Materials Science and Engineering C 43 (2014) 109–116

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Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Evaluation of the healing activity of therapeutic clay in rat skin wounds

Giordana Maciel Dário a, Geovana Gomes da Silva a, Davi Ludvig Gonçalves a, Paulo Silveira a,
Adilson Teixeira Junior a, Elidio Angioletto b, Adriano Michael Bernardin b,⁎
a
Health Sciences Post-Graduation Program, UNESC, Av. Universitária 1105, Cricúma, Santa Catarina 88806-000, Brazil
b
Materials Science and Engineering Post-Graduation Program, UNESC, Av. Universitária 1105, Cricúma, Santa Catarina 88806-000, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The use of clays for therapeutic practice is widespread in almost all regions of the world. In this study the
Received 1 February 2012 physicochemical and microbiological healing characteristics of a clay from Ocara, Brazil, popularly used for
Received in revised form 24 April 2014 therapeutic uses, were analyzed. The presence of Ca, Mg, Al, Fe, and Si was observed, which initially indicated
Accepted 29 June 2014
that the clay had potential for therapeutic use. The average particle size of the clay (26.3 μm) can induce the
Available online 6 July 2014
microcirculation of the skin and the XRD analysis shows that the clay is formed by kaolinite and illite, a swelling
Keywords:
clay. During the microbiological evaluation there was the need to sterilize the clay for later incorporation into the
Clays pharmaceutical formula. The accelerated stability test at 50 °C for 3 months has showed that the pharmaceutical
Pharmaceutical uses formula remained stable with a shelf life of two years. After the stability test the wound-healing capacity of the
Wound-healing formulation in rats was evaluated. It was observed that the treatment made with the formulation containing the
Therapeutic clay Ocara clay showed the best results since the formula allowed greater formation of collagen fibers and consequent
regeneration of the deep dermis after seven days of treatment and reepithelialization and continuous formation
of granulation tissue at the 14th day.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
The use of clay with medicinal purpose is an ancient and widespread
practice. However, the modern use of clay in pharmaceuticals or
cosmetics needs a rigorous study regarding the preparation processes
and evaluation techniques [1–6]. Substances for pharmaceutical use
are organic or inorganic and they can be used as active ingredients for
the production of medicines, which may be obtained from natural
sources or not. These substances can also be used as such or as part of
a drug formulation [7–9].
About half the medicines used today are of natural origin and many
are still unexplored. However, before becoming an active ingredient in a
pharmaceutical formula, it is essential to check the medicines' purity
and to characterize them biologically, chemically and physically [7,10,
11].
Among the products of dermatological interest those with topical
action incorporated into pharmaceutical formulas deserve particular
attention because they can restore the skin integrity after possible
aggressions. Therefore, they must restore normal conditions of the
skin and for that the healing process is of fundamental importance.
⁎ Corresponding author. Tel./fax: +55 48 3444 3735.
E-mail address: amb@unesc.net (A.M. Bernardin).
The healing process involves the migration of inflammatory cells, the
synthesis of granulation tissue, deposition of collagen and proteo-
glycans and maturation of the scar, being associated with intense refur-
bishment. Complementary medicine has been used as an alternative to
their treatment [4,12–15].
Many natural materials have been used in dermatological products;
within this group are the so called medicinal clays [2,16]. Clays are
composed of aluminosilicate particles and several trace elements that
promote the absorbent, healing and antiseptic action [5]. The use of
clays for therapeutic and cosmetic purposes must comply with certain
physical, physicochemical and microbiological requirements [7].
The study of the stability of pharmaceutical and cosmetic formulas is
of fundamental importance for the safety of those who consume them;
therefore, in order to these formulations fit the quality standards
imposed by regulators, it is necessary to evaluate the same through sta-
bility tests [3,7,8,17]. Considering the importance that medicinal clays
have acquired and the complexity of the different components present
in cosmetic preparations – that can interfere with the effectiveness of
the active ingredients and these in vehicle stability – it is necessary to
evaluate the real activity that the incorporated principle will have on
cutaneous wound-healing, therefore the proposed formulation could
be viable for commercialization and use.
The use of geological nanomaterials to heal skin infections has been
evident since the earliest recorded history and specific clay minerals
may prove valuable in the treatment of bacterial diseases, including

