Comp Clin Pathol
DOI 10.1007/s00580-014-1887-9
ORIGINAL ARTICLE
The effect of honey-impregnated human placenta membrane
on burn wound healing in rat
Fatemeh Samiee rad & Akram Beheshti &
Amir Abdollah Zangivand & Younes Shafigh
Received: 17 December 2013 / Accepted: 16 January 2014
# Springer-Verlag London 2014
Abstract Burn is a severe form of thermal injury. Placenta
membrane has been introduced as a rapid physiological
wound healer; honey has been known for its antibacterial
activity and silver sulfadiazine (SSD) as one of the most
commonly used topical treatment for partial thickness burns.
Present study has been performed to compare the placenta
membrane, honey-impregnated placenta membrane, and
SSD-impregnated placenta membrane in the healing of burn
wounds in rat. For this study, 64 male Wistar rats were
randomly divided into four groups: control, placenta mem-
brane, honey-impregnated placenta membrane, and SSD-
impregnated placenta membrane groups. For all animals, un-
der the general anesthesia, deep partial thickness burn was
created. At the end of the first, second, third and fourth weeks
of treatments, biopsies were taken from burn and adjacent
normal areas. In the prepared slides, the number of polymor-
phonuclear (PMN) leukocytes, vascular channels, and fibro-
blasts were examined. In the third group treated by honey-
impregnated placenta membranes, PMN leukocyte count was
significantly lower than that of the other three groups
(P<0.05). The amount of granulation tissue formation and
F. S. rad
Department of Pathology, Qazvin University of Medical Sciences,
Qazvin, Iran
A. Beheshti (*)
Department of Dermatology, Clinical Research Develop Unit,
Qazvin University of Medical Sciences, Qazvin, Iran
e-mail: beheshti13025@gmail.com
A. A. Zangivand
Qazvin University of Medical Sciences, Qazvin, Iran
Y. Shafigh
Department of Surgery, Qazvin University of Medical Sciences,
Qazvin, Iran
the number of fibroblasts in this group were greater than those
of the other three groups (P<0.05). The organization of gran-
ulation tissue in the abovementioned group was significantly
better (P<0.05). The reepithelialization, containing horny lay-
er, in the second and third groups was completely formed. The
honey-impregnated placenta membrane was an ideal tissue for
temporary wound coverage and repair surface injuries after
partial thickness burns.
Keywords Burn . Honey . Placenta membrane . Silver
sulfadiazine
Introduction
Burn is an especially severe form of thermal injury including a
hypermetabolic condition associated with immune response
depression and alternation in wound healing pathways. Due to
irreversible cellular injury including cell membrane destabili-
zation and ATP depletion, tissue necrosis happens (Zambruno
et al. 1995; Atiyeh et al. 2003).
Wound healing followed by burn does not differ from other
forms of wound healing (Atiyeh et al. 2005a). Wound healing
includes dynamic interaction of biological components like
cellular and blood-borne growth/chemokine factors, multiple
signaling cascades, and also extracellular matrices (Atiyeh
and Hayek 2004; Diegelmann and Evans 2004).
The best therapeutic approach to accelerate burn healing is
to block the immediate inflammatory response and many
components of inflammatory cascade regulated in molecular
level by growth hormones and cytokines (Zambruno et al.
1995). Therefore, cytokine modulation by local application of
therapeutic agents is a high topic modality. Interpretation of
these interactions, drug effect assessment, and determination
of exact significance of specific cellular and cytokine modu-
lation on wound recovery, individually studied or in

