Cosmetic efficacy claims in vitro using a three-dimensional human skin model

A tissue engineered human skin equivalent is successfully used for the testing of raw materials and cosmetic formulations. This reconstructed skin is supported by a collagen-glycosaminoglycan-chitosan biopolymer in which human keratinocytes and dermal fibroblasts were co-cultured to form a tissue that closely reproduces the in vivo architecture of normal human skin and takes into account the complex interactions between epidermis and dermis. On the other hand, dermal and epidermal responses can be assessed separately in the dermal or skin equivalent. The three-dimensional model has important advantages compared to monolayer cell cultures and epidermis models in efficacy testing: (i) the possibility of long-term cultivation with repeated application of cream formulations containing bioactives and (ii) the similarity to human skin concerning the interaction between dermis and epidermis. These similarities include the expression of keratinocyte differentiation markers such as cytokeratin 10, filaggrin and transglutaminase, as well as proteins of the basal lamina (laminin, collagen type IV) and extracellular matrix proteins such as elastin. The efficacy of selected bioactives was determined using different endpoints, for example, stimulation of collagen synthesis in the dermal and skin equivalents was shown in comparison to vitamin C as a positive control. On skin equivalents using immunofluorescence techniques we also demonstrated stimulation of the differentiation marker filaggrin, which is important for skin moisturization. The results could be used for claim substantiation, e.g. for the treatment of dry and aged skin.     

Oligogalacturonides improve tissue organization of in vitro reconstructed skin

The aim of this study was to analyse the effects of oligogalacturonides obtained from apple pectin enzymatic hydrolysis (mainly composed of galacturonic acid and oligogalacturonides; OGA) on normal human keratinocytes behaviour using different in vitro models. We demonstrate that 0.01% OGA promotes epidermal growth, organization and stratification in an in vitro reconstructed skin. The presence and the in vivo-like location of epidermal differentiation markers (i.e. keratin 10, involucrin, desmoglein 1 and 3, and cathepsin D) confirms the histological analysis, and underlines the cohesion of the treated epidermis. On the opposite, 0.05% OGA delays epidermal growth and disturbs differentiation, showing that the positive effects of OGA are dependent on its concentration. In parallel, using collagen IV and laminin 332 substrates, two relevant components of dermal-epidermal basement membrane, we demonstrate that the presence of 0.01% OGA clearly stimulates keratinocytes spreading out, paralleled by a well-organized microfilament network. Keratinocytes develop more focal adhesions with the substrates, implicating α6β4 on laminin 332. Cellular cohesion is also promoted by 0.01% OGA through the over-expression of integrins α2β1 on collagen IV, and α3β1 on laminin 332 at cell-cell junctions. Thus, by modulating integrins expression and organization, OGA 0.01% should improve cell-cell interactions and therefore dermal-epidermal cohesion. In conclusion, 0.01% OGA stimulates epidermal spreading and promotes keratinocytes attachment to basement membrane components by reorganizing cytoskeleton and modulating integrins recruitment. Furthermore, 0.01% OGA promotes epidermal differentiation and regulates epidermis homeostasis. Considering that OGA has a beneficial effect on parameters playing a key role in ageing, OGA can be presented as a new anti-ageing active ingredient.     

Cervi cornus Colla (deer antler glue) induce epidermal differentiation in the reconstruction of skin equivalents

In the reconstruction of skin equivalents (SEs), keratinocyte differentiation is important because epidermal differentiation is closely related with barrier function. The aim of this study was to investigate the effects of Cervi cornus Colla (CCC) on the stem cell activity and epidermal differentiation in the reconstruction of skin equivalent. Four different models were constructed according to different composition of dermal substitute. Results showed similar morphologic findings when hyaluronic acid (HA) and/or CCC was added. But, immunohistochemical staining showed that p63 was significantly increased by addition of HA and/or CCC. Increased staining of integrin α6 and β1 was variably observed when HA and/or CCC was added to make dermal substitute. These finding showed that addition of HA and/or CCC may affect the stem cell activity in the reconstruction of skin. Furthermore, filaggrin expression was much increased when CCC was added. It showed that epidermal differentiation was significantly improved by addition of CCC. In conclusion, simultaneous presence of HA and CCC contributed to the stem cell activity and epidermal differentiation in the reconstruction of SE. Legislation in the EU prohibits marketing cosmetics and personal care products that contain constituents that have been examined through animal experiments. To avoid these limitations, SEs can be used for testing the safety or the efficacy of cosmetic ingredients. Therefore, our results showed that combined use of HA and CCC can be helpful for the reconstruction of SE with good stem cell activity and epidermal differentiation.     