http://dx.doi.org/10.1016/j.msec.2014.06.024
0928-4931/© 2014 Elsevier B.V. All rights reserved.
110 G.M. Dário et al. / Materials Science and Engineering C 43 (2014) 109–116
infections for which there are no effective antibiotics. Clay minerals can
affect bacterial metabolism indirectly by altering the physicochemical
properties of a specific environment or directly through surface interac-
tions. Thus, physicochemical properties of hydrated clays, mainly illite
or montmorillonite indirectly kill bacteria by generating an unfavorable
environment to them [18].
Therefore, the main objective of this study was to evaluate the ther-
apeutic effect of natural clay from the Northeast region of Brazil. The
clay was prepared as an emulsion and its therapeutic effect was studied
on the healing of rat wounds.
2. Experimental
2.1. Characterization
The sample used was a black clay coming from a lake in the munic-
ipality of Ocara, Ceará State, Brazil. The sample was obtained from a
batch of 200 kg extracted manually from the lake and the sample under-
gone a sieving step (100 μm sieve) to eliminate the non-clay fraction
before characterization. After characterization and after the initial mi-
crobiological analysis – where the sample showed 300 CFU/g for bacte-
ria and 100 CFU/g for fungi – the sample was sterilized. All samples
were kept in a dry controlled environment for at least 48 h before test-
ing. After the microbiological evaluation there was the need to sterilize
the clay for later incorporation into the pharmaceutical formula. The
chemical composition was determined by X-ray fluorescence (XRF,
Philips PW2400, sample melted with lithium tetraborate). The particle
size distribution was determined by laser diffraction (Cilas 1064, 20 s
sonication). The mineralogical composition was determined by X-ray
diffraction (Shimadzu XRD-6000, CuKα radiation, scan rate of 2°/min,
from 0° to 90°). The transmission spectroscopy analysis was performed
by Fourier Transform Infrared (FTIR) analysis (Shimadzu IR Prestige 21,
KBr pellets, from 400 to 4000 cm−1). The microbiological tests were:
agar diffusion and determination of bacteria and fungi by total score
[19].
2.2. Development of the formulation containing the Ocara clay
The emulsion preparation process and the incorporation of the
active principle – the Ocara clay – were performed according to the pre-
cepts of the Good Handling Practices RDC n. 67 [20]. The emulsions were
prepared by heating in a water bath at 75 °C plates of aqueous and oily
phases separately, then joined and kept under manual stirring and
heating for 15 min. After this time, the emulsions were removed from
the water bath, keeping the manual agitation for 5 min at room temper-
ature. The emulsions prepared were kept at rest for 24 h at room tem-
perature after sealing the containers in which they were potted. After
24 h, the samples were observed. Table 1 shows the neutral emulsion
and the emulsion containing the Ocara clay. Polawax was used as
the emulsion base, whose formulation and preparation process are
described by Eccleston [21]. The production of a neutral emulsion with-
out the addition of the Ocara clay was performed in order to evaluate
possible interferences related to the emulsion base.
2.3. Accelerated stability test and determination of the validity
The preliminary/accelerated stability test (T = 50 °C) was used, and
the samples were collected and evaluated at 0, 5, 10, 15, 30, 60 and
Table 1
Composition of the neutral emulsion and the emulsion containing the Ocara clay.
Emulsion Neutral talc (g)
Neutral emulsion 40
Emulsion/clay 40
90 days. The organoleptic, centrifugation, rheological, density, pH and
microbiological characteristics were analyzed [22]. In order to deter-
mine the organoleptic characteristics, the appearance of a small amount
of the homogenized sample was checked in a transparent container and
observed against a white background for the presence of residues,
degree of turbidity and phase separation. The color was determined
by visual identification and the odor by the sense of smell.
The relative density of the sample was determined using a calibrated
pycnometer (25 mL, 20 °C). The rheological analysis was performed in a
viscometer (Bohlin Visco 88). The centrifugation test was performed at
3000 rpm for 30 min with a small sample of the emulsion. The pH was
determined in a calibrated pH meter (Quimis Q400, 1:10 dilutions).
For the microbiological evaluation, the Ocara clay and the prepared
emulsion samples were diluted in BHI (Brain Heart Infusion) at a ratio
of 1:9 and three dilutions were prepared. For each dilution, inoculations
