EFFECT OF BENTONITE ON SKIN WOUND HEALING:

EXPERIMENTAL STUDY IN THE RAT MODEL

S. H. Emami-Razavi1, N. Esmaeili1, S. K. Forouzannia2, S. Amanpour1, S. Rabbani1, A. M. Alizadeh1
and M. A. Mohagheghi1

1) Experimental Laboratory, Cancer Research Center, Cancer Institute, Imam Khomeini Hospital,
Tehran University of Medical Sciences, Tehran, Iran
2) Department of Cardiovascular Surgery, Afshar Hospital, School of Medicine, Yazd University
of Medical Sciences, Yazd, Iran

Abstract- Wound healing in the skin depends upon the availability of appropriate trace metals as
enzyme cofactors and structural components in tissue repair. The present study is a part of a series of
experimental investigations to examine the influence of Bentonite on skin wound healing. Surgically
induced skin wounds in 48 young adult male rats were exposed topically to Bentonite (12 round wound
and 12 incisional wound) and control wounds (12 round wound and 12 incisional wound) received de-
ionized water only. Skin wounds (round and incisional) treated with Bentonite exhibited no significant
difference in margins with erythema and edematous changes. Scab and wound debris was more
extensive and persisted for at least 7 days after surgery in control group (P < 0.05). Skin wounds
exposed to Bentonite exhibited a mild retarded re-epithelialization, the treatment wounds were
characterized by a prominent central mass of inflammatory cells, cell debris and wound exudate. The
intense infiltrate of lymphocytes, macrophages, monocytes and fibroblasts extended from the wound
margin into the region of the panniculus carnosus muscle and hypodermis. Vascular dilatation and
dermal oedema were prominent features of these wounds. External utilization of Bentonite for wound
healing is safe and feasible, and we finalized that macroscopic healing of wound that treated by
Bentonite was superior versus control group.
Acta Medica Iranica, 44(4): 235-240; 2006
© 2006 Tehran University of Medical Sciences. All rights reserved.

Key words: Wound healing, trace metals, re-epithelialization

INTRODUCTION In the inflammatory phase, the recruitment of

leukocytes such as neutrophils and macrophages into
Wound healing immediately starts after an injury the wound site is a hallmark. In the proliferative
and proceeds with a complicated but well-organized phase, the migration and proliferation of
interaction among various types of tissues and cells. keratinocytes, fibroblasts, and endothelial cells result
Skin wound healing is composed of the in re-epithelialization and tissue granulation. In the
inflammatory, proliferative, and maturation phases. maturation phase, excess collagen in the wound site
is degraded by several proteolytic enzymes, leading
Received: 5 Sep. 2005, Revised: 13 Dec. 2005, Accepted: 18 Mar. 2006
to the completion of tissue repair (1, 2). It is well
* Corresponding Author: known that biological substances such as cytokines,
S. K. Forouzannia, Department of Cardiovascular Surgery, Afshar
Hospital, School of Medicine, Yazd University of Medical Sciences, chemokines and growth factors are closely involved
Yazd, Iran in every phase of wound healing process. Trace
Tel: +98 351 5254067
Fax: +98 351 5255011 metals have a major role in tissue repair systems as
E-mail: drforouzan_nia@yahoo.com
cofactors in metalloenzyme systems and as structural
Effect of bentonite on skin wound healing

