Long term culture of normal skin to test the efficacy of a hydroxy acid-containing cream

ARTICLE

Alpha-hydroxy acids (AHAs) applied topically at low concentrations, are now well-known to reduce the thickness of hyperkeratotic stratum corneum by reducing corneocyte cohesion at lower levels of the stratum corneum, increasing viable epidermal thickness and consequently thickening both the epidermal and dermal compartments of treated skin [1-3]. They have been shown to increase dermal glycosaminoglycans and thus counteract the atrophogenic effects of topical corticosteroids [4]. During the past few years, numerous articles written about AHAs have described their cosmetic and dermatologic efficacy but little work been performed on normal human skin particularly concerning the hypothetical stimulation of collagen synthesis. So, the aim of this work was to analyze the efficacy of a cosmetic cream containing alpha-hydroxy acids, beta-hydroxy acid and palmitate of vitamin A, applied topically over 3 weeks to human skin, by histological, immunohistological and biochemical studies. An ex vivo technique was used. Previous works [5, 6] have shown that it is possible to maintain skin for several weeks in a defined medium. This model consisted of full-thickness, normal human skin explants obtained during plastic surgery and maintained in long term organ culture for 21 days. Modifications at the epidermal and dermal levels (collagen and elastic fibers) were studied in surviving skin after 21 days of treatment with the AHAs cosmetic cream.

Materials and methods

Organ culture of human skin specimens

Our original culture method is based on previous studies [5, 6]. We adapted these methods to obtain full-thickness skin surviving 21 days such that the structure of the epidermis and dermis closely resembled skin in vivo.

Eight, normal, human skin fragments were obtained by plastic surgery, from women 35 to 45 years of age. Skin fragments were cut into 1 cm², full-thickness samples and washed three times with an antibiotic solution. Subcutaneous fat and lower dermis were mechanically removed under a stereomicroscope using a surgical scalpel.

Skin biopsies were placed with the epithelium uppermost, at an air/liquid interface, on culture inserts (filter pore size 0.45 µm; Costar, Poly-Labo Paul Block, France). These inserts were set on 12 well plates (Costar) for 21 days at 37° C in a humidified incubator with 5% CO₂. Cohesion between skin and the insert was obtained with polysiloxane vinyl seal in such a way that no skin retraction or lateral passage of the cream towards the dermis was possible.

Medium was added to the wells so that the surface of the medium was level with the filter. Organ cultures were performed with Dulbecco's minimal essential medium (Gibco BRL) containing antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; Gibco BRL, USA), 200 µg/ml L-glutamine (Gibco BRL), bovine pituitary extract, growth factors and fetal calf serum (DAP, France) [5-8]. All supplements were freshly prepared at each medium change, every two days.

The viability of our organ culture had been verified previously at different times (0, 5, 10, 15 and 21 days) by using histological, immunohistological and biochemical techniques (data not shown).

Histologically, the characteristic differentiation pattern of epidermis was reproduced, after 21 days of culture, on hemalun-eosin stained sections. Sometimes, after 15 days, we noticed a slight decrease in the tinctotrial affinity of the nuclear chromatin. No modification of collagen and elastic fibers was noticed in the dermis.

Immunohistologically, the dermal-epidermal junction integrity was verified with anti-laminin and anti-collagen IV staining; no disruption of this junction was seen. Anti-profilaggrin/filaggrin antibody demonstrated that keratinization was always present. Anti-total cytokeratins antibody showed a normal expression of the differentiation markers throughout the 21 days of culture. A normal number of fibroblasts in the dermis was demonstrated by means of an anti-vimentin antibody and of endothelial cells with anti-human von Willebrand factor. For all these parameters, no differences between 0 and 21 days were noticed.

Biochemically, cell viability evaluated by MTT conversion showed no change between 0 and 21 days [9].

Study of a cosmetic formulation containing alpha-hydroxy acids, beta hydroxy acid and vitamin A palmitate in long term human skin culture

A water in silicone emulsion containing alpha-hydroxy acids (10% fruit acids complex and 0.5% pure glycolic acid), beta-hydroxy acid (0.5% salicylic acid) and lipomicrons of vitamin A palmitate (6%) or its vehicle was applied to the epidermis every two days and compared with non-treated skin. The cream was applied directly to the skin, for 21 days at the dose of 8 mg/cm², followed by gently massage. This dose was calculated to be equivalent to the dose currently applied in vivo, i.e. 2 mg/cm².

After 21 days of culture, the effects of this cosmetic formula on normal human skin were studied at differents levels:

* Study of epidermal changes. Skin fragments were removed and fixed in Bouin's liquid for hemalun-eosin staining. Stratum corneum thickness and the number of Malpighian cell layers were determined.

* Study of elastic fibers. Elastic fiber network was revealed with (+)-catechin staining [10].