were made in duplicate using PCA (Plate Count Agar) culture media for
bacteria and Sabouraud dextrose agar for fungi. In samples containing
preservatives 1% Tween 80 (polyoxyethylene sorbitan monooleate)
was used for inactivation by adding it to the BHI. Petri dishes were
used with an adequate amount of medium to achieve a thickness of
4 mm. 0.1 mL of each dilution was spread with the aid of the Drigalski
handle. The plates were incubated for 24 h at 35 °C for bacteria and
for seven days at 25 °C for fungi, and after the specific time the colonies
were counted [23].
The count of colonies was performed as described by the Farmacopeia
Brasileira [24] based on the United States Pharmacopeial Convention
[25]. The results were expressed as colony forming units (CFU) per
gram of sample, divided into two categories: type I within a limit of 5 ×
102 CFU/g and type II within a limit of 5 × 103 CFU/g. For both categories,
the absence of Pseudomonas aeruginosa, Staphylococcus aureus and total
fecal coliforms was also observed in 1 g of product.
Finally, the provisional validity of the products was determined
using Eq. (1) [24]:
.
t90 ðT1 Þ
T90 ðT2 Þ ¼ ΔT
=10 ð1Þ
Q 10
where T90(T2) is the estimated shelf life, t90(T1) is the expiration date at
a certain temperature, Q is the activation energy and ΔT is the difference
between temperatures T1 and T2. The commonly used values for Q are 2,
3 and 4 regarding the activation energies for temperatures close to room
temperature (25 °C). In general, there is a reasonable estimate using the
value 3 (activation energy: Ea = 19.4 kcal/mol). In this study 25 °C was
used for T2, which represents the temperature for the estimated shelf
life [23].
2.4. Evaluation of the healing activity
The research was conducted in accordance with international stan-
dards for biomedical research on animals, according to the Brazilian
Federal Law N. 6638 (1979). A total of 45 male Wistar rats of adult age
and average body weight of 300 g (ranging between 250 and 350 g)
were used. The animals were kept in proper cages with food and
water ad libitum, in a semi-controlled macro-environment at room
temperature (21 °C), while the brightness, noise and humidity were
that of the general environment [23]. The animals underwent a burning.
After intramuscular anesthesia – consisting of 0.04 mL/100 g
Rompun dose (which has a relaxing, sedative and anesthetic effect)
Glycerin (g) Polawax cream (g) Ocara clay (g)
112.5 500 –
112.5 500 75
 G.M. Dário et al. / Materials Science and Engineering C 43 (2014) 109–116
and 0.08 mL/100 g 10% ketamine Agener dose (with an anesthetic
effect) – trichotomy of the dorsal region of the rats was performed
and the burn was caused with an 2.5 cm2 aluminum foil heated to
130 °C that was pressed into the skin of the back for 20 s. Initially, the
animals were randomly divided into three groups according to the
treatment to be performed, totaling 15 rats for each group, as follows:
group 1B (control), treatment with saline; group 2B, treatment only
with vehicle (base emulsion); and group 3B, treatment with the emul-
sion containing the Ocara clay. Letter B was used to define the burning
procedure that was performed. Each group was divided into subgroups
according to the date of euthanasia (guillotine), which was held on the
7th, 14th and 21th days of the experiment and five animals were used in
each time point [23].
The injuries were treated and protected with bandages and evaluat-
ed on daily basis. The treatment for each group was administered topi-
cally, on the injured area, around the same time of the day (20:00 h),
with the products assigned to each group. The wounds were cleaned
with 0.9% saline solution before each new application of the products
under study [23]. The animals (anesthetized using ethyl ether) were
observed on daily basis for injury repair regarding phenotypic changes
such as presence or absence of edema, exudation, crust and color
of the wound. All variables were registered for further analysis. In
sequence, the lesions were measured with the aid of a digital caliper
and the results were collected [23].
Finally, surgical samples of the skin were taken and sent for histo-
logical and morphometric studies. A fragment of the skin with 0.5 ×
2.0 cm2 rectangular area was resected from each animal. Each skin seg-
ment had the central area injured and the peripheral area non-injured
in order to serve as control. All skin samples with lesions were fixed in
a solution containing formalin (Karnovsky solution) for a minimum of
48 h [23]. After fixation, the samples were gradually dehydrated in in-
