Skincare: Making good connections

Valérie André, Françoise Pivard, Corinne Reymermier and Eric Perrier introduce a new corn extract believed to have an impressive effect on the cutaneous tissue during the ageing process

The connective tissue of the skin has been extensively studied but some fundamental information is still missing. The main scaffolds, namely the collagen and the elastic networks, are now more or less elucidated as far as their three-dimensional organisation and functions are concerned. Collagen molecules for instance can be assembled into collagen fibrils and then fibres that are responsible for the mechanical properties of the skin. On the other hand, the complex elasticity fibres network is also tremendously important for the plasticity of the skin, playing a pivotal role in the intrinsic and UV-related ageing phenomena. A third network has been poorly studied until now but is also essential to the connective tissue organisation as well as the cell-cell and cell-matrix interactions - the fibronectin network.

Fibronectin designates a family of glycoproteins (of which there are about 20 members) synthesized by alternative splicing. Produced in insoluble form by the connective tissue's fibroblasts, it is found within cells at the cell surface and on extracellular levels. Fibronectin is capable of contracting lesions, not only with most of the other connective tissue molecules but also with numerous cellular types through the integrins present on their surface. It also combines with the components of the cytoskeleton such as actin, through surface proteoglycans to promote cellular adhesion (Ruoslahti, 1989).

There is some controversy over the status of fibronectin during ageing. Several scientific teams have observed an increase in fibronectin production according to age, while an equivalent number of authors have demonstrated that the synthesis of this glycoprotein diminishes over time in vivo or ex vivo (Pierragi Figure 1 - The fibronectin molecule and its different linking areas: a multipurpose connector

et al, 1984; Ashcroft et al, 1995). However, it has been demonstrated that fibronectin content is decreased under wrinkles and that its enzymatic degradation increases with age (Rocquet et al, 2002).

Based on the authors’ experience with skin engineering culture models, the status of fibronectin was examined during the course of skin ageing. The expression of this protein was investigated using monolayer and 3D fibro-blast-based cell cultures such as reconstructed dermis (Mimederm, Engelhard Lyon) and reconstructed full-thickness skin (Mimeskin, Engelhard Lyon) using cells extracted from human biopsies from young and mature donors. How is fibronectin expressed during ageing? Are there some age-related relationships regarding the level of fibronectin that are relevant and comparable between in vitro and ex vivo experiments?

Working with a hospital research team, the researchers first analysed in a standardized way the evolution of the fibronectin expression in skin human biopsies from donors of different ages. Using immunohistological techniques, it was observed that fibronectin was slowly reduced in human tissue with age.

Fibronectin synthesis was then evaluated in different cell culture models: fibroblasts in monolayers, equivalent dermis (Mimederm) and reconstructed skin models (Mimeskin). Each model was composed of cells from young or mature subjects (young fibroblasts, mature fibroblasts, young Mimederm, mature Mimederm, young Mimeskin, mature Mimeskin). In each case, the quantity of fibronectin produced was quantified in incubatory environments using a specific sensitive Elisa dosage. The differential expression of the fibronectin gene in the young and mature models was also analysed by Northern Blot.

It has been discovered that fibronectin should be studied while using reconstructed skin models only, the other models being irrelevant compared to the results obtained ex vivo. Using such models able to more closely mimic the skin, a reduction of 20% of fibronectin was observed while ageing, using a sensitive and specific Elisa method. Fibronectin protein but also RNAm coding for this protein was strongly reduced using the same model (-43% after Northern Blot analysis).

Active ingredient offer

Using such observations, it was then possible to build a miniaturised test able to screen best ingredients, plant extract or pure chemicals able to stimulate fibronectin synthesis in order

to counteract the effects of ageing

(figure 4). It was possible to demonstrate the ability of one specific active compound, a corn extract (Deliner, Engelhard Lyon), to stimulate fibro-blast proliferation in particular. Migration using a wound healing model was performed. To achieve this, normal human dermal fibroblasts were cultured in mono-layers in

6-dish culture plat in absence or in presence of Deliner and reference product. Selected product promoted the cell migration and proliferation (up to +65% vs the non treated

control), such ability being even stronger than that observable with TGFbeta used at 10nglml (figure 5). Dose effect has been performed on monolayered fibloblasts and efficacy demonstrated when Deliner was used at 1-5% on fibronectin synthesis by immuno-labelling (figure 6).