Heat shock proteins in the skin

Heat shock proteins (hsp) are expressed in all cells and organisms. Their expression is induced by heat shock (temperatures above 42 degrees C) and other forms of pathophysiological stress. Elevated levels of hsp protect cells from further stress exposure. Hsp are expressed intracellularly. They are highly conserved throughout evolution indicating hsp being necessary for survival under potentially harmful environmental conditions. Hsp are divided into families according to their molecular weight. The majority of hsp function as molecular chaperones. Chaperone function is characterized by binding to other proteins and mediating their folding, transport and interaction with other molecules. In human epidermis hsp are abundantly expressed and have been linked with functions in cell differentiation and photobiology. Recent research has mainly focused on the 27 and 72 kD hsp that are constitutively expressed in human keratinocytes. ultraviolet radiation (UV)-induced cell death and sunburn cell formation can be inhibited by previous heat shock exposure and UV itself can induce hsp expression. The expression of the 27 kD hsp (hsp27) in epidermal keratinocytes in situ and in culture correlates with differentiation. Expression of hsp27 increases simultaneously with keratinocyte differentiation. For that reason, hsp27 is described as a marker of epidermal differentiation. Changes in the expression and inducibility of hsp have been linked with ageing. In the skin, recent data indicate that hsp72 expression remains remarkably stable with intrinsic ageing. In contrast, levels of hsp27 have been found to be elevated in sun-protected aged skin indicating a link between hsp27 expression and age-dependent epidermal alterations. Regulation of hsp can be modified by pharmacological intervention and the development of safe topical and systemic treatments for the prevention of skin damage and disorders of keratinocyte differentiation can be expected for the future.     

Zinc l-pyrrolidone carboxylate inhibits the UVA-induced production of matrix metalloproteinase-1 by in vitro cultured skin fibroblasts, whereas it enhances their collagen synthesis

Reduced collagen matrix in the dermis constitutes one of the characteristic features of chronologically aged skin, which is further enhanced on the sun-exposed portions of the body by chronic ultraviolet light (UV) irradiation, inducing the unique changes associated with skin photoageing. The zinc salt of l-pyrrolidone carboxylate (Zinc PCA) has long been used as a cosmetic ingredient, because of its astringent and anti-microbial properties. In the present study, by employing cultured normal human dermal fibroblasts, we found that Zinc PCA suppressed UVA-induced activation of activator protein-1 (AP-1) and reduced matrix metalloproteinase-1 production in these cells, which is thought to be involved in collagen degradation in photoaged skin. Moreover, Zinc PCA treatment of the cells increased the expression of an ascorbic acid transporter mRNA, SVCT2, but not SVCT1, resulting in the enhanced production of type I collagen. Based on these in vitro findings, we consider Zinc PCA to be a promising candidate for an anti-skin ageing agent.     

No. 5Vol 42, No. 4, pp. 313–318, 2008A New Water-in-Powder Technology A Novel Structure for Creating Unique Cosmetic Products

A Water-in-Powder cosmetic is one encapsulating a large amount of water with powders. In this study we explain why the cosmetic is possible. First we focused on hydrophilic and hydrophobic balance of nano-sized powder surface. We looked into the surface state of powders with the gas adsorption test and discovered the functional powders that have the optimized balance of hydrorepellency due to fractal surface and hydrophilicity due to free hydroxyl groups. By using this material, we created a new powder technology that can encapsulate a large quantity of water stably. We also looked into how the powders encapsulated water by observation with wet-SEM and how the encapsulated water is stabilized by measuring water mobility with ¹7Q-M\IJR As a result, we developed a new technology with water and powder. By encapsulating a large amount of water, we achieved a cosmetic with many unique characteristics : the powder cosmetic can change into liquid by application of friction on skin, it gives us comfort in use and provides a watery, cool feel. It also provides excellent makeup function by spreading smoothly on skin to form a uniform makeup coating.
Keywords:  water-in-powder, hydrorepellency, hydrophilicity, fractal, encapsulate, technology     