creasing concentrations of ethanol (70 to 100%), cleared in xylene, and
embedded in paraffin blocks, so that the dermoepidermal side could
face the cut surface (transversal section) of the block. The fragments
embedded in paraffin were cut using microtome and sections with
5 μm thickness were obtained. The histological slides were kept in an
oven to dry, and later the cuts were stained with hematoxylin and
eosin (HE) and Gomori's trichrome for histological analysis. The analysis
was held in an optical microscope with 100× magnification in order to
observe the inflammation and scarring [23].
3. Results and discussion
3.1. Ocara clay characterization
3.1.1. Chemical analysis
Table 2 shows the XRF results for the Ocara clay. Silica (SiO2) is the
main compound and trace elements as Mg, Ti and P are present. The
presence of trace elements can induce different therapeutic actions,
including wound-healing. Haydel et al. [18] showed the possibility
that the antibacterial activity could be attributed to a combination of el-
ements and/or chemical compounds that work in concert to mediate
toxicity in bentonite and kaolinite clays. Standard cation exchange can
occur on some clays, where the natural exchange sites (primarily inter-
layer ions in the expandable smectite mineral) were replaced by K+. As
an example, the incubation of Escherichia coli with K+-exchanged clay
resulted in complete loss of bactericidal activity.
Table 2
Chemical analysis (FRX) of the Ocara clay.
Oxide SiO2 Al2O3 Fe2O3 TiO2 CaO MgO Na2O K2O P2O5
Quantity 68.6 7.9 2.4 0.8 6.5 0.3 0.3 1.4 0.1
(mass %)
111
When compared to the chemical composition presented by other
authors [4,12,26], the amount of silica found in the sample is far above
the average values. This is an undesirable feature in the clay mineral, es-
pecially if quartz is present, because quartz presents low absorption and
could be unpleasant to the skin if the particle size is larger. The amount
of alumina (Al2O3) is far below the values found by other authors [4,12,
26] that put a range of 40 to 48% of aluminum oxide for medicinal clays.
In clay minerals the loss on ignition (LOI) is mainly due to the presence
of interspersed, coordination and zeolite water, hydroxyls present on
clay minerals and hydroxides. However, the volatile components
evolved from organic matter, sulfides, sulfates and carbonates, when
present, are included in this value [6]. Therefore, the loss on ignition is
due to the presence of organic matter and constitutional water.
3.1.2. Particle size distribution analysis
Fig. 1 shows the particle size distribution of the Ocara clay. Around
10 μm the distribution is trimodal, with particles coarser than the clay
size, probably due to quartz contamination. The clay particle size is
in the range of 0.1 to 0.4 μm and the average diameter of the clay is
26.3 μm due to the contamination with quartz. Clays with particle
sizes near 70 μm apparently act on the microcirculation of the skin,
increasing the blood flow and acting as a vasomotor stimulator [1,
15–17]. As mentioned, coarser particles are unpleasant to the skin.
3.1.3. X-ray diffraction analysis
Fig. 2 shows the mineralogical analysis of the Ocara clay. The clay
presents as main phases illite (JCPDS 00-009-0343) with general formu-
la K0.5(Al, Fe, Mg)3(Si, Al)4O10(OH)2; kaolinite (JCPDS 01-0782110),
Al4(OH)8(Si4O10); quartz (JCPDS 01-07-3755), SiO2; calcite (JCPDS 01-
071-3699), CaCO3; and dolomite (JCPDS 00-009-0343), CaCO3·MgCO3.
Some clay properties are a direct result of their mineralogical composi-
tion. Mineral phases containing Al and Mg as interchangeable cations
could present healing effects during treatment [4,10,11,13].
3.1.4. Fourier transform infrared (FTIR) analysis
Fig. 3 shows the FTIR analysis of the Ocara clay. The peaks occurring
at 3699 and at 3626 cm−1 are related to OH stretches. According to
Hunt [1], the band at 3431 cm−1 is related to H\OH hydrogen bonds.
The band at 3626 cm−1 is related to the internal hydroxyl octahedra
and the peak at 1042 cm−1 is related to the Si\O asymmetric stretch.
Finally, Al\OH deformation at 912 cm−1 and Si\O\Al vibrations at
794 and 692 cm−1 are also observed.
3.1.5. Microbiological analysis
The microbiological evaluation of the Ocara clay shows results
above 300 CFU/g for bacteria and 100 CFU/g for fungi. The plates with
100
80
Cumulative distribution, %
Histogram, ×20
60
40
20
0.1 1.0 10 100
LOI Particle size, µm
7.4
Fig. 1. Particle size distribution of the Ocara clay (D50 = 26.3 μm): the Ocara clay acts on
the microcirculation of the skin.
112 G.M. Dário et al. / Materials Science and Engineering C 43 (2014) 109–116