combination with other inflammatory cells and cytokines
needs to be clearly made (Jurjus et al. 2007; Amendt et al.
2002).
Daily, around the word, the silver sulfadiazine (SSD) is
the most popular topical agent applied for treatment of
partial thickness burns. However, results of many studies
have been recently disclosed that SSD application may
delay healing partial thickness burns (Demling RH: Burns
and other thermal injuries. In: Lawrence W et al. 2002;
Gracia 2001).
The application of human placenta membrane in China and
Japan as a potent medication has long been known.
Tyszkiewicz et al. (Tyszkiewicz et al. 1999) believed these
materials have “magical strength of youth.” The use of human
placenta membrane as a temporary biologic dressing for
wounds was first described by Douglas in 1952; then Sabella
(1913) demonstrated the use of placenta membrane in burn
wounds. The results of many studies showed that the amniotic
membrane inhibited the migration of polymorphonuclear
(PMN) leukocytes, stimulated epithelialization, and accelerat-
ed granulation tissue formation at the wounded area. There-
fore, they believed the amniotic membrane promoted rapid
healing (Rinastiti et al. 2006; Efem 1993).
The antibacterial effect of honey has been contributed to
the exclusive composition of honey including high osmo-
larity, low pH, and presence of glucose oxidase enzyme
and phytochemicals (Molan 1992). Many previous studies
about wound and burn healing on animal models demon-
strated that honey as a topical agent could be used. The
beneficial effects of topical honey use were supported in
many researches. Topical application of honey was said to
have improved granulation tissue formation and epithelial-
ization and reduced the amount of fibrinoleukocyte exu-
dates. Some studies compared honey to other topical ther-
apy such as sterile gauze, SSD, and placental membrane.
The results denoted that topically applied honey showed
favorable outcome compared to other treatments, not only
in improving the healing speed of partial thickness burns
but also in controlling of bacterial infections during healing
(Ndayisaba et al. 1993).
Various attempts to introduce materials to accelerate burn
wound healing and reconstruction of skin tissue damage has
been made. As previously described, many studies have been
conducted for evaluation of clinical usefulness of SSD, honey,
and placenta member in the treatment of burns; however, from
the clinical benefits point of view, comparison between SSD-
containing amniotic membrane and honey-containing amni-
otic membrane as a temporary dressing and selection the
better choice between them are an area of active debate. The
present study is performed to compare the effect of placenta
membrane, honey-impregnated placenta membrane, and
SSD-impregnated placenta membrane in the experimental
burn model in rats.
Comp Clin Pathol
Method
For this study, 64 male Wistar rats (weight ranged from 200 to
250 g) were obtained from Razi Institute (Karaj, Iran) and
were randomly divided into four groups (each consisting 16
animals). Rats were kept at constant environmental status
(room temperature 21±2 °C) with a normal 12 L/12D and
free access regimen under the standard laboratory conditions.
Prior to the experimental burn, the hair on neck back of the
rats were shaved with a standard electric shaving machine. All
guidelines and recommendations of the European Communi-
ties Council Directive dated on 24 November 1986 (86/609/
EEC) were followed for minimizing the rat numbers and their
inflictions. All animals were anesthetized by intraperitoneal
injection of 40 mg/kg ketamine (Rotexmedica, GmbH, Ger-
many) and 5 mg/kg xylazine 2 % (Loughrea Co., Galway,
Ireland). If necessary, for pain-free status during the experi-
mental procedure, booster injections up to one-half of the
initial dose were administered again. Burn injury was created
by a heated (to 80 °C) round iron stamp, 1.75 cm in diameter.
It was then applied for 1 second to produce a deep partial
thickness (second-degree) burn (Shahabi et al. 2005). This
concept was confirmed by pathologic review of hematoxylin
and eosin stain (H&E) prepared slides.
Group 1 was control group whose burn wounds were
covered by a simple saline dressing; in the second, third, and
fourth groups, the applications of placenta membrane, honey-
impregnated placenta membrane, and SSD-impregnated pla-
centa membrane (SSD 1 %, Sobhan Pharmaceutical Compa-
ny, Iran ) were done, respectively.
Daily, the covered ulcer area was cleansed with saline, and
any eschar and necrotic tissue were removed and the dressings
were exchanged after sedating the rats with intramuscular
injection of 0.4 ml/kg of 2 % xylazine.
In order to avoid any risk of transplanting infectious mate-
rial, we used placentas from pregnant women with completed
and normal prenatal care including medical history (cancer,
infectious diseases, and drug abuse) and laboratory studies
(hepatitis B and C, syphilis, and HIV). As soon as, after
delivery (elective cesarean section, CS), under a lamellar-
flow hood, placentas and associated fetal membranes were
washed with Ringer’s solution, then under sterile conditions,
chorion and amnion were separated by trained staff. The
placenta membranes were then transferred to sterile and cool
