Experimental dermatology Original article

CED
Clinical and Experimental Dermatology

Effect of topical clay application on the synthesis of collagen in skin: an experimental study
D. M. Z. Valenti, J. Silva, W. R. Teodoro,* A. P. Velosa* and S. B. V. Mello*
Department of Physiology, ABC Faculty of Medicine, and *Department of Internal Medicine, Rheumatology Division, School of Medicine, o Paulo, Sa o Paulo, Brazil University of Sa
doi:10.1111/j.1365-2230.2011.04216.x

Summary

Background. Clay is often used in cosmetic treatments, although little is known about its action. Aim. To evaluate the effect of topical clay application on the histoarchitecture of collagen bres in rat skin. Methods. Animals received a daily application of clay and retinoic acid (RA) 0.025% to the dorsal skin over 7 and 14 days, under vaporization at 37 C for 40 min. Control skin was not vaporized. Samples from each region were excised, and stained with picrosirius red for collagen evaluation. Results. Seven days after clay treatment, an increase in the number of collagen bres was observed in treated skin compared with control skin (51.74 1.28 vs. 43.39 1.79%, respectively, P < 0.01), whereas RA did not alter the collagen level (45.66 1.10%). Clay application over 14 days did not induce a further increase in skin collagen, whereas treatment with RA did (58.07 1.59%; P = 0.001 vs. control). Conclusion. Clay application promotes an increase in the number of collagen bres, which may account for its benecial effects.

Introduction
The skin is the bodys rst barrier against environmental insults. Skin ageing is affected by chronic exposure to ultraviolet radiation, among other stressors. Extrinsic factors induce the generation of harmful compounds called reactive oxygen species (ROS), which damage cell DNA and cell walls by altering their structural and morphological characteristics.1 The shift in the constitution of the extracellular matrix (ECM) is one important factor in ageing. Collagen bres

Correspondence: Dr Suzana B. V. Mello, Faculdade de Medicina da o Paulo, Av Dr Arnaldo 455, Sa o Paulo, Sa o Paulo Universidade de Sa 01246903, Brazil E-mail: svmello@usp.br Conict of interest: none declared. Accepted for publication 1 August 2011

associated with proteoglycans are important components of the dermis, and healthy skin is dependent on a balance between collagen synthesis and degradation. Collagen degradation is primarily controlled by the activity of matrix metalloproteinases (MMPs), and inactivation of MMPs reduces the formation of wrinkles.2 The increase in life expectancy has led to an increase in research into the maintenance of a youthful appearance. Among the therapeutic options for skin revitalization, topical compounds and particularly alltrans-retinoic acid (RA, or tretinoin) are commonly used in the treatment of photoaged skin.3 Tretinoin prevents collagen loss by inhibiting MMPs2,4 and stimulating new collagen formation.5 Retinoids can also inuence DNA repair and gene expression to increase ECM production.6 Application of RA 0.05% for 2 weeks promoted wrinkle reduction in rats.7

164

The Author(s) CED 2012 British Association of Dermatologists Clinical and Experimental Dermatology, 37, 164168

Effect of topical clay application on the synthesis of collagen in skin D. M. Z. Valenti et al.

Natural products are becoming more popular in cosmetic clinics,8 with clay being a popular option.9 However, there is little information about the functional effects of clay upon the skin layers to support its use. One study that examined the mechanism of action of clay reported that keratinocytes incubated with microalgae derived from black mud had overexpression of collagen genes and upregulation of MMP-1 expression in broblasts.10 Clay and other biomaterials, when implanted in skin lesions, have been shown to stimulate collagen synthesis.11 The present study was conducted to compare the in vivo effects of topical application of a commercially available clay mask with RA on the collagen histoarchitecture of the skin.

treated with clay or RA. All samples used for histological analyses were xed in 10% buffered formalin, embedded in parafn wax, and sectioned at 4 lm. The sections were stained with picrosirius red for collagen evaluation under polarized light microscopy.12
Collagen evaluation

Methods
The animal ethics committee (Protocol #1114 08 CAPPESQ) of the Brazilian College of Experimental Animals approved all experimental procedures performed on animals, in accordance with The Universities Federation for Animal Welfare.
Animals

