International Journal of Surgery 56 (2018) 94–101

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International Journal of Surgery

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Original Research

Propolis extract a new reinforcement material in improving bone healing: T

An in vivo study
Abdolhamid Meimandi-Parizia,∗, Ahmad Oryana, Emad Sayahia, Amin Bigham-Sadeghb
a
Clinical Sciences Department, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
b
Department of Veterinary Surgery and Radiology, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Background: Propolis is known for its antioxidant, immune response modulating, and wound healing effects. In
Bone the present study in order to determine the bone healing capacity of the propolis extract, a critical sized,
Healing nonunion, radial bone defect model was repaired in rat, using chitosan and demineralized bone matrix (DBM)
Propolis scaffolds along with propolis extract.
DBM
Materials and methods: Seventy-two radial bone defects in 36 healthy male rats were randomly divided into 6
Tissue engineering
groups (n = 12/group). The groups included autograft, defect or untreated group, chitosan, DBM, chitosan and
Chitosan scaffold
propolis (chitosan-propolis), and DBM and propolis (DBM-propolis). The bone repairing capability was char-
acterized using radiography at 28th, 42nd and 56th postoperative days. Gross morphologic, histopathologic,
histomorphometric and biomechanical examinations were performed following euthanasia at the 56th post-
operative day.
Results: The DBM-propolis group, showed better structural and biomechanical properties compared to the un-
treated, DBM, chitosan and chitosan-propolis groups. The defect site in the chitosan and untreated groups were
mainly restored by fibrous connective tissue while the lesions in the autograft group were mostly filled by
cartilage and a lesser amount of woven bone. The woven bone, and the hyaline cartilage were the main con-
stituents of the newly formed tissues in the DBM-propolis group, at the 56th day after injury.
Conclusion: The results of this study showed that percutaneous injection of diluted aqueous propolis extract in
the bone defect (25 mg/defect) can improve bone formation in the critical radial bone defect in rat. Since there
was no significant difference between the autograft and DBM-propolis group, probably this therapeutic strategy
has high potential in augmentation of autologous bone grafting.

1. Introduction
The autogenous bone graft is the gold standard in treatment of bone
defects, bone fractures/loss and in cases of delayed union or non-union
[1]. Due to the limited source and morbidity of harvesting the auto-
graft, the allograft and xenograft were developed [2,3]. Tissue en-
gineering is an ultimate option in treatment of bone fractures and in-
cludes scaffolds, bone healing promoting factors, and stem cells [4].
Demineralized bone matrix (DBM) is a natural scaffold produced by
demineralization of cortico-medullary bone pieces leading to elimina-
tion of the surface antigens which reduces the immune response to the
scaffold [5]. Chitosan is a natural polysaccharide obtained after dea-
cetylation of chitin [6]. With its unique combination of biocompat-
ibility, biodegradability, and low toxicity, chitosan attracted much at-
tention in regenerative medicine and pharmaceutical fields [7]
Propolis is collected by honey bees and is then combined with wax
to make hives. This adhesive and gum-like material has a specific scent
and its color could be in a range of yellow to dark brown. There are
commercially produced extracts of propolis in the aqueous, ethanol,
glycol and oily types [8]. Propolis is composed of over 300 chemical
compounds. Flavonoids are the main components of propolis which
form 25% of its compounds [9]. These polyphenolic compounds apply
their antioxidant properties through inhibition of lipid peroxidation
[10]. Intra-articular injection of propolis has been found useful in
treatment of infectious arthritis [11,12]. Moreover, many studies re-
ported the beneficial effects of propolis alone or in combination with
materials such as silver sulfadiazine or honey in healing of burn wound
injuries [13–15].
The present investigation was designed to study the effects of
chitosan and DBM scaffolds with and without propolis on clinical,
radiological, histopathological and biomechanical performance of a
nonunion critical defect in the radial bone of rat [17]. The hypothesis of

∗
Corresponding author.
E-mail address: meimandi@shirazu.ac.ir (A. Meimandi-Parizi).

