsfromCallendulaofficinalis • Risethefibroblastsproliferation, but activate themetalloproteinasesfromextracellularmatrix; significanteffects on tissuerepairandphotoageingtreatment. • Dermo – U – phytocompoundfromSalvia officinalis • “Estrogen-like” effecton fibroblasts, quicklyactsonproliferationstimulationandcollagensynthesis; • Dermo – ET - biocomplex fromTrifoliumPratense • “Estrogen-like” effecton fibroblasts, quicklyactsoncellular turn-over (augment D N A synthesisandproliferation) COLAGEN METABOLISM • Materials: • Human TGF beta1 Single Plex Flex Set (BD CBA) • Human Soluble Protein Master Buffer Kit (BD CBA) • Method :beads - based flow cytometry assay for the quantization of soluble proteins (TGF- β) Sandwich Complex sample bead with specific antibodies Detection antibodies , PE stained L.Rhein – Ageing Skin: Current and future Therapeutic strategies –Allured Bussiness Media -2010 • MATRIX METALOPROTEINASES activation ←MMP-9 (92kDa) ←MMP-2 (72kDa) The MMPs are members of the unique family of proteolytic enzyme which contain a zinc ion at their active sites and can degrade native collagens and other ECM components. Gelatin-based zymography of the culture medium of HS27 cells treated with COSMETIC INGREDIENTS. CONCLUSIONS: Hee-Jae Cha, Soo-Kyung Bae, Ho-Young Lee, Ok-Hee Lee, Cancer Research 56, 2281-2284, May 15, 1996 • All anti-ageing skin care products have pharmaceutically effect, including the enhanced wound healing effects, acting at cellular / molecular level, so scientific testing methodologies are strictly recommended • A cosmetic may be characterized in the light of increasing knowledge of its effect on normal or damaged skin • “in vitro” tests provide a relevant and complex quantification regarding anti-ageing ingredients effect at cellular/molecular level – a necessary preliminary report about product efficacy • “In vivo” relevance of “in vitro” tests are strong connected with the efficacy of future anti-ageing skin care products • The regulatory agencies should protect the public from unproven claims and unsafe materials, imposing scientific test for cosmetic effects • The implementation of a skin functional parameters scanning in respect of the active ingredients action will have a deep impact in: • the quality of final product formulation • a better treatment scheme for dermatological dysfunctions • rising performances for the new skin care anti-ageing ingredients • COLAGEN synthesis Collagen is one of the more important structural proteins in the body being of particular importance in connective tissues by providing their durability. As such, knowledge of at least the amonunt of collagen in a particular tissue is essential for the complete understanding of the structural and mechanical proprities of that tissue. Fields and Dunn have suggest that it is the structural proteins, collagen primarily, which are responsible for the tissue’s echographicvisualizability. Collagen contains a unique aminoacid in its structure: Hydroxyproline. Hyp level is strong correlated with the total collagensynthetised. (1mg of collagen contain 0.0122mg Hyp) G. KesavaReddyandChukuka S. Enwemeka, ClinicalBiochemistry, Vol.29, No. 3. 225-229, 1996 Project title: POS CCE ID 383 SMIS CSNR 6009 CTR 107/2010 - DERMOLAB “International standards implementation for the organisation of a dermato-cosmetic research and testing core” Material editor: SC BIOTEHNOS SA Date of publication: 11.05.2011 Experimental developement and inovation activities in dermatocosmetics are done with financial help from POS CCE ID 383 SMIS CSNR 6009 CTR 107/2010 This report doesnotnecessarilyrepresenttheofficialposition of the European Union or the Romanian Government