quartz
Intensity, a.u.

dolomite
calcite
illite kaolinite

10 20 30 40 50 60 70
2θ, deg

Fig. 2. X-ray diffraction patterns of the Ocara clay, indicating the presence of illite, kaolinite, calcite, dolomite and quartz: illite is a swelling clay and can absorb pathogenic species.

bacterial growth were tested for gram and the results showed that the white throughout the period of analysis. Although color and odor can-
microorganisms were not bacteria, but fungi. In order to continue the not be characterized as analytical measures, they significantly contrib-
experiment it was necessary to sterilize the Ocara clay [16,26] since, ute to quality assurance. These criteria are factors that have a major
as reported by López-Galindo [7], a high microbial content could jeopar- psychological effect on patients [7]. Therefore, the acceptance by the cli-
dize the stability of a product, causing the loss of therapeutic efficacy by ent is undoubtedly the most important reason which explains the pop-
degradation of the active ingredient or due to the change in fundamen- ularity of topical emulsions, since they possess some elegance and are
tal physical parameters for its activity, such as pH. Contaminated raw easily cleaned when it is desirable [7].
materials can also harm the consumers' health.
3.2.2. Emulsion density
The samples' density showed a slight variation over the period of
3.2. Accelerated stability test and determination of the validity of emulsions
analysis (Table 3) due to mass reduction attributed to the incorporation
with and without the Ocara clay
of air into the emulsion. Density is a critical parameter for maintaining
the physical stability of an emulsion. It may indicate incorporation of
3.2.1. Organoleptic characteristics
air on the sample and can result in growth of microorganisms and
The samples showed the same initial characteristics at all evaluated
oxidation, affecting the stability of the formulations.
periods, indicating the stability of the formulas [23]. The emulsion con-
taining the Ocara clay showed grayish color, the same odor of the raw
materials, homogeneous aspect and no presence of bubbles. The same 3.2.3. Emulsion viscosity
features were observed in the base emulsion, but its color remained Fig. 4 shows the rheological behavior of the emulsion made with the
Ocara clay. Initially, the emulsion showed a viscosity of 28 Pa·s that
sharply decreased to less than 5 Pa·s when the applied shear rate was
25 s−1. This feature of a fast decreasing on viscosity with the application
of a stress is an important feature when the product should be applied
2849
2920

2344

on skin and on wounds, since it avoids the effect of removing cell layers
4565

40 that are in recovery [12,13,15,27].