https://doi.org/10.1016/j.ijsu.2018.06.006
Received 19 October 2017; Received in revised form 27 April 2018; Accepted 5 June 2018
Available online 11 June 2018
1743-9191/ © 2018 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.
A. Meimandi-Parizi et al.
this study was the probable positive effects of propolis along with
chitosan or DBM scaffolds in healing of the radius bone defect in rat.
2. Materials and methods
2.1. Preparation of scaffolds
2.1.1. Chitosan scaffold
A medium molecular weight chitosan powder (Sigma-Aldrich) was
completely dissolved in acetic acid (0.5 M) (Merk, Germany) and its pH
was neutralized by gradual addition of 1 M NaOH [30]. The porous
chitosan scaffold was obtained by freeze drying of the chitosan solution
(2% w/v) and cross-linked by glutaraldehyde [5,18].
2.1.2. DBM scaffold
The DBM scaffold was prepared as previously described [5] from the
fresh long bones of a slaughtered healthy 2-year-old Holstein cattle. The
bone pieces were demineralized using 0.5 M HCl [19]. The deminer-
alized bone matrices were fixed in absolute ethanol for 24 h until the
alcohol was completely evaporated and then stored at 4 °C aseptically.
The propolis extract used in this experiment was produced according to
Fathi Najafi and colleagues [20]. The aqueous propolis extract
(250 mg/mL) had a pH = 6.5 and was yellowish in color.
2.2. Animals and surgical operation
In choosing an animal model, different factors such as animal
availability, cost, easy handling and care, animal behavior, community
acceptance, surgical resilience, infections and diseases, similarity to
human, bone structure and composition, as well as characteristics of the
bone modeling and its remodeling, are considered. Rats are useful for
both long bone and calvarial models. They are hygienic and low cost to
use. In our study we used male rats due to lack of menstrual cycle which
could affect bone healing process (Kylan et al., 2010). It is well ac-
cepted that the critical size defect of long bones is approximately two
times that of the bone diameter (Bolander et al., 1986). As the diameter
of the radius of adult Wistar rats is about 2–3 mm, a radial defect should
be no less than 5 mm long. A total of 39, eight-weeks-old, 200–250 g,
male Wistar rats were purchased from a local animal house. Before
initiating the study process the animals were moved to new cages to get
familiar with the new environment and diet. The water and food were
freely accessible throughout the experiment. During the experiment,
animal care was taken for all rats in accordance with the animal and
health guidelines published by the National Institutes of Health (NIH
Publication No. 85-23, revised 1985). The animal studies were ap-
proved by the Ethics Committee of our school of veterinary medicine.
In order to achieve a critical non-union bone defect, a 5 mm bone
piece was harvested by approaching the midshaft of radius, under
general anesthesia using ketamine (10%, 75 mg/kg BW) and xylazine
(2%, 10 mg/kg BW) (both from Alfasan Co., Woerden, Holland)) [21].
Based on the implanted material, the radial bone defects were randomly
divided into six groups (n = 12/group). The first two groups were: the
bone defects remained empty; the autograft in which the defects were
filled with the extracted bone pieces from the contralateral radius.
Groups 3–6 were: the chitosan, DBM, chitosan-propolis, and DBM-
propolis, in which the bone defects were implanted with the chitosan or
DBM scaffolds with similar size to the harvested radial bone segments
(dimensions = 2 × 2 × 5 mm3). After implanting the scaffolds and
closing the approach, 0.1 ml of the diluted propolis extract (250 mg/ml)
was injected percutaneously into the defect site, in the chitosan-pro-
polis and DBM-propolis groups. Injection of the propolis extract was
similarly repeated in the third postoperative day. Analgesic and anti-
biotic treatment regimens were administered by intramuscular injec-
tion of enrofloxacin (Enrofan 5%) and Flunixin meglumine (2.5 mg/kg)
for 5 days. Three rats (n = 6; number of radial bones) were randomly
selected and considered as the normal bone group to compare the
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International Journal of Surgery 56 (2018) 94–101
biomechanical performance of the intact radius and ulna bone com-
plexes with the injured treated or untreated bones of other groups. The
animals were euthanized at the 56th postoperative day.
2.3. Clinical examinations
Clinical behavior, physical activities, and weight bearing on the
injured limb were blindly evaluated throughout the experiment, in
terms of pain expression at digital touch and presence of inflammatory