containers (4 ° C) in the delivery room and immediately sent
to the processing site where placenta membranes were rinsed
four times and soaked in normal saline, then once with
Dakin’s solution (0.25 % sodium hypochlorite solution), next
four times with normal saline in order to remove blood and
other contaminants. The HPM samples were sliced into de-
sired sizes. After that, they were refrigerated in sterile con-
tainers at 4 °C. By coordination between team subjects, all
stages were performed in less than 2 h.
Comp Clin Pathol
Rats were sacrificed (by high dose of ether gas) at the end
of first, second, third, and fourth weeks of treatments (Four
rats of each group were sacrificed at the indicated time). The
tissue biopsies were taken from burn and adjacent normal
areas. The tissue biopsies were fixed in 10 % neutral formalin
buffer, passaged, processed, embedded in paraffin, and sec-
tioned serially at 5 μm. The sections were prepared by H&E
staining and examined under the light microscope. At the high
magnification (×400) for vascular channels and×1,000, oil
Wound surface percentage ¼ ðthe area of woundonparticular day  100Þ the area of woundonthe first day:
Healingpercentage ¼ ð100−woundsurface percentageÞ
Ethical approval
The study was approved by the ethics committee of Qazvin
University of Medical science before initiation. All the proto-
cols used in this study conformed to the ethical guidelines of
1975 Helsinki Declaration.
Data analysis
The collected data were analyzed using the Statistical Package
for the Social Sciences (SPSS) software (version 11.0, SPSS
Inc, Chicago, Ill, USA). Data were described as mean±stan-
dard error of the mean. Multiple comparisons were made
among and within each group of rats at intervals. The signif-
icance degree of differences among these groups was analyzed
by using one-way ANOVA and the Bonferroni post hoc
Tukey’s test. A level of P<0.05 was accepted as significant.
Results
Review of four group slides on day 7 displayed large amounts
of necrotic tissues admixed with inflammatory cells mainly
composed of PMN leukocytes (fibrinoleukocyte exudates)
associated with epidermal destruction. Underlying dermis
partially was replaced by the first stages of granulation tissue
formation including proliferation of new vascular channels in
markedly edematous and acutely inflamed loose connective
tissue. In this step, statistical analysis revealed that PMN
leukocyte count in animals treated with honey-impregnated
placenta membrane (group III) was significantly lower than
that of the other groups (P<0.001) (Table 1).
Histopathological appearance of burn-struck tissues from
four groups of animals on day 14 showed diminished
fibrinoleukocyte exudates, replacement of acutely inflammatory
emersion, for PMN leukocytes and fibroblasts, all samples
were examined by Cellsens Entry software. The related im-
ages were taken by the Olympus DP25 camera. Then, the
number of PMN cells, fibroblasts, and new blood vessels were
counted. The observer other than the authors was blinded to
the treatment groups. For evaluation of the wound healing
percentage, wound surface was measured on the 7th, 14th,
21th, and 28th days. For measuring the percentage of wound
healing, we applied following formula (Talas et al. 2002):
cell infiltration by chronically kinds (including lymphocytes,
plasma cells and macrophages) and progressive granulation
tissues formation, which was constituted of new blood ves-
sels and many numbers of fibroblasts. The amounts of
fibrinoleukocyte exudates were less in the third and fourth
groups than those of the other groups, and between them (III
and IV), in the third group, was significantly lower
(P<0.001) (Table 2). The amount of granulation tissue for-
mation and numbers of fibroblasts were greater in the second
and third groups than those of the other two groups, and
between them (II and III), in the third group, was significantly
higher (P<0.001) (Tables 2 and 3). Wound healing percent-
age was calculated 13, 23, 34, and 21 %, respectively, in the
control group and other groups treated by placenta mem-
brane, honey-impregnated placenta membrane, and SSD-
impregnated placenta membrane.
On the 21th day, the cellularity of ulcer bed continued to
increase in four groups. Except control group that revealed
high density of fibroblastic proliferation associated with im-
mature and unorganized collagen fiber deposition,
reepithelialization was started in the other three groups. The
horny layer of epidermis, was not present in SSD-impregnated
placenta membrane group, while in two other groups was
formed. The organization of granulation tissue in the second
and third groups was more obvious than that of the other two
groups, and between them (II and III), in the third group, was
significantly higher (P<0.001) (Tables 2 and 3). Meanwhile,
the percentage of PMN leukocyte was decreased and percent-
age of macrophages started to rise (Fig. 1). The percentage of
wound healing was calculated 29, 65, 83, and 65 %, respec-
tively, in the control, placenta membrane, honey-impregnated
placenta membrane, and SSD-impregnated placenta mem-
brane groups.
At the end of the fourth week, in control group,
reepithelialization was incomplete in the central portion of
 Comp Clin Pathol