components (3). Recent studies in the rat model have Table 1. Analysis of bentonite by X-ray distribution.
demonstrated that as wounds heal, local Major phase
concentrations of zinc, calcium, copper and Montmorillonite
magnesium change according to the phase in the Minor phase
wound healing cascade and associated biochemical 1. Illite-Montmorillonite [K-Al4(SiAl)8O20(OH)4,XH2O]
events (4). Clinical observations in humans and 2. Nontronite-15A [Na0.3Fe2Si4O10(OH)2,4H2O]
experimental studies have demonstrated that 3. Albite [(Na,Ca) Al(Si3Al)O8
deficiencies in the availability of these metals, 4. Hslloysite-7A [Al2Si2O5(OH)4]
imbalance in local concentrations, or defects in
metabolism are potential causes of defective or Full-thickness wounds
nonhealing wounds (5, 6). Rats were anaesthetized by Ketamine 50mg/Kg
The present study is a part of a series of and Xylazine HCL 5mg/Kg and all subsequent
experiments designed to investigate the action of wounding procedures were carried out on a heated
bentonite on skin wound healing in the rat model. table. The dorsal region of the animal was shaved
and sterilized using 70% alcohol. Using sterile 1 cm
MATERIALS AND METHODS punch biopsies, a full-thickness excisional wound
(round wound) and 1 cm longitudinal full thickness
Animals wound (incisional wound), (4, 10) were created on
Male young adult rats, SD (250+/-50 g body each dorsolateral flank equidistant from the midline.
weight) were used in all experiments. Rats were Wounds were separated by a margin of at least 15
housed in groups of four in plastic solid-bottomed mm. Each wound region was digitally photographed
cages provided with sterile dust-free bedding and at the indicated time intervals, and the areas of the
temperatures of 22 ± 2 °C, relative humidity of 45– wounds were calculated by PhotoShop software,
55%, and 12 h:12 h day/night cycles. All version 7.0. Changes in the wound areas were
experiments on animals were performed in expressed as the percentage of the initial wound
accordance with UK legal requirements. areas. After preparation of Bentonite (2 gr/5cc H2o),
the covering was applied in round and incisional
Chemicals wound in case group and the treatment was repeated
Type of mineral elements of the bentonite was 10 days until the end of the experiment.
distinguished in previous studies: commercial term Measurements of the inner wound edge were
for clays containing montmorillonite type minerals repeated on days 3, 5, 7 and on day 10 to enable
(7), Na 0:33[Al1: 67 Mg 0:33] Si4 [OH]2 (8), Native calculation of wound closure as a result of re-
hydrated colloidal aluminum silicate clay (9). epithelialization. Bentonite treated and control rats
The principle constituent is montmorillonite. after 10 days and wound sites excised. The tissue
However, other minerals such as illite, kaolinite and was preserved in 10% phosphate buffered formalin
nonargillaceous detrital minerals can be present. for histology and haematoxylon and eosin stained
Most bentonites appear relatively pure and other sections. Comparable numbers of control animals
mineral contributions rarely exceed 10%. received de-ionized water only.
Cristobalite is often present. Montmorillonite
compositions frequently vary either in its lattice Clinical assessment of wounds healing
structure or in the exchangeable ions present. We During and the end of the treatment wound sites
analyzed Bentonite by X ray distribution that were examined daily for evidence of hemorrhage,
summarized in Table 1. Bentonite functions as an ulceration, scab formation, suture loss or other
absorbent, bulking agent, emulsion stabilizer, changes. For this, wound outlines were transferred to
opacifying agent, suspending agent-nonsurfactant, software capable of determining the area and the
and viscosity-increasing agent-aqueous in cosmetic extent of wound closure expressed as a percentage of
formulations (9). the original wound size: % wound closure = 100×

236 Acta Medica Iranica, Vol. 44, No. 4 (2006)

 S. H. Emami-Razavi et al.

[(area on day 0- area of open wound)/area on day 0] Microscopic observations

(11). Differences in the histological profile between
wounds treated with Bentonite and controls were
Pathological assessment of wound healing marginal through the 10 days following incision.
At time end of the study, animals were scarified After 10 days, wound sites were re-epithelialized but
by cervical dislocation and the wounded skin was some epidermal hyperplasia was present in most
excised and fixed overnight in a 4% buffered sections. Skin wounds exposed to Bentonite
formalin solution. The tissue was washed in PBS, exhibited a mild retarded re-epithelialization, the
embedded in fibrowax, and sections (10 µm) were treatment wounds were characterized by a prominent
stained using standard procedures with central mass of inflammatory cells, cell debris and
haematoxylon and eosin for morphological wound exudate (Fig. 2). The intense infiltrate of
assessment. Using planimetry software, captured lymphocytes, macrophages, monocytes and
images of the stained tissue were analyzed. Analysis fibroblasts extended from the wound margin into the
of stained tissue comprised of cell degeneration, region of the panniculus carnosus muscle and
giant cell, epidermis, collage, infiltration and vessel hypodermis. Vascular dilatation and dermal oedema
formation. Granula-tion tissue thickness was were prominent features of these wounds.
assessed by dividing the measured granulation tissue
area (mm2) by the length measured (mm) for each DISCUSSION
treatment. Re-epithelialization was assessed by
measuring the length, area, and average thickness of Skin injury immediately causes clot formation
the epidermal ‘tongue’ for each wound site (12). and local inflammation characterized by an
infiltration of neutrophils and macrophages into the
Statistical methods wound sites. These pathological changes are
Statistical differences were determined using the hallmarks of the inflammatory phase of wound
Student’s t test. All data are presented as the mean ± healing. The inflammatory response is believed to be
standard deviation. P value < 0.05 was accepted as instrumental in supplying the growth factors,
statistically significant. cytokines, and chemokines that orchestrate the cell
movement necessary for wound repair (1, 2).
RESULTS Recent studies have demonstrated that
concentrations of essential trace metals change in
Macroscopic observations skin wounds to reflect their requirements in
Wound healing in control rats progressed with metalloenzyme complexes in sequential events in the
crusting, scab formation and re-epithelialization wound healing cascade (4). Since bivalent trace
around the round and incisional wound for metals compete for binding sites on carrier proteins
approximately 10 days. Skin wounds (round and and in metabolic events, the balance of trace metals
incisional) treated with Bentonite exhibited no in the wound site is critical for the stage in healing.
significant difference in margins with erythema and Imbalances in the relative concentrations of
edematous changes. Scab and wound debris was calcium:zinc, zinc:copper, etc., or the presence of a
more extensive and persisted for at least 7 days after xenobiotic ion like cadmium, are potential causes of
surgery in control group (P < 0.05) (Table 2, Fig 1). impaired wound healing (6, 13, 14).