The function of polyglyceryl erucate/isostearate/ricinoleate

Today, it is said that the formula design of cosmetics from ingredients of plant origin is an indispensable way and trend. From this consideration, cosmetic materials made from animal and synthetic petroleum ingredients are becoming less usable. Instead, cosmetic materials are designed from ingredients of plant origin and many and various botanical ingredients are being developed. Lanolin, which is one of the animal-based ingredients, is said to have ideal functions as a cosmetic oil, and it has been used in many fields such as make-up cosmetics as well as hair and skin care products for a long time. However, unfortunately, lanolin is an animal-based ingredient; therefore, the development of a botanical ingredient to replace lanolin was desired. Polyglyceryl-8 decaerucate/isostearate/ricinoleate, which we have developed, is an ester oil originating from plants and has an equivalent or higher function than lanolin. We have confirmed that our developed ester oil has various excellent characteristics such as a water-holding capability 2.5 times higher than that of lanolin, high air permeability, moisture keeping in dermal layers, protection of hair from changes in external environmental humidity, and excellent gloss and excellent dispersability of pigments. Thus, this newly developed ester oil is expected to be a promising new botanical cosmetic ingredient which can be applied in various fields.     

Oat-based complex stimulates skin barrier protein synthesis and reduces skin ageing

Citation: IFSCC Magazine , 11 (2008) (3) 225–229
The dermis is considered a highly dynamic structure that determines the biomechanical properties of the skin. It is composed of two dermal compartments separated by a vascular plexus: the papillary dermis and the reticular dermis. In the last few years, several studies have demonstrated the role of the dermal epidermal junction in the cutaneous ageing process. Recently, teams specialized in the study of the dermal matrix have focused their studies on the superior dermis in close contact with the dermal epidermal junction: the papillary dermis. They defined the role of matrix proteins in this area. Collagens XII and XVI, non-fibrillar collagens specific to the papillary dermis, are responsible for skin deformability and extensibility. Oxytalan fibres are related to elastic properties of the skin. Ubiquitous collagens such as collagens I and VI are associated with the cohesion and the resistance of the dermis. As the papillary dermis is the primary site of intrinsic dermal ageing, we studied expression of these molecules in our own in vitro model of intrinsic ageing of the papillary dermis. The results of this innovative approach confirmed that their expression was reduced. Nevertheless, active molecules may exist in nature that are capable of restoring a normal expression profile of these markers for a cosmetic anti-ageing application.
Keywords:  Anti-ageing, papillary dermis, collagens XVI and XII, oxytalan fibres     

In vitro cultured human skin cells as alternatives to animals for skin irritancy screening

In the last few years a lot of attention has been paid to the development of the in vitro models which would substitute for animals in cutaneous irritancy studies. These models explore either organ or explant cultures using freshly excised skin or serial cultures of isolated skin cells (epidermal keratinocytes or dermal fibroblasts). The organ or explant models are suitable only for short exposures of skin samples to the compounds tested and the use of it will always be restricted by the limited availability of fresh human skin. The model that uses submerged cultures of keratinocytes or fibroblasts permits the production of a large number of cells, and permits large scale toxicity screening tests with many substances, that can be applied in a broad concentration range. Since the stratum corneum is absent in conventional (submerged) keratinocyte culture systems, this model is mainly suited for testing of water soluble compounds and it is less suitable for poorly soluble compounds and for topical products consisting of complex formulations which are made of active ingredients and their vehicles. This shortcoming can be overcome by using ‘organotypic cultures’in which keratinocytes are grown at the air-liquid interface on a suitable dermal substrate. Under these conditions, the culture forms a multilayered epidermis showing an overall structure which resembles that of a native epidermis. The presence of a coherent stratum corneum layer in these cultures permits the application of potential irritants at concentrations and in formulations as applied in vivo. For the evaluation of toxicity a number of tests have already been developed: assessment of cell viability, changes in cell morphology, modulation of cell proliferation and differentiation, monitoring of membrane damage, the measurements of the uptake or incorporation of radioactive precursors, establishment of the modulation of cell metabolism, determination of the release of inflammatory mediators, etc. All these in vitro techniques are still in a state of validation as far as their predictive value for in vivo skin irritancy is concerned.     

The regulatory role of the tetrapeptide AcSDKP in skin and hair physiology and the prevention of ageing effects in these tissues – a potential cosmetic role