responses such as hyperemia and edema. The animals were euthanized
at the 56th day after injury by rapid intracardiac injection of potassium
chloride after inducing deep anesthesia [22]. The radius and ulna
complex were extracted and the healing area of the radius was eval-
uated macroscopically.
2.4. Evaluation of the diagnostic images
Lateral radiography was performed at 0, 28th, 42nd and 56th post-
surgical days from the injured forelimbs in the anesthetized animals.
Blind evaluation of radiographs was performed by two orthopedic
surgeons and scored for bone formation, union and remodeling by Lane
and Sindhu system which has been described in Table 1.
2.5. Histopathological evaluations
The extracted radius and ulna complexes (n = 6/group) were fixed
in 10% neutral buffered formalin solution, decalcified, routinely pro-
cessed and 5 μm in thickness tissue sections were stained by
Hematoxylin and eosin staining. Blind assessments of tissue sections
were performed by two experienced pathologists. The cells including
fibroblasts/fibrocytes, chondroblasts/chondrocytes, osteoblasts/osteo-
cytes, osteoclasts and also inflammatory mononuclear and poly-
morphonuclear cells as well as blood vessels were counted on three
different microscopic field in each tissue sections (×400, magnifica-
tion), using a digital camera (Olympus, Tokyo, Japan) attached to an
ordinary light microscope (Olympus, Tokyo, Japan). The density of fi-
brous connective tissue (DFT), cartilaginous tissue (DCT) and osseous
tissue (DOT) were also assessed in the same magnification.
2.6. Biomechanical evaluations
The samples intended for biomechanical assessments (n = 6, in each
group) were wrapped by PBS-soaked sterile gauze and kept at −20 °C.
The three-point bending test was performed by a universal tensile
testing machine (Instron, London, UK) [23]. The ultimate load, yield
Table 1
Modified Lane and Sandhu radiological scoring system.
Bone formation
No evidence of bone formation 0
Bone formation occupying 25% of the defect 1
Bone formation occupying 50% of the defect 2
Bone formation occupying 75% of the defect 3
Bone formation occupying 100% of the defect 4
Union (proximal and distal evaluated separately)
No union 0
Possible union 1
Radiographic union 2
Remodeling
No evidence of remodeling 0
Remodeling of medullary canal 1
Full remodeling of cortex 2
Total point possible per category
Bone formation 4
Proximal union 2
Distal union 2
Remodeling 2
Maximum score 10
A. Meimandi-Parizi et al.
load, maximum stress, bending stiffness, ultimate strain, and yield
strain were measured and calculated. All the extracted data from the
load-deformation curve were presented as Mean ± SD, as previously
described [24].
2.7. Statistical analysis
The quantitative data such as the biomechanical and histomorpho-
metric results were presented as mean ± SD and were analyzed sta-
tistically by one-way ANOVA and followed by Tukey post-hoc test. The
qualitative or scored data were presented as mean (min-max) and
subjected to Kruskal-Wallis, non-parametric ANOVA, and subsequent
Mann-Whitney U test. P-values lower than 0.05 were considered sta-
tistically significant. The whole statistical analysis was done by SPSS
software, version 19.0 (SPSS, Inc., Chicago, USA).
3. Theory
Considering the beneficial effects of Propolis extract on the immune
system, wound healing, and its antioxidant and antibacterial properties,
propolis may have a potential role to enhance bone healing. In fact,
some studies on systemic administration of propolis have shown in-
teresting results in bone healing, which has greatly encouraged appli-
cation of propolis and its main ingredients, such as Caffeic acid phe-
nethyl ester in wound healing. The DBM and chitosan scaffolds have
mainly been used for their osteoconductive characteristics to examine
the actual capacity of propolis to improve healing and regeneration of
the critical-sized bone defects in rats.
4. Results
4.1. Radiographic evaluation
4.1.1. Day 28
Radiographic evaluation did show significantly better results in the
DBM-propolis than the chitosan-propolis treated group, at the 28th
postoperative day (see Table 2). There was no significant difference
among the chitosan-propolis and the defect groups. However, there was
a significant difference in the statistical analysis between the autograft
and DBM-propolis with other groups at the 28th day after surgery,
which indicated the superiority of the radiological results in the auto-
graft and DBM-propolis groups. There was statistically significant su-
periority in the autograft group over the DBM-propolis group at the