The resulting selected product was then used in a 3D reconstructed cellular model in which artificial wounds were created to mimic the loss of matrix components observed in a wrinkle structure. In these models, cell migration via fibronectin connections and cell multiplication can be observed and quantified. The results showed that the selected active compound induced a significant increase (p<0.01) in the number of fibroblasts having colonized the artificial wound (+21% compared to the untreated control). It also stimulated the cellular proliferation of fibroblasts in this area (+26% compared to the untreated control). Parallel to this stimulation of the re-colonization, used at 1%, this selected material increased fibroblastic fibronectin production in this area (+56% compared to the untreated control, p<0.05).

A correlation could be foreseen between the different parameters measured. The increase in fibronectin quantities present in the reconstructed skin could induce cellular migration and proliferation through the creation of a veritable intra-dermal scaffolding action. Selected active compounds therefore leads, through the stimulation of cutaneous fibronectin production, to increased fibroblastic re-colonization of areas from which these cells have been eliminated. Logically this re-colonization should result in the origin of an extracellular new matrix formation.

In vivo efficacy

The anti-wrinkle effects of Deliner was evaluated in an in vivo study on healthy volunteers. Twenty healthy female volunteers were used for this half-face study. For 56 days, each volunteer applied a placebo formulation to half the face and a formulation containing 3% of the selected active compound to the other half. Before and after the 56-day treatment, the wrinkle depth of all volunteers’ crow’s feet was performed without modifying the volume of the analysed area using a fringe projection analysis method.

The results showed that a 56-day treatment was able to significantly improve the cutaneous relief of the volunteers in the crow's feet area. Under the same experimental conditions, the placebo formulation did not induce any significant reduction of the measured parameters.

One of the characteristics of physiological ageing of the skin is the reduction of skin thickness with age. In an ultrasound study of skin ageing, Escoffier and De Rigal (1989) demonstrated a clear-cut atrophy of skin tissue after the age of 65. The same kind of evaluation was performed after Deliner treatment. Five of the 20 women between 40 and 45 years of age who participated in the preceding study applied the product to the face daily for four months. Measurements of the skin thickness were taken in the eye contour area after two and four months (high resolution skin ultrasound, 20 MHz).

After analysis and comparison of ultrasound measurements at different kinetic time points, it was found that the thickness of the skin at the crow's feet (outer eye contour) increased by +6% after two months and by +14% (p<0.05) after four months of application. Thus the effect was observed after two months and became significant after four months of use.

In conclusion, the selected active compound through identified metabolic effects offers a significant reduction in wrinkle network and increases the skin thickness on the crow’s feet area of volunteers.

Deliner is obtained from Zea mays (honey bantam) cultivated in the Abashiri area on Hokkaido island in Japan. Seeds are first ground, then the powder is extracted into a water/butylene glycol solution giving rise to a clear yellow preserved xanthane gel. By stimulating the synthesis of fibroblast fibronectin, Deliner reorganises the extracellular matrix and renders it denser in the dermis. The effects are shown by an increased thickness of the skin over time, which is the confirmation in vivo of the efficacy of the material on skin ageing.

References

1. Ruoslati E, 2002, Proteoglycans in cell regulation, J. Biol. Chem. 264, 13369-13372

2. Pieraggi M T et al, 1984, Dermal aging. Immunofluorescence study of collagens I and III and fibronectin. Ann. Pathol. 4(3), 185-194

3. Rocquet C & Bonte F, 2002, Molecular aspects of skin ageing - recent data. Acta APA, 11(3), 71-94

4. De Rigal J et al, 1989, Assessment of aging of the human skin by in vivo ultrasonic imaging, J Invest Dermatol. 93(5):621-625

Authors

Valérie André, Françoise Pivard, Corinne Reymermier & Eric Perrier, Engelhard Lyon, France

Contacts

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