1. INTRODUCTION As part of our ongoing search for predictive tools to understand skin responses, we are evaluating tissue engineered human skin equivalent cultures. Of particular interest is understanding of skin cell responses to compounds, such as N-acetyl glucosamine and niacinamide, that are included in cosmetic skin care compositions. Such compositions include those intended for use by individuals with signs of facial skin aging. To define in vitro responses of epidermal-dermal and melanocyte- containing human skin equivalent cultures to compounds used in anti- aging cosmetic products. Rosemarie Osborne, Ph.D., Lisa A. Mullins, B.S., Bradley B. Jarrold, M.S., Sara McPhail, B.S., Larry Robinson, Ph.D. The Procter & Gamble Company, Cincinnati, Ohio USA Evaluation of Cosmetic Ingredients in Tissue Engineered Human Skin In Vitro In human skin equivalent cultures (Fig. 1), compounds used in anti-aging cosmetic products, including N-acetyl glucosamine and niacinamide, increased production of skin matrix components. N-Acetyl glucosamine increased hyaluronan and collagen (Fig. 2a & b), acting synergistically with niacinamide to increase collagen (Fig. 3). Skin equivalent cultures containing melanocytes transfer melanin from the melanocytes to keratinocytes, and pigment over time (Fig. 4). Niacinamide, an inhibitor of melanocyte transfer from melanocytes to keratinocytes, and N-acetyl glucosamine, which inhibits activation of tyrosinase (the rate-limiting enzyme in melanin synthesis) act synergistically to reduce pigmentation (Fig. 5). These results indicate that in vitro human skin equivalent skin models provide a useful approach to understand skin care product compounds. CONCLUSIONS RESULTS OBJECTIVE In Vitro Human Skin Equivalents The MatTek Human Skin EpiDermFT and Melanoderm Skin Models (MatTek Corp.) were treated with solutions of test compounds (100 ul/culture) applied topically to the stratum corneum surface of the skin equivalent cultures for 1 to 7 days at 37°C. Biomarker Assays Hyaluronan and Procollagen 1 in cell extracts were measured by ELISA (Corgenix; Takara Bio Inc.), and normalized to total protein (Coomassie blue). mRNA levels were measured by RT-PCR (Qiagen Inc.). Melanin was measured spectrophotometrically (450 nm, VMax, Molecular Devices Corp.), and compared to a standard curve of synthetic melanin (Sigma Chemicals) for quantification. Mean biomarker values from n=4 cultures and representative of at least 3 independent experiments are shown; SEM values were <10% of mean. METHODS Stimulation of Hyaluronan and Collagen Expression with N-Acetyl Glucosamine and Niacinamide in Full-Thickness Epidermal-Dermal Skin Equivalent Cultures Full thickness skin equivalent cultures contain a cornified stratified squamous epithelium and a fibroblast-containing dermis (Fig. 1). Following a 24- hr topical treatment with N-acetyl glucosamine, hyaluronan (Fig. 2a) and procollagen-1 (Fig. 2b) increased significantly relative to vehicle control, and in a dose-responsive manner. In addition, the combination of low concentrations of N-acetyl glucosamine and niacinamide acted synergistically to increase procollagen-1 (Fig. 3). Decreased Melanin with N-Acetyl Glucosamine and Niacinamide in Melanocyte-containing Epidermal Cultures The cultures evaluated contain a cornified stratified squamous epithelium and melanin producing melanocytes (Fig. 4). A top view reveals stellate melanocytes together with cuboidal keratinocytes (Fig. 4a). In cross-section, the melanocytes are located on the basement membrane and transfer melanin to the differentiating keratinocytes (Fig. 4b, paraffin sections with Fontana-Masson stain). The cultures pigment over a 13 day growth period (Fig. 4c, cultures are 1 cm in diameter). Individual treatments with niacinamide and N-acetyl glucosamine decreased melanin formation, and acted synergistically to reduce melanin (Fig. 5). This work was funded by P&G Beauty 3a3b SC E Stratum Corneum Viable Fibroblasts Epidermis Dermis Figure 1. Human Full-Thickness Skin Cultures Figure 2. Increased Hyaluronan (a) and Procollagen-1 (b) with N-Acetyl Glucosamine 2a. Hyaluronan 2b. Procollagen-1 * * * * * *p < 0.05 vs control; t-test. Figure 3. Synergy between N-Acetyl Glucosamine and Niacinamide to Increase Procollagen-1 * *p < 0.01 vs control; t-test. Figure 4. Human Melanocyte-Epidermal Cultures 4b.4a.4c. 3 Days 6 Days 10 Days 13 Days *p < 0.01 vs control; t-test. Figure 5. Deceased Melanin Production with N-Acetyl Glucosamine and Niacinamide * * *