Table 2. Clinical observation of wound healing (ring and long) that expose to Bentonite compared to control group after 7 days.
Round wound Wound healing Inflammation
Bentonite group 10 (83.3%) 4 (33.3%)
Control group 5 (41.6%) 4 (33.3%)
Incisional wound Wound healing Inflammation
Bentonite group 11 (91.6%) 5 (41.6%)
Control group 4 (33.3%) 6 (50%)

Acta Medica Iranica, Vol. 44, No. 4 (2006) 237

Effect of bentonite on skin wound healing

Bentonite group. A Control group

Bentonite group B Control group

Fig 1. Macroscopic observations of Skin round (A) and incisional (B) wounds exposed to Bentonite compared to control group after
7 days.

Bentonite is widely used in many, many successful treatment for exposure to chemical
industries. However, what make it unique for warfare. One Army emergency livestock protocol
consideration in natural medicine are its very unique calls for immediate administration of bentonite
properties. When properly hydrated, it creates and internally to counter effects of radiation poisoning in
sustains its own subtle electromagnetic field livestock (food sources). The immunotoxicity of
(negatively charged particles). It has extremely Bentonite through environmental and dietary
powerful Absorptive properties, i.e. it attracts and exposure is recognized in humans and experimental
holds to its surface many toxic substances. U.S. animals (15-17). Beck and Bignon dosed peritoneal
Army studies show that bentonite may be a macrophages with two samples of Bentonite and the

A B
Fig 2. Microscopic observations of skin wounds exposed to Bentonite exhibited a mild retarded re-epithelialization, the treatment
wounds were characterized by a prominent central mass of inflammatory cells, cell debris and wound exudates (A) and control group
(B).

238 Acta Medica Iranica, Vol. 44, No. 4 (2006)

 S. H. Emami-Razavi et al.

triphenyltetrazolium chloride (TTC) reduction, LDH between two groups and we concluded that
activity, and methylene blue absorption were used to Bentonite was not very efficient for improving of
assess cytotoxicity (18). Bentonite inhibited TTC wound. Further work by more cases needs to be done
reduction similar to the fibrogenic dusts such as on these.
quartz. However, the extracellular LDH activity was
not increased and methylene blue absorption was Conflict of interests
very high. Hatch examined the cytotoxicity of We have no conflict of interests.
Bentonite to rabbit alveolar macrophages (19).
The viability percentage of the macrophages and
the ATP content of the cells as index of cytotoxicity
were determined. Bentonite caused a large reduction
REFERENCES
in both the viability and ATP levels.
TTC reduction, LDH activity, and methylene 1. Martin P. Wound healing--aiming for perfect skin
blue absorption were measured as indexes of regeneration. Science. 1997 Apr 4; 276(5309):75-81.
cytotoxicity in a study by Adamis (20). TTC 2. Singer AJ, Clark RA. Cutaneous wound healing. N
reduction was much greater and proved Bentonite to Engl J Med. 1999 Sep 2; 341(10):738-746.
be cytotoxic. Extracellular LDH was almost half for 3. Lansdown AB. Physiological and toxicological changes
Bentonite compared to control values. Methylene in the skin resulting from the action and interaction of
blue absorption was significantly higher for metal ions. Crit Rev Toxicol. 1995; 25(5):397-462.
Bentonite. Murphy, Roberts, and Horrocks 4. Lansdown AB, Sampson B, Rowe A. Sequential
investigated the cytotoxicity of Bentonite to human changes in trace metal, metallothionein and calmodulin
umbilical vein endothelial (HUVE) cells, concentrations in healing skin wounds. J Anat. 1999
undifferentiated N1E-115 neuroblastoma cells, and Oct; 195 ( Pt 3):375-386.
ROC-1 oligodendroglial cells (21). Indices of 5. Moynahan EJ. Letter: Acrodermatitis enteropathica: a
cytotoxicity used in this study were morphological lethal inherited human zinc-deficiency disorder.
examination, LDH activity, and fatty acid release. In Lancet. 1974 Aug 17; 2(7877):399-400.
a separate study by Murphy, the cytotoxicity of 6. Heng MK, Song MK, Heng MC. Reciprocity between
Bentonite was examined in two cell lines: primary tissue calmodulin and cAMP levels: modulation by
murine spinal cord neurons and differentiated N1E- excess zinc. Br J Dermatol. 1993 Sep; 129(3):280-285.
115 neuroblastoma cells and no cytotoxicity were 7. Gamble JF. Silicate pneumoconiosis. In: Merchant JA,
recorded as a result of Bentonite treatment (22). editor. Occupational respiratory diseases. Cincinnati:
When used immediately in severe trauma US Department of Health and Human Services, Public
situations (externally), clay packs significantly Health Service, CDC; 1986. DHHS publication no.
reduce tissue damage associated with swelling. The (NIOSH). p. 86-102.
protective mechanism of Bentonite is likely to be a 8. Rheox Inc. The benefits of hectorite clay and safety
combination of allergen absorption and skin barrier data sheet on Bentone MA 1999 (purified hectorite).
function improvement. This may result in Unpublished data submitted by Rheox Inc. 3 pages.2
significantly reduced percutaneous allergen 9. Wenninger JA., Canterbery RC, McEwen Jr. GN, eds.
penetration (23). International Cosmetic Ingredient Dictionary and
In this observation we studied the effect of Handbook. 8th ed. Baltimore, Maryland: Cosmetic
Bentonite on skin of rat on ring and long wound Toiletry & Fragrance Assn; 2000.
healing. Wound size both in ring and long group was 10. Lansdown AG. Influence of zinc oxide on the closure
rapidly improved in Bentonite treated wound of open wounds: an experimental study. Int J Cosmet
compared to control group, but inflammation Sci. 1993; 46: 83-85.
response was not significantly different between two 11. Bevan D, Gherardi E, Fan TP, Edwards D, Warn R.
groups. Moreover, in pathology examination we did Diverse and potent activities of HGF/SF in skin wound
not show significant differences in wound healing repair. J Pathol. 2004 Jul; 203(3):831-838.