* Study of collagen synthesis. Fibroblastic activity for collagen synthesis was analysed after 21 days survival of an ex vivo culture. With this aim, skin biopsies were removed from inserts, placed directly in the wells and 20 µCi/ml of L-proline-(2, 3-³H) (Amersham, France, 1 mCi/ml, specific activity 43 Ci/mmol) with 100 µg/ml ascorbic acid and 50 µg/ml beta-aminopropionitrile were added to the culture medium for 24 h.

Extracellular ³H-proline-labelled-collagen was extracted by the addition of 1 mg/ml pepsin in 0.5 M acetic acid to the biopsies over 48 h at 4° C. Then, the ³H-proline-labelled-collagen was purified by Webster's method, consisting of successive salt precipitations at acid and neutral pH [11]. Radioactivity in each precipitate was measured in a liquid scintillation counter and expressed as disintegrations per minute (dpm). Total protein concentration was measured by spectrophotometric determination with the Pierce BCA protein Assay Reagent kit and finally results were expressed in dpm per mg protein.

Mean values and standard deviations were calculated for collagen analysis. The statistical significance of changes recorded for this parameter was determined with Student's t-test.

Results

Epidermal changes

After 21 days treatment, no change was observed in skin tissue treated with the vehicle (Fig. 1A) when compared to untreated skin as studied histologically after hemalun-eosin staining (Fig. 1B).

However, significant changes were observed on skin treated with the cream (Fig. 1C). Indeed, in all cases, there was a noticeable decrease in stratum corneum thickness and in some cases even a disappearance in comparison with untreated skins. When Malpighian layers were compared by counting the number of cell layers, the treated samples showed an increase (7 to 8 layers) whereas in the untreated skin 3 to 5 layers were identified. Budding of the epithelium in the dermis was observed in treated skin (10 to 12 layers).

Elastic fibers network

In the dermis, the elastic fiber network appeared to be better organized in treated skin in both the superficial and the upper part of the reticular dermis. Indeed, elastic fibers stained intensely positive for catechine and tended to be longer and thicker in all treated skin
(Fig. 2A) in comparison with untreated skin (Fig. 2B). Moreover, the fragmentation of elastic fibers occasionally observed in untreated dermis was less evident in treated skin.

Collagen synthesis

Extracellular ³H-proline-labelled-collagen measured by Webster method in treated skin (49.1 ± 12.65 cpm/mg protein) reached a higher level than in untreated skin (38.2 ± 12.85 cpm/mg protein) or in skin treated with vehicle (31.4 ± 7.45 cpm/mg protein). This analysis demonstrated an increased collagen synthesis of 28.5% in the treated skin as compared to untreated skin (p < 0.05).

Discussion

We demonstrated the efficacy of an AHA formulation on normal human skin using an ex vivo method of long term skin culture, at the epidermal and dermal level. In spite of the fact that the formulations are different, these results may be compared with results obtained in vivo in human skin treated with AHAs and also in an in vitro skin model.

Indeed, we noticed an important decrease in the stratum corneum cell layers; the same result was obtained by Van Scott et al. [2, 3] when they demonstrated that topical application of AHAs on human skin diminished corneocyte cohesiveness at the lower, newly forming levels of stratum corneum.

We found an increase of the number of Malpighian layers. Ditre et al. [12] showed in a recent study, that human skin treated with a 25% lotion of lactic, glycolic or citric acid showed a 30-50% increase in epidermal thickness. Moreover, they showed that the mean thickness of the papillary dermis in AHA-treated skin was greater than in control skin. With reference to elastic fibers of the reticular dermis, Ditre described fibers which appeared less clumped, less fragmented and with an apparent lengthening of the individual fibers compared with controls. Our results are consistent with this earlier report.

In our work, synthesis of collagen was increased as demonstrated by a higher incorporation of tritiated proline by dermal fibroblasts. Donnelly et al. [13] demonstrated an increase of ³H-proline incorporation in an in vitro skin model consisting of a cultured dermis and stratified epidermis grown on nylon mesh treated with topical application of AHAs. This study corroborates our results.

The ex vivo method of long term skin culture used in this work presents numerous advantages. This technique is similar to the in vivo environment; human biopsies can be avoided and animals spared. Finally, cream can be applied directly to the epidermis, reproducing the exact conditions of application of topical products.

Alternatively, human epidermis reconstructed on a matrix of collagen of type I and III with fibroblasts [14, 15] is already used. Other works [5, 6] have demonstrated the possibility of maintaining complete skin for several weeks. Using these techniques, we have verified the maintenance of skin viability and the fully differentiated state of normal human skin ex vivo.

As in vivo, topically applied AHAs on ex vivo skin can cause significant increases in viable epidermal thickness and in dermal extracellular matrix components such as collagen [12, 16]. This study confirms the benefit of using ex vivo, normal human skin for the evaluation of epidermal and dermal modifications after topical application of an AHA cream. In the future, this ex vivo method of skin culture could be useful in examining the efficacy of other compounds and their modulation by various vehicles.

CONCLUSION

Acknowledgments

We wish to thank Mrs A. Lesot and N. Vignot for their technical assistance in immunochemistry.

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