A quantitative assessment of collagen bres in the skin was conducted using a microscope (BX-51; Olympus, Center Valley, PA, USA) tted with a camera (Q Color 5; Olympus). Micrographs were processed using Image ProPlus software (version 6.0; Media Cybernetics, Inc., Bethesda, MD, USA) on a computer (Pentium IV PC with 3300 MHz processor). Collagen content was assessed via the selection of red orange tones, which corresponded to collagen bres. We randomly selected 10 high-resolution ( 400) microscopic elds for analysis. Ten elds of 68.26 lm2 each were randomly selected for evaluation from the epidermis, supercial dermis and deep dermis. The collagen amount was expressed as the percentage of the total tissue area that was birefringent.13
Statistical analysis

Adult man Wistar rats weighing 200250 g were used. The animals were given a standard pellet diet and water ad libitum.
Topical treatment with clay and retinoic acid

Results are expressed as means SE. To compare the differences between means, we used a one-way ANOVA, followed by the StudentNewmanKeuls test. P < 0.05 was considered signicant.

Depilation of dorsal hair was performed under anaesthesia to minimize stress to the animals. The skin was cleansed, and two square areas of 4 cm2 each were dened for topical application of bandages with a commercial clay mask [water, kaolin, propylene glycol, carbomer, triethanolamine, methylchloroisothiazolinone, methylisothiazolinone, methylparaben, and biomin (Saccharomyces copper, iron, magnesium, silicon, and zinc ferments at physiological pH)] and with RA 0.025%. Groups of six animals were treated for 7 or 14 days. The treatment was performed daily in nonanaesthetized animals, which were kept in their boxes under water vaporization for 40 min at approximately 37 C.
Morphological analysis

Results
Untreated representative skin biopsies displayed preserved ECM histoarchitecture, and a dense birefringent net of collagen bres in the dermis layer (Fig. 1a). Clay treatment for 7 days increased the collagen content (Fig. 1b; red staining) and preserved the bre-network architecture. The collagen content in skin treated with RA for 7 days (Fig. 1c) was similar to that of the untreated group. Clay treatment for 7 days also promoted a signicant (P < 0.01) increase in the percentage area of collagen bres (51.74 1.28%) compared with untreated skin (43.39 1.79%) (Fig. 1d). RA treatment for 7 days had no effect on the percentage area of collagen bres compared with controls (45.66 1.10%; P = 0.16) (Fig. 1d). Extending the duration of treatment to 14 days (Figs 2b,d) did not further increase the percentage area

The animals were killed in a CO2 chamber, then punch biopsies (5 mm) were taken of untreated skin and skin

The Author(s) CED 2012 British Association of Dermatologists Clinical and Experimental Dermatology, 37, 164168

165

Effect of topical clay application on the synthesis of collagen in skin D. M. Z. Valenti et al.

(a)

(a)

(b)

(b)

(c)

(c)

(d)

(d)

Figure 1 (a) Untreated skin and skin treated topically with (b) clay

or (c) retinoic acid (RA) for 7 days, with a network of birefringent collagen bres (arrowed) (picrosinus, original magnication 200). (d) Effect of clay and RA treatment for 7 days on the amount of collagen in rat skin; results are expressed as means SE (n = 6).

Figure 2 (a) Untreated skin and skin treated topically with (b) clay

or (c) retinoic acid (RA) for 14 days, with a network of birefringent collagen bres (arrowed) (picrosinus, original magnication 200). (d) Effect of clay and RA treatment for 14 days on the amount of collagen in rat skin; results are expressed as means SE (n = 6).

166

The Author(s) CED 2012 British Association of Dermatologists Clinical and Experimental Dermatology, 37, 164168

Effect of topical clay application on the synthesis of collagen in skin D. M. Z. Valenti et al.

of collagen bres in the clay-treated skin, but it did in the RA-treated skin (58.07 1.59%; P = 0.04 vs. Control) (Figs 2c,d). Notably, after 14 days, both treatments had improved the number of collagen bres compared with control skin (Fig. 2d).

Whats already known about this topic?

Natural products are becoming more popular in cosmetic clinics. Clay is one of the accepted options; however, little is known about its functional effects on skin layers to support its use.

