effect of topical clay application on the synthesis of collagen in skin

7/30/2019 Effect of Topical Clay Application on the Synthesis of Collagen in Skin

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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,

University of Sao Paulo, Sao Paulo, Brazil

doi:10.1111/j.1365-2230.2011.04216.x

Summary Background. Clay is often used in cosmetic treatments, although little is knownabout its action.

Aim. To evaluate the effect of topical clay application on the histoarchitecture of

collagen fibres 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. Controlskin 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 fibres

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 fibres,

which may account for its beneficial effects.

Introduction

The skin is the bodys first barrier against environ-

mental insults. Skin ageing is affected by chronic

exposure to ultraviolet radiation, among other stres-

sors. 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 fibres

associated with proteoglycans are important compo-

nents 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 skinrevitalization, topical compounds and particularly all-

trans-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 influence

DNA repair and gene expression to increase ECM

production.6 Application of RA 0.05% for 2 weeks

promoted wrinkle reduction in rats.7

Correspondence : Dr Suzana B. V. Mello, Faculdade de Medicina da

Universidade de Sao Paulo, Av Dr Arnaldo 455, Sao Paulo, Sao Paulo

01246903, Brazil

E-mail: [email protected]

Conflict of interest: none declared.

Accepted for publication 1 August 2011

Experimental dermatology Original article CEDClinical and Experimental Dermatology

The Author(s)

164 CED 2012 British Association of Dermatologists Clinical and Experimental Dermatology, 37, 164168


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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 fibro-

blasts.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 histo-

architecture of the skin.

Methods

The animal ethics committee (Protocol #1114 08CAPPESQ) of the Brazilian College of Experimental

Animals approved all experimental procedures per-

formed on animals, in accordance with The Universities

Federation for Animal Welfare.

Animals

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

Depilation of dorsal hair was performed under anaes-

thesia to minimize stress to the animals. The skin was

cleansed, and two square areas of 4 cm2 each were

defined for topical application of bandages with a

commercial clay mask [water, kaolin, propylene glycol,

carbomer, triethanolamine, methylchloroisothiazolin-

one, 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 non-

anaesthetized animals, which were kept in their boxesunder water vaporization for 40 min at approximately

37 C.

Morphological analysis

The animals were killed in a CO2 chamber, then punch

biopsies (5 mm) were taken of untreated skin and skin

treated with clay or RA. All samples used for

histological analyses were fixed in 10% buffered

formalin, embedded in paraffin wax, and sectioned at

4 lm. The sections were stained with picrosirius red

for collagen evaluation under polarized light micro-

scopy.12

Collagen evaluation

A quantitative assessment of collagen fibres in the skin

was conducted using a microscope (BX-51; Olympus,

Center Valley, PA, USA) fitted 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 fibres. We randomly selected

10 high-resolution ( 400) microscopic fields for anal-ysis. Ten fields of 68.26 lm

2each were randomly

selected for evaluation from the epidermis, superficial

dermis and deep dermis. The collagen amount was

expressed as the percentage of the total tissue area that

was birefringent.13

Statistical analysis

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 significant.

Results

Untreated representative skin biopsies displayed pre-

served ECM histoarchitecture, and a dense birefringent

net of collagen fibres in the dermis layer (Fig. 1a).

Clay treatment for 7 days increased the collagen

content (Fig. 1b; red staining) and preserved the

fibre-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 significant (P < 0.01)

increase in the percentage area of collagen fibres(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 fibres

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 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.


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(a)

(b)

(c)

(d)

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 fibres (arrowed) (picrosinus,original magnification 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).

(a)

(b)

(c)

(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 fibres(arrowed) (picrosinus,originalmagnification 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).

The Author(s)




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of collagen fibres 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 treat-

ments had improved the number of collagen fibres

compared with control skin (Fig. 2d).

Discussion

We found that topical clay application for 7 days

promotes an increase in collagen fibres 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 findings are the first

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 sameanimal. The positive control with RA produced similar

findings 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 sufficient to reduce wrinkles.7

Our experimental model allowed maintenance of ideal

conditions of temperature and humidity for clay ther-

apy, producing optimum results from this treatment.

The therapeutic effects of mud are mainly due to the

temperature changes and, consequently, increased

blood flow to the skin,15

with consequent absorption

of ions or substances that could be responsible for their

efficacy.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

flow for the efficacy of clay therapy, it has been shown

previously that an increase in temperature can influ-

ence collagen fibril formation.16

Conclusion

Our data provide a scientific 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.

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.

What does this study add?

The present study was conducted to compare the

in vivo effects of topical application of a commer-

cially available clay mask with those of RA on the

collagen histoarchitecture of the skin.

Our data provide a scientific 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.

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