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52 ANTI-AGEING 300


*** Real


250


value: 471


200 150 **


100 * 50 * ** * * * * ** *** **


0


Retinyl linoleate


Retinyl sunflowerseedate


Retinyl palmitate


Retinol


Figure 2: Cell proliferation by BrdU incorporation assay.


on skin, including redness, dryness and flaking.11 Esters of retinol (e.g. Retinyl palmitate) are also used in anti-ageing skin care products because they are more stable than retinol. However, retinyl palmitate has a reputation as being less effective as an anti-ageing ingredient than retinol.6 To combine the effectiveness of retinol,


but without the irritation, and the stability of retinyl esters, a different class of retinol derivatives was developed. These ingredients are retinol derivatised with either purified linoleic acid (INCI: Retinyl linoleate) or with a natural blend of unsaturated fatty acids derived from sunflower seed oil (INCI: Retinyl sunflowerseedate). The fatty acid component of both retinoids is rich in unsaturated and polyunsaturated fatty acids, proposed to offer better solubility in skin lipids and penetration efficacy.


The aim of this study was to compare the


in vitro effects of different retinoids retinol, retinyl palmitate, retinyl linoleate, and retinyl sunflowerseedate on cell turnover/proliferation, and on production of HA in Normal Human Dermal Fibroblasts (NHDFs).


Materials and methods Cell culture and treatments: Normal Human foreskin-derived Dermal Fibroblasts (NHDF) were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco/Life Technologies, 31885) supplemented with 10% of Fetal Bovine Serum (FBS; Gibco/Life Technologies, 10270) and antibiotics (Penicillin/Streptomycin, Gibco/ Life Technologies, 15140). Cells were maintained in a humidified incubator at 37°C with a 5% CO2 atmosphere. The NHDF fibroblasts were plated on 24-well plates in complete culture medium 24h before the treatments. Then, the cells were


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treated during 72h with the test compounds in a culture medium without FBS. Transforming growth factor-beta 1 at 20ng/ml (TGF-β1; R&D systems, 240-B), ascorbic acid at 500µM (vitamin C, VWR, 83568.180) and oleoyl-L-alpha- lysophosphatidic acid sodium salt at 3µM (LPA sodium salt; Sigma Aldrich, L7260) were used as reference conditions. All the treatments were performed in triplicate of culture (n=3). The test compounds were pre-solubilised in DMSO except for retinol which was solubilised in chloroform. BrdU incorporation assay: cell proliferation


was measured by the colorimetric Cell Proliferation ELISA kit (Roche, 11 647 229 001) based on the use of 5-bromo-2’-deoxyuridine (BrdU), a synthetic nucleoside incorporating newly synthetized DNA instead of thymidine, during the cell cycle. After 56h of treatment by the test compounds (in medium without FBS), BrdU was added to the culture medium and incubated overnight at 37°C. Afterwards, ELISA test with an anti-BrdU antibody was performed to measure BrdU incorporation into DNA of mitotic cells following the manufacturer’s instructions. Complete medium (containing 10% FBS) and mitomycin C (2µg/ml) were used in parallel respectively as positive and negative controls. Quantification of HA production: at the end


of the treatments, all the supernatants were collected and stored at -20°C until further use for the quantifications of the extracellular release of hyaluronan (HA; R&D systems, DHYAL0) according to the supplier’s specifications.


Results Test compounds were initially screened to estimate an effective range of treatment concentrations for each test retinoid. To assess the effects on cell survival and proliferation,


NHDF cells were treated with the test retinoids at dilutions from 0.1% to 0.001%. Figure 1 shows representative images of fibroblast cultures after 72h of treatment under control conditions and with test retinoids. Cells treated with the three retinyl esters showed no signs of cytotoxicity at 0.01% and below, while retinol required a 10-fold further dilution to 0.001% before normal cell growth was observed. These screening tests established the maximum treatment concentration for each test retinoid. We first sought to determine if the test


retinoids were able to modulate the cell proliferation potential of dermal fibroblasts. Cell proliferation was measured using a BrdU assay, in which the thymidine analog BrdU (5-bromo- 2’-deoxyuridine) following its incorporation into newly synthesised DNA and subsequent detection with an anti-BrdU antibody. To quantify relative cell proliferation potential, NHDF cells were treated for 72h with the test retinoids at five different concentrations in the pre-determined ranges, along with the untreated control, medium supplemented with 10% FBS was used as proliferative control, and mitomycin C at 2µg/ml as anti-proliferative molecule.13


The


solvents used for the preparation of the test items were assessed in parallel at 0.1% for each test compound. The proliferative potential (BrdU incorporation) of each condition is presented in Figure 2 relative to the untreated control which was set at 100%. A statistical analysis was performed to compare the effects of each treatment to respective controls including solvent alone. As expected, treatment with Mitomycin C (2µg/ml) and FBS (10%) significantly decreased and increased cell proliferation, respectively, relative to the untreated control. When cells were treated with the different retinoids, a significant


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Cell proliferation (% reative to untreated control)


Untreated control


Mitomycin C 6h 2µg/ml FBS 10%


DMSO control 0.1% 0.01%


0.005% 0.001% 0.0005% 0.0001% 0.01% 0.005% 0.001% 0.0005% 0.0001% 0.01% 0.005% 0.001% 0.0005% 0.0001% Chloroform control0.1% 0.01% 0.005% 0.001% 0.0005% 0.0001%


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