The naturally occurring tetrapeptide acetyl‐N‐Ser‐Asp‐Lys‐Pro (AcSDKP) recognized as a potent angiogenic factor was shown recently to contribute to the repair of cutaneous injuries. In the current article, we report the ability of AcSDKP to exert a beneficial effect on normal healthy skin and scalp and to compensate for the ageing process. In vitro AcSDKP at 10^?11–10^?7 M significantly stimulates the growth of human keratinocytes, fibroblasts and follicle dermal papilla cells. Moreover, it enhances the growth of human epidermal keratinocyte progenitor and stem cells as shown in a clonogenic survival assay. Topical treatment of ex vivo cultured skin explants with 10^?5 M AcSDKP increases the thickness of the epidermis and upregulates the synthesis of keratins 14 and 19, fibronectin, collagen III and IV as well as the glycoaminoglycans (GAGs). In the ex vivo‐cultured hair follicles, AcSDKP promotes hair shaft elongation and induces morphological and molecular modifications matching the criteria of hair growth. Furthermore, AcSDKP at 10^?11–10^?7 M was shown to improve epidermal barrier, stimulating expression of three protein components of tight junctions (claudin‐1, occludin, ZO‐1) playing an important role in connecting neighbouring cells. This tetrapeptide exercises also activation of SIRT1 implicated in the control of cell longevity. Indeed, a two‐fold increase in the synthesis of SIRT1 by cultured keratinocytes was observed in the presence of 10^?11–10^?7 M AcSDKP. In conclusion, these findings provide convincing evidence of the regulatory role of AcSDKP in skin and hair physiology and suggest a cosmetic use of this natural tetrapeptide to prevent skin ageing and hair loss and to promote the cutaneous regeneration and hair growth.     

A comparison of biological activities of a new soya biopeptide studied in an in vitro skin equivalent model and human volunteers

During aging, the epidermis and dermis become thin and an efficient anti-aging product should be able to stimulate the metabolism of senescent fibroblast and keratinocytes, in order to increase the quantity of extra-cellular matrix components such as collagen and glycosaminoglycans. A study performed in parallel on an in vitro skin equivalent model, and in vivo, with human volunteers, demonstrated the efficacy of one specific soya biopeptide for anti-aging properties. Such a biopeptide induces a significant increase of glycosaminoglycans synthesis in vitro and in vivo after a one-month treatment. We also showed that this new cosmetic ingredient is able to stimulate favourably the collagen synthesis in vitro and in vivo. This study provided the proof for anti-aging properties of a new soya biopeptide and also validated the skin equivalent model developed for this experimentation as an alternative method to animal or human testing for some cosmetic efficacy evaluations.     

Free internal lipids in hair from pre- and post-menopausal women

Plant secondary metabolites such as flavonoids, isoflavones and phytosterols have been proposed as cosmetic ingredients displaying anti-aging effects. On the cellular level, however, the activity profiles of these ingredients are only partially understood. In this study we analyzed the effects of apple seed phytosterols on age-related structural and functional parameters using cell biochemical, molecular biological and bioengineering techniques. The expression of age-related genes was studied using skin equivalents and cDNA microarrays. Incubation of skin equivalents with apple seed phytosterols had significant consequences: (i) differential regulation of a set of genes associated with keratinocyte proliferation and differentiation, (ii) stimulation of hyaluronic acid synthesis, and (iii) increase of epidermal thickness. In vivo studies revealed that apple seed phytosterols improve skin elasticity and decrease skin roughness. In conclusion, apple seed phytosterols display distinct biological effects and significantly improve the structure and function of mature skin.     

No. 2Vol. 42, No. 4, pp. 289–296, 2008An influence of cosmetic formula on thermal characteristics and its relationship with the sensation of freshness during application

Polyglycerol fatty acid esters (PGFE) , a food grade emulsifier has been used in cosmetic products in recent years due to its safety and low stimulation of skin. Cosmetic formulation containing CoQ1O to provide a stable and clear pre-paration is difficult because it has a high crystalline property. We have tried the development of CoQ10-soluble pre-paration with PGFE applied for cosmetics. The present study is summarized as follows: This CoQ10-soluble pre-paration has a bicontinuous microemulsion structure that provides high solubilization capacity and thermodynamic stability. Cosmetics using this CoQ1O soluble pre-paration tend to improve the skin permeability and also to involve the change in skin conditions (moisturization and elasticity), compared to the non-bicontinuous microemulsion pre-paration.
Keywords:  coenzyme Q10, solubilization, bicontinuous, microemulsion, polyglycerol fatty acid ester, cosmetics, food, emulsifier, safety, low stimulation, skin, moisturization, elasticity     