Table 1 Comparison of PMN leukocytes count (per 1,000) in four groups

Parameters Control )mean±SD) Placenta membrane Honey-impregnated placenta Silver sulfadiazine-impregnated P value
(mean±SD) membrane (mean±SD) placenta membrane (mean±SD)

1th week 130±2.4 118±2 102±2.1 107±2.3 <0.001

2th week 79±2.7 47.1±2.5 32±2.1 38±1.4 <0.001
3th week 28±2.2 18±1.7 9±1.2 12±1.7 <0.001
4th week 0 0 0 0 0

ulcer. The epidermis including horny layer was well formed in
peripheral parts of ulcer. The reepithelialization, containing
horny layer, in the second and third groups was completely
formed. The horny layer was not found in the fourth group.
The well-formed epidermal appendages were found in the
second and third groups (Fig. 2). In the SSD-impregnated
placenta membrane group, upper dermis occupied by fibrous
stroma including dense and well-organized collagen fibers
was detectable. This phenomenon was also present in the
central areas of the other groups. At this time, however,
limited amount of chronically inflammatory cell infiltration
was seen. The percentage of wound healing was calculated 71,
100, 100, and 100 %, respectively, in the control, placenta
membrane, honey-impregnated placenta membrane, and
SSD-impregnated placenta membrane groups.
Discussion
The main aim of this study was to evaluate the effectiveness of
treatment with honey-impregnated placenta membrane com-
pared with placenta membrane and SSD-impregnated placen-
ta membrane in the healing of partial thickness burn in 64
male Wistar rats randomly divided into four groups. The
results of present study showed that the honey-impregnated
placenta membrane was a suitable and effective topical agent
for burn healing and reconstruction of partial thickness burns.
Any disruption in the integrity of tissue structure is wound
and one of the most common forms of wounds is burn. Wound
healing is a complex and continual activity that divides into
three distinct, but overlapping stages: inflammation, prolifer-
ation, extracellular matrix deposition and reorganization.
Wound healing is a different biological event due to dy-
namic interactions among inflammatory and growth-
promoting mediators, resident and infiltrating cellular compo-
nents, and the extracellular matrix. The inflammatory media-
tors’ signals initiate wound healing process through different
events, including the production of some components in ex-
tracellular matrix, boosting of inflammatory response and
granulation tissue formation. However, different inflammato-
ry cells that are recruited to the wound focus may do more
harm than useful effects. Therefore, by regulation of leukocyte
infiltration, new therapeutic strategies can be introduced for
modulating the inflammatory reaction (Al-Waili et al. 2011).
Today, burn management or care is one of the most chal-
lenging concepts in health care field and in the world wide
searches are being carried out for finding ideal topical prepa-
ration to access better burn healing and controlling infection in
bum wound. Some biological skin substitutes contain certain
anti-inflammatory cytokines that inhibit inflammatory re-
sponse, leading to a promotion of wound healing (Halim
et al. 2010).
Results of our study showed that SSD-impregnated placen-
ta membrane and honey-impregnated placenta membrane
may inhibit the migration of PMN leukocytes thereby sup-
pressing inflammatory response.
The main underlying pathogenesis by which placenta
membrane decreases the migration of PMN leukocytes is
unknown, but it may work via the initiation of cell apoptosis,
inhibition of chemokine, production of anti-inflammatory cy-
tokine (interleukin 10), and prevention of microbial coloniza-
tion due to its ability of powerful attachment on the wound
surface (Park and Tseng 2000).
As a wound dressing, honey provides a wet healing micro-
environment with antibacterial activity. Because of its

Table 2 Comparison of fibroblasts count in four groups

Parameters Control (mean±SD) Placenta membrane Honey-impregnated placenta Silver sulfadiazine-impregnated P value
(mean±SD) membrane (mean±SD) placenta membrane (mean±SD)