transmitance, %

1684

3.2.4. Centrifugation test

1636

Centrifugation, when used carefully, is a useful tool for the predic-

tion of the emulsion stability. The centrifugation test produces stresses
3431

795

in the sample, simulating an increase in the gravitational force, thus in-

692

creasing the mobility of the particles and anticipating possible instabil-

3699
3628

ities. These may appear in the form of precipitation, phase separation,

20
coalescence, formation of exudates, cremation, and flocculation,
912

among others [7,19]. The samples did not show any abnormality
when compared with the recommendations of not showing any insta-
bility, as stated before [7,19].
4000 2000 1000
wavenumber, cm-1
3.2.5. Determination of pH
Fig. 3. Spectrum in the infrared region of the Ocara clay: the OH, H\OH and Al\OH groups pH for emulsions should be between 5.0 and 6.5. Once observed that
show the swelling characteristics of the Ocara clay. the pH of the neutral emulsion was higher than recommended, the pH
 G.M. Dário et al. / Materials Science and Engineering C 43 (2014) 109–116
Table 3
Emulsion density evaluation in function of time (days).
Time of analysis (days) 0 5
Emulsion with Ocara clay 0.9344 0.9342
Neutral emulsion 1.0400 1.0254
was corrected using citric acid solution at 10%, in order to achieve the
required reduction. Table 4 shows that the pH over time has not
changed significantly for both base emulsion and for the emulsion
with the Ocara clay. Observing the averages and individual values of
pH (Table 4) all pH values are suitable for emulsions. Regarding the in-
dividual values, small variations were observed; however, taking into
account that small variations in temperature can influence the final
result of pH analysis, the variation was considered insignificant [7,27].
3.2.6. Microbiological evaluation of base emulsion and emulsion prepared
with the Ocara clay
The microbiological quality of pharmaceutical products is one of the
essential attributes for its proper performance, especially regarding
safety, efficacy and acceptability of these products. The samples subject-
ed to time analysis remained within the limits for products intended for
infant use, eye area, and contact with mucous membranes [27,28].
Therefore, the pharmaceutical product under study, the emulsion with
the Ocara clay, can be used safely. Normally, low levels of microbial con-
tamination are inevitable and do not represent a significant threat.
However, microbial growth can cause deterioration of a specific formu-
lation and can exacerbate other problems to the final user [23,24,27,28].
3.2.7. Determination of the provisory validity
Products subjected to the accelerated stability test that endure a
three month period have the shelf life determined for two provisional
years [23]. The results obtained for the emulsion with the Ocara clay
showed that the product has a long shelf life of two years, but for further
evaluation the calculation described in Eq. (1) was used. Applying the
formula of the Q10 method the provisional shelf life of 1402 days or
3.8 years was obtained. Most important, it is necessary to continue the
studies, performing tests of long-term stability (under normal con-
ditions of storage), or shelf tests, in order to confirm the durability re-
sults initially obtained for two years or more. New evaluations can
supplement the information about the stability of the product, confirm
the estimated shelf life and recommend storage conditions for the for-
mulation [23].
Fig. 4. Viscosity behavior of the emulsion with the Ocara clay as a function of shear stress: the viscosity behavior shows that the emulsion can be applied on skin and on wounds.
113
10 15 30 60 90 Average
0.9289 0.9291 0.9286 0.9301 0.9296 0.9307
1.0358 1.0374 1.0371 1.0368 1.0366 1.0355
3.3. Evaluation of the healing activity
3.3.1. Macroscopic evaluation of the results
Fig. 5 shows the histological evolution (before sacrifice) at 7, 14 and
21 days in function of treatment type. The animals showed yellowish
brown wounds and few of them showed exudates/pus or swelling in
the lesions during the course of the experiment. The crust formation
was observed in the first to second week of analysis and these lesions
were moving over time for all groups (saline, Fig. 5a, pure emulsion,
Fig. 5d and emulsion + Ocara clay, Fig. 5g). Also, the neoepithelization
around the lesion over time, indicating a reduction in the extent of the
injury was noticed. The color of the neoepithelization was lighter than
the color of the not injured skin because deep burns can cause depig-
mentation of the skin (saline, Fig. 5b, pure emulsion, Fig. 5e and emul-
sion + Ocara clay, Fig. 5h). Visually, a slow reduction in the size of the
wounds was observed; however, the burns inflicted on the animals
have been profound, affecting all layers of the skin and muscles, so
they were considered third-degree burns (Fig. 5a, d, g). A sharp recovery
of the lesion in relation to depth was observed, especially in the group of
animals treated with the emulsion with Ocara clay, where drier wounds
were observed too (Fig. 5g, h, i for 7, 14 and 21 days of treatment,
respectively).
Taking into account all animals evaluated during treatment, only
two of the emulsion group showed edema formation added to the inter-
nal injury, forming a large bag filled with liquid (not shown in Fig. 5).
The presence of chemicals inside the bubble may slow the healing pro-
cess. Injuries associated with burns should be protected against me-
chanical trauma and possible contaminants, but the cage where the
animals were kept could present both possible situations, becoming a
negative factor for the test in question.
Table 5 shows a description of the histological characteristics
(before sacrifice) at 7, 14 and 21 days in function of treatment type.
The most statistically significant reduction of injuries, when measured
with calipers, was those in which animals were treated with emulsion
with the Ocara clay, followed by base (neutral) emulsion and saline.
Fig. 6 shows the evolution of the healing activity (reduction of injuries)
for saline, base emulsion and Ocara clay with emulsion before clay
114 G.M. Dário et al. / Materials Science and Engineering C 43 (2014) 109–116