Potential cosmetic applications of reconstructed epidermis

Various models of reconstructed epidermis already provide useful tools for safety and efficacy assessment of cosmetic products. However, the majority of these in vitro models are composed of keratinocytes only. Recently, the introduction of melanocytes into epidermal reconstructs has considerably enlarged their field of application. Depending on the melanocyte donor, the different phototypes (I-VI) as well as the racial specific pigmentation, caucasian, Asiatic or black epidermis can be reproduced in vitro. The reconstructed pigmented epidermis allows the evaluation of modulators of melanogenesis such as the depigmenting agent kojic acid. In contrast to conventional melanocyte cultures, the pigmented reconstructed epidermis is air-exposed and covered, as in vivo, with a stratum corneum. This allowed us to evaluate the effect of UV-irradiations on the epidermis and its protection by topically applied sunscreens. The introduction of resident epidermal Langerhans cells into the reconstructed epidermis remained an important challenge. We succeeded by seeding blood derived CD34+ hematopoietic progenitors onto a reconstructing epidermis composed of keratinocytes and melanocytes. The resulting pigmented epidermis shows melanocytes in the basal layer and resident epidermal Langerhans cells suprabasally. As in normal skin, the melanocytes transfer melanin to the neighboring keratinocytes, and the Langerhans cells express major histocompatibility complex class II molecules, CD1a antigen and Birbeck granules. This reconstructed epidermis, comprising for the first time the three major epidermal cell types, has the potential to serve in the near future as a predictive model for immuno-pharmaco-toxicological in vitro studies.     

Polyphenols as active ingredients for cosmetic products

Polyphenols are secondary plant metabolites with antioxidant, anti‐inflammatory and anti‐microbial activity. They are ubiquitously distributed in the plant kingdom; high amounts contain, for example, green tea and grape seeds. Polyphenolic extracts are attractive ingredients for cosmetics and pharmacy due to their beneficial biological properties. This review summarizes the effects of polyphenols in the context of anti‐ageing activity. We have explored in vitro studies, which investigate antioxidant activity, inhibition of dermal proteases and photoprotective activity, mostly studied using dermal fibroblasts or epidermal keratinocytes cell lines. Possible negative effects of polyphenols were also discussed. Further, some physicochemical aspects, namely the possible interactions with emulsifiers and the influence of the cosmetic formulation on the skin delivery, were reported. Finally, few clinical studies, which cover the anti‐ageing action of polyphenols on the skin after topical application, were reviewed.     

An analysis of semantic structure of KAITEKI-KAN and development of its measuring method using emotional words

Conspicuous facial pores are one of the more serious aesthetic defects of concern to most women. However, the mechanism that causes the conspicuousness of facial pores remains unclear. We observed the epidermal structure around the conspicuous pores on cheeks in vivo using a confocal microscope in detail, and found that there were peculiar epidermal structures around the conspicuous facial pores. These epidermal structures were characterized by a thick epidermis, which reached the deep dermis, and elongated dermal papillae. As the shape and the size of these epidermal structures at more than one hundred micrometers below the surface were similar to those of the surface hollow area around the conspicuous pores, we thought that this epidermal structure was one cause for facial pore conspicuousness on cheeks. To improve these epidermal structures through contraction of keratinocytes, we developed l-isostearylglycerol-3-phosphate, which promoted the gel contractile ability of keratinocytes and suppressed the keratinocytes' growth in vitro. The treatment of lotion containing l-isostearylglycerol-3-phosphate could alter the epidermal structures and decrease the hollow area around the conspicuous facial pores.
Key words:  pores, conspicuousness, hollow, epidermis, structure, dermal papillae, keratinocytes, contraction, follicle, cheek.     

Two new lipoaminoacids with complementary modes of action: new prospects to fight out against skin aging

The mode of action of two cosmetic active ingredients (AIs), palmitoyl glycine (PG) and cocoyl alanine (CA) was studied with cDNA array experiments and quantitative PCR confirmations, which were performed on experimentally aged human fibroblasts. These preliminary studies revealed complementary profiles. Thus, specific supplementary investigations were then carried out for each AI. Protocols used were based either on in vitro models: (i) biochemical assays, (ii) monolayer cell culture (primary human fibroblasts and keratinocytes) and (iii) the model of capillary-like tube formation by human endothelial cells or on ex vivo models, i.e. topically treated skin explants and both immunohistochemical and Chromameter^TM investigations. New prospects are proposed to fight out against skin aging. Indeed, PG and CA showed complementary properties and thus enabled a regulation or a restoration effect on main aging-associated disorders. Thus, they can not only act on tissue architecture, cell–cell interactions and extracellular matrix protection but also on inflammation, cell longevity, skin immune system protection, skin radiance and stem cell survey. Finally, a clinical trial performed on Caucasian women confirmed AI anti-wrinkle efficacy, which was superior to that of a market reference ingredient. In the future, complementary experiments enabling a better understanding of the aging-induced decline of epidermal stem cells would be of a great interest.