1th week 8±0.8 12.±0.9 18±0.8 11±1 <0.001

2th week 27±0.8 38±1 43±1.3 32±0.7 <0.001
3th week 37±1.2 43±0.9 56±1.3 38±1.2 <0.001
4th week 39±1 41±1.1 57±2.1 38±0.8 <0.001
Comp Clin Pathol
Table 3 Comparison of new vascular channels count (per 400) in four groups
Parameters Control (mean±SD) Placenta membrane
(mean±SD)
1th week 6±1.2 12±0.8
2th week 12±1.4 25±1.6
3th week 7±0.8 11±1.2
4th week 4±0.7 4±0.8
capability of inhibiting prostaglandin synthesis, it also dimin-
ishes inflammatory response by decreasing edema, exudation,
and infiltration of inflammatory cells mainly composed of
PMN leukocytes and macrophages (AL-Waili 2005). There-
fore, topical application of honey-impregnated placenta mem-
brane induces a synergy including antibacterial and anti-
inflammatory effects due to some common properties of ma-
terials, placenta membrane, and honey. This fact was ap-
proved by our study.
One of the main causes of unregulated inflammation in
burn wound is microbial colonization and infection. By using
of topical SSD preparations, burn infections may be prevented
or treated (Bowler 2002). Therefore, PMN leukocytes infiltra-
tion is reduced through a decrease in inflammatory response.
This issue was also documented by our findings, when ther-
apeutic effects of topical SSD were added to placenta mem-
brane. Our results demonstrated that at the end of the second
week, the amount of granulation tissues formation and the
number of fibroblasts in the honey-impregnated placenta
membrane group were greater than those of the other groups.
Placenta membrane may accelerate granulation tissues for-
mation via fibroblastic proliferation and angiogenesis. Placen-
ta membrane produces many cytokines and growth factors
including fibroblast growth factor (FGF), epidermal growth
Fig. 1 The organization of granulation tissue with decreasing of PMN
leukocyte percentage in the group treated with honey-impregnated pla-
centa membrane, the third week (H & E stain; ×400)
Honey-impregnated placenta Silver sulfadiazine-impregnated P value
membrane (mean±SD) placenta membrane (mean±SD)
13±1.1 9±1.1 <0.001
32±1.5 24±1.1 <0.001
12±1 7±0.9 <0.001
5±1 3±0.6 <0.001
factor (EGF), transforming growth factor beta (TGF-β), and
interleukin-1 (IL-1) that promote proliferative stage of wound
healing. This property of placenta membrane in stimulation of
neovascularization has been suppressed in the wounded cor-
nea (Kobayashi et al. 2002).
Honey has low glycosaminoglycan and proteoglycan con-
centrations, so it stimulates collagen synthesis with higher
quality and better degree of cross-linking between fibers. It
also promotes rapid epithelialization of wounds. Honey can
accelerate granulation tissue formation and angiogenesis in
burn healing processes (Iftikhar et al. 2010). This issue was
documented by our research, in which proliferative effects of
honey was added to placenta membrane. Our results have
suggested that by the end of the third week, the organization
of granulation tissue in the honey-impregnated placenta mem-
brane group was the most obvious and this group showed
initiation of reepithelialization.
Placenta membrane contains TGF-β and tissue inhibitors
of metalloproteinase polypeptides (TIMPs), which stimulate
fibroblasts for rapid synthesis of collagen fibers. Placenta
membrane also contains FGF that promotes collagen matura-
tion (Yam et al. 2002).
Another issue about application of SSD is the development
of antibiotic resistance, so silver sulfadiazine decreases wound
Fig. 2 The reepithelialization, containing horny layer and epidermal
appendages in the group treated with honey-impregnated placenta mem-
brane, the fourth week (H & E stain; ×400)

healing (Cho Lee et al. 2005). Also SSD dressings have
cytotoxic effect on dermal fibroblasts (Du Toit and Page
2009). Therefore, according to our findings, therapeutic ef-
fects of topical honey application were added to therapeutic
effects of placenta membrane.
Our results have demonstrated that at the end of the fourth
week, the reepithelialization, and reconstruction of skin struc-
tures, in the placenta membrane and honey-impregnated pla-
centa membrane groups were completely formed. Placenta
membrane containing growth factors enhance rapid epithelial
cell proliferation and differentiation, leading to rapid
reepithelialization and reconstruction of skin. It may also
inhibit scare formation. Honey seems to cause more rapid
epithelialization, compared with placenta membrane, presum-
ably because it provides a moist environment which acts as a
viscous barrier to wound infection and high trans-epidermal
water loss (Atiyeh et al. 2005b).
Ultimately, we found that in the honey-impregnated pla-
centa membrane group, the percentage of wound healing was
significantly more than the other three groups, during the
second, third, and fourth weeks. At the end of the fourth week,
except for control group, the other groups showed complete
healing.
In conclusion, the honey-impregnated placenta membrane
can be an ideal tissue for temporary wound coverage and
repair surface injuries after partial thickness burns. It is found
to be cost effective since it accelerates wound healing and
decreases liability to infections compared with other described
modalities.
Acknowledgments The study was made possible due to a special grant
from the Research Board at the Qazvin University of Medical Sciences,
Iran. The authors are grateful for its financial support. We would also like
to thank Mr. Majid Eshsgi (From Rajaee Hospital, pathology ward) for
his kindness and help in data collection and slide preparation.
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