Acta Medica Iranica, Vol. 44, No. 4 (2006) 239

Effect of bentonite on skin wound healing

12. Low QE, Drugea IA, Duffner LA, Quinn DG, Cook 18. Beck EG, Bignon J, editors. In vitro effects of mineral
DN, Rollins BJ, Kovacs EJ, DiPietro LA. Wound dusts. NATO ASI Series, Vol. G3. Berlin: Springer-
healing in MIP-1alpha(-/-) and MCP-1(-/-) mice. Am J Verlag; 1985.
Pathol. 2001 Aug; 159(2):457-463. 19. Hatch GE, Boykin E, Graham JA, Lewtas J, Pott F,
13. Klevay LJ. The relationship of zinc to copper of diets Loud K, Mumford JL. Inhalable particles and
in the United States. Nutr Rep Int.1975; 11: 237. pulmonary host defense: in vivo and in vitro effects of
14. Vicanova J, Boelsma E, Mommaas AM, Kempenaar ambient air and combustion particles. Environ Res.
JA, Forslind B, Pallon J, Egelrud T, Koerten HK, 1985 Feb; 36(1):67-80.
Ponec M. Normalization of epidermal calcium 20. Adamis Z, Timar M, Kofler L, Tatrai E, Ungvary G.
distribution profile in reconstructed human epidermis is Biological effects of the respirable dusts from ore
related to improvement of terminal differentiation and mines. Environ Res. 1986 Oct; 41(1):319-326.
stratum corneum barrier formation. J Invest Dermatol. 21. Murphy EJ, Roberts E, Horrocks LA. Aluminum
1998 Jul; 111(1):97-106. silicate toxicity in cell cultures. Neuroscience. 1993
15. Descotes J. Immunotoxicology of cadmium. Jul; 55(2):597-605.
IARC Sci Publ. 1992;(118):385-390. 22. Murphy EJ, Roberts E, Anderson DK, Horrocks LA.
16. Stelzer KJ, Pazdernik TL. Cadmium-induced Cytotoxicity of aluminum silicates in primary
immunotoxicity. Int J Immunopharmacol. 1983; neuronal cultures. Neuroscience. 1993 Nov; 57(2):483-
5(6):541-548. 490.
17. Bernier J, Brousseau P, Krzystyniak K, Tryphonas H, 23. Fowler JF Jr. A skin moisturizing cream containing
Fournier M. Immunotoxicity of heavy metals in Quaternium-18-Bentonite effectively improves chronic
relation to Great Lakes. Environ Health Perspect. 1995 hand dermatitis. J Cutan Med Surg. 2001 May-Jun;
Dec;103 Suppl 9:23-34. 5(3):201-205.

240 Acta Medica Iranica, Vol. 44, No. 4 (2006)