Table 4 differences (p b 0.05) between groups for the healing evolution of the
pH results for the neutral emulsion and emulsion with the Ocara clay in function of time animals treated with the Ocara clay with emulsion for 7, 14 and 21
(days).
days of treatment in comparison with the saline solution and the base
pH (average) 0 5 10 15 30 60 90 Average emulsion for the same period of analysis. In addition, the Ocara clay
Emulsion with Ocara clay 6.36 6.28 6.32 6.31 6.34 6.32 6.32 6.32 with the base emulsion shows a progressive and constant evolution of
Neutral emulsion 6.46 6.48 6.49 6.48 6.46 6.46 6.49 6.47 the healing activity for the testing period, differently regarding the sa-
line solution and the base emulsion, that show no significant results
for 7 and 14 days of treatment (Fig. 7).
sterilization for 7, 14 and 21 days of treatment (see Section 3.1.5). The It is believed that the base emulsion was able to mediate the constit-
reduction of injuries (lesions) was not significant (p N 0.05) when com- uents of the Ocara clay to the sites of action. Also, according to Girase
pared between different treatments before clay sterilization. The Ocara et al. [28,29], the stacked layered structure of clay minerals promotes
clay apparently showed no healing activity, probably due to the higher the controlled diffusion of active substances with antimicrobial activity.
silica content and higher concentration of fungi. Kokabi et al. [30] have found that the quantity of clay added to a nano-
Fig. 7 shows the evolution of the healing activity (reduction of inju- composite hydrogel is the key factor in obtaining suitable properties re-
ries) for 7, 14 and 21 days of treatment for saline, base emulsion and quired for wound dressing. Also, Ul-Islam et al. [31] have shown that the
Ocara clay with emulsion after clay sterilization. There are significant slow release of water from clay composites can be useful in enhancing