Discussion
We found that topical clay application for 7 days promotes an increase in collagen bres in rat skin. Extending the duration of treatment to 14 days did not further increase the collagen response for clay, whereas this length of time was necessary to observe an effect of RA treatment. To our knowledge, these ndings are the rst description of an in vivo effect of clay on the collagen content of uninjured skin with side-by-side comparisons with RA. Experimental models allow simultaneous comparison of two treatment substances in the same animal. The positive control with RA produced similar ndings to those previously reported. For example, a similar protocol applying retinoid at a higher dosage (0.1%) in human skin led to a slow and gradual increase in collagen.14 Contrarily, RA has been described as a potent and rapid stimulator of collagen in photoaged human skin,3 however in mice, application of RA 0.05% for 2 weeks was sufcient to reduce wrinkles.7 Our experimental model allowed maintenance of ideal conditions of temperature and humidity for clay therapy, producing optimum results from this treatment. The therapeutic effects of mud are mainly due to the temperature changes and, consequently, increased blood ow to the skin,15 with consequent absorption of ions or substances that could be responsible for their efcacy.9 We would have preferred to know the precise composition of the commercial clay treatment used in our study. Clays are a complex mixture of iron, quartz crystals and other minerals, which have a large capacity to store energy. These features could affect the biological response to the clay. Reinforcing the importance of heat ow for the efcacy of clay therapy, it has been shown previously that an increase in temperature can inuence collagen bril formation.16

What does this study add?

The present study was conducted to compare the in vivo effects of topical application of a commercially available clay mask with those of RA on the collagen histoarchitecture of the skin. Our data provide a scientic basis for clay use in cosmetic therapy. The rapid effects of clay on the collagen network, while not affecting the skin histoarchitecture, suggest that clay may be a good therapeutic option for facial rejuvenation.

References
1 Zouboulis CC, Makrantonaki E. Clinical aspects and molecular diagnostics of skin aging. Clin Dermatol 2011; 29: 314. 2 Lateef H, Sevens MJ, Varani J. All-trans-retinoic acid suppresses matrix metalloproteinase activity and increases collagen synthesis in diabetic human skin in organ culture. Am J Pathol 2004; 165: 16774. 3 Uitto J. The role of elastin and collagen in cutaneous aging: intrinsic aging versus photoexposure. J Drugs Dermatol 2008; 7: S126. 4 Schiltz JR, Lanigan J, Nabial W et al. Retinoic acid induced cyclic changes in epidermal thickness and dermal collagen and glycosaminoglycan biosynthesis rates. J Invest Dermatol 1986; 87: 6637. 5 Griffiths CE. The role retinoids in the prevention and repair of aged and photoaged skin. Clin Exp Dermatol 2001; 26: 6138. 6 Varani J, Fay K, Perona P. MDI 301, a non-irritating retinoid, induces changes in human skin that underlie repair. Arch Dermatol Res 2007; 298: 43948. 7 Bhattacharyya TK, Linton J, Mei L, Thomas JR. Profilometric and morphometric response of murine skin to cosmeceutical agents. Arch Facial Plast Surg 2009; 11: 3327. 8 Chanchal D, Swarnlata S. Novel approaches in herbal cosmetics. J Cosmet Dermatol 2008; 7: 8995.

Conclusion
Our data provide a scientic basis for clay use in cosmetic treatments. The rapid effects of clay on the collagen network, although not affecting the skin histoarchitecture, suggest that clay may be a good therapeutic option for facial rejuvenation.

The Author(s) CED 2012 British Association of Dermatologists Clinical and Experimental Dermatology, 37, 164168

167

Effect of topical clay application on the synthesis of collagen in skin D. M. Z. Valenti et al.

9 Clijsen R, Taeymans J, Duquet W et al. Changes of skin characteristics during andafterlocalparafango therapy asusedin physiotherapy. Skin Res Technol 2008; 14: 23742. 10 Grether-Beck S, Mu hlgerg K, Brender H et al. Bioactive molecules from the Blue Lagoon: in vitro and in vivo assessment of silica mud and microalgae extracts for their effects on skin barrier functions and prevention of skin ageing. Exp Dermatol 2008; 17: 7719. 11 Cohen IK, Diegelmann RF, Wise WS. Biomaterials and collagen synthesis. J Biomed Mater Res 1976; 10: 965 70. 12 Junqueira LC, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 1979; 11: 44755.

13 Gundersen HJ, Bendtsen TF, Korbo L et al. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 1988; 96: 37994. 14 Tucker-Samaras S, Zedayko T, Cole C et al. A stabilized 0.1% retinol facial moisturizer improves the appearance of photo damaged skin in an eight-week, double-blind, vehicle-controlled study. J Drugs Dermatol 2009; 8: 9326. choz C, Gasparini S. Effects of 15 Poensin D, Carpenter PH, Fe mud pack treatment on skin microcirculation. Joint Bone Spine 2003; 70: 36770. 16 Williams BR, Gelman RA, Poppke DC, Piez KA. Collagen fibril formation. Optimal in vitro conditions and preliminary kinetic results. J Biol Chem 1978; 253: 657885.

168

The Author(s) CED 2012 British Association of Dermatologists Clinical and Experimental Dermatology, 37, 164168