Fig. 5. Histological evolution (before sacrifice) at 7, 14 and 21 days in function of treatment type.
 G.M. Dário et al. / Materials Science and Engineering C 43 (2014) 109–116
Table 5
Description of the histological characteristics (before sacrifice) at 7, 14 and 21 days in function of treatment type.
Animal group (day of sacrifice) Treatment
Saline (group 1B)
7th Rare fibroblasts with small
amounts of collagen fibers
14th Moderate amount of collagen
fibers, macrophages
21th Organization of collagen fibers
the biomedical efficacy of cellulose–montmorillonite composites as
dressing material. Moreover, the composite has wound healing proper-
ties when applied as a wet paste on wounds and skin. Finally, Salcedo
et al. [32] have studied the interaction between chitosan and montmo-
rillonite, producing a new biohybrid material that can be considered as
promising candidate for modified drug delivery formulations.
El-Arnaouty et al. [33] suggested that PVA/PVP/clay nanocomposite
showing good swelling and barrier capability against microbe penetra-
tion can act as a wound dressing. Kim and Yeum [34] showed that PVA/
PU/montmorillonite nanocomposites are hydrophilic, non-toxic and
biocompatible materials that can be used as wound dressing materials.
Oh et al. [35] have combined polyurethane foam with protein drug-
loaded pH-sensitive alginate–bentonite hydrogel for wound dressings.
Sirousazar et al. [36] have prepared non-toxic nanocomposite hydrogel
that could be used as a biocompatible wound dressing.
These previous results agree with the results obtained here, where
the Ocara clay emulsion when tested in animal models, have shown
healing activity (Section 3.3.2). The main difference between this study
and the previous ones is the carrier of the healing clay, polymers ore
hydrogels in the previous studies [30–36] and a simple emulsion in here.
3.3.2. Histological evaluation
The wound-healing process consists of four highly integrated and
overlapping phases: hemostasis, inflammation, proliferation, and tissue
remodeling or resolution. These phases and their biophysiological func-
tions must occur in the proper sequence, at a specific time, and continue
for a specific duration at an optimal intensity [37].
Table 5 shows the evolution of injury healing in animal models. An-
imals treated with the emulsion containing the Ocara clay have present-
ed more expressive results than others. A healing process goes through
some steps, the first of which occurs in the early hours (hemostasis).
This process is the activation of platelets, with consequent platelet ag-
gregation and coagulation cascade. In the second phase, which occurs
in days, the inflammatory process is present, and there is recruitment
of neutrophils and macrophages, which among other things help to
Fig. 6. Evolution of the healing activity for saline, base emulsion and Ocara clay with
emulsion before clay sterilization for 7, 14 and 21 days of treatment.
115
Neutral emulsion (group 2B) Emulsion + Ocara clay (group 3B)
Few fibroblasts with few polymorphonuclears Few collagen fibers with moderate
amounts of polymorphonuclears
Moderate number of Epithelium overlying the lesion;
collagen fibers, macrophages numerous bundles of collagen fibers;
presence of macrophages
Organization of collagen fibers Thick bundles of collagen fibers;
regeneration of the deep dermis
break down the devitalized tissue. In addition, the macrophages stimu-
late the growth of new tissue, as observed in animals on the 7th day,
with presence of small collagen fibers. During the third stage of healing,
which occurs around days or weeks, there is a more intensified process
of reepithelialization and continuous formation of granulation tissue;
this could be seen in animals related to the 14th day. The presence of
macrophages at this stage encourages internal production of fibroblasts
and deposition of connective tissue, while the migratory fibroblasts pro-
duce collagen. It was noticed that in the group of animals from day 21
the third stage was still present; this is explained by the depth of the
lesion, which may be a limiting factor for the evolution of healing [1].
Clays can be a very simple and effective way to remove heavy metals
and chemical contaminants from the surface tissue of the body. Most
chemical and metal toxins have a positive charge, whereas the clay
has a negative charge. Therefore, the toxins are drawn towards the
clay surface. As an example, bentonite clays have a great capacity for
absorbing many times its own weight in toxins. When bentonite clay
absorbs water and swells, it is stretched open like a highly porous
sponge; the toxins are drawn into these spaces by electrical attraction
and bound fast. In fact, bentonites can absorb pathogenic viruses, afla-
toxin and pesticides and herbicides. The clay is eventually eliminated
from the body with the toxins bound to its multiple surfaces [38].
Finally, clays are extremely affordable if one has access to a clay
source; clay treatment causes no harm when properly employed; rapid
results are often achieved even when all other courses of action have
been exhausted; use of clays often results in a rapid reduction of pain
and irritation; clays can often be used with other forms of treatment.
4. Conclusions
According to the results of this study the Ocara clay showed a large
amount of microbial and silica content, which is a negative factor to
the healing activity of this clay. Therefore, a more detailed study is need-
ed regarding the exploitation of the clay in its original deposit. The
Fig. 7. Evolution of the healing activity for 7, 14 and 21 days of treatment for saline, base
emulsion and Ocara clay with emulsion after clay sterilization (*significant (p b 0.05) at
7 and 14 days; **not significant; ***significant at 7, 14 and 21 days).
116 G.M. Dário et al. / Materials Science and Engineering C 43 (2014) 109–116
chemical and physical analyses showed that the Ocara clay must be sub-
mitted to a prior processing step with the purpose of improving its qual-
ity. However, when incorporated into a pharmaceutical formula, the
Ocara clay was not able to modify adversely the stability of the formula-
tion, thus allowing a validity of 2 years for the emulsion with the Ocara
clay. This formulation when tested in animal models showed healing ac-
tivity, even though the reduction of injuries has not been significant
among the groups. However, the histological evaluation was able to
demonstrate differences between the recovery of the deep dermis be-
tween the different groups, and the best results were assigned to the
group treated with the emulsion containing the Ocara clay.
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