28th post-operative day. There were no significant differences between
other groups at the 28th day post injury.
4.1.2. Day 42
Radiographic evaluation did not show a significance difference in
statistical analysis between the DBM-propolis and chitosan-propolis, at
the 42nd post-operative day. There were no significant differences be-
tween the chitosan-propolis, chitosan and DBM groups with the defect
group at this stage. There was no significant difference between the
DBM-propolis and the autograft group, while the chitosan-propolis
Table 2
Sum of the radiographically bone healing scores at the 28th, 42nd, and 56th postoperative days.
Postoperative days Mean (min-max)
Autograft (n = 12) Defect (n = 12) Chitosan (n = 12)
28 5.27 (3–8)b 1.58 (1–2)c 1.2 (0–4)d
42 5.90 (4–10)g 2.91 (1–4)h 2.5 (1–4)i
56 6.36 (4–10)k 3.00 (1–5)l 3.7 (2–5)m
DBM (demineralized bone matrix). aKruskal-wallis non-parametric ANOVA. bp < 0.05 (1 vs. 2, 3, 4, 5, 6).cp = 0.019(2 vs. 6).dp = 0.004(3 vs. 6).ep = 0.001(4 vs.
6).fp = 0.010(5 vs. 6).gp < 0.05 (1 vs. 2, 3, 4, 5, 6). hp = 0.023(2 vs. 6). ip = 0.004(3 vs. 6).jp = 0.006(4 vs. 6). kp < 0.05 (1 vs. 2, 3, 4). lp=0.027, p=0.006 (2 vs.5,
6).mp = 0.013(3 vs. 6).
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International Journal of Surgery 56 (2018) 94–101
group showed a significant difference with the autograft group group
indicating inferior result of chitosan-propolis group. There was a sig-
nificantly higher radiological results in the autograft and DBM-propolis
groups over the chitosan, DBM and defect groups (p < 0.05). There
were no significant differences between other groups at this stage.
4.1.3. Day 56
Radiographic evaluation at the 56th day post-operation did not
demonstrate any considerable difference in statistical analysis between
the chitosan-propolis and DBM-propolis groups. Both chitosan-propolis
and DBM-propolis groups showed statistically significant differences
with the defect group (p=0.027, p=0.006, respectively). There were no
significant differences between the chitosan-propolis and DBM-propolis
with the autograft group at this stage. The radiographic evaluations did
not show any significant differences between the chitosan-propolis with
the DBM and chitosan groups. There was a significant superiority in the
DBM-propolis group over the chitosan group at the 56th post-operative
day. There were no significant differences between other groups at this
stage. (see Fig. 1).
4.2. Gross and histopathological findings
During the 56 days of study, there were no surgical site complica-
tions, toxemia or local foreign body reactions. All animals had normal
activity and mild weight gain at the end of examination. The lesions in
the DBM-propolis groups demonstrated a mild inflammatory reaction in
the first 10 days which was subsided in the third week. The defects in
the DBM-propolis group were filled with a relatively firm tissue, at the
56th day, and palpating the bone edges was difficult. A high in-
flammatory reaction was observed, in the chitosan-propolis group,
which started to subside in the second week. The inflammatory re-
sponse in the autograft group was higher than the DBM-propolis group
during the first two post-injury weeks but started to subside from the
third week. The edges of bone couldn't be palpated, in the autograft
group, at the 56th postoperative day. A severe inflammatory response
was observed in the untreated group which subsided up to the 56 days
of experiment and at the end of the study the defects were filled with
soft connective tissues and the bone edges could be easily palpated.
While in the DBM and chitosan groups there was a severe inflammatory
response which subsided at the end of the experiment. The defect area
was filled with a semi-firm tissue, in these groups, and some movable
parts could be palpated and the bone edges could be found after 56 days
of injury (see Fig. 2).
Histopathologic evaluation revealed that the distance of autograft
and bone edges was filled with woven bone and hyaline cartilage. While
these two hard connective tissues made the most part of the regenerated
tissue, some dense fibrous connective tissue also was visible in the
middle portion of the defect. The least regeneration and osteosynthesis
was seen in the untreated defects and major part of the defects were
filled with loose areolar connective tissue, and some cartilage and
minimum amounts of bone tissue was observed near the cut edges of
radial bone. Some disintegration had happened, in the case of chitosan
scaffold, and fibrous connective tissue enveloped the scaffold remnants.
pa
DBM (n = 12) Chitosan-propolis (n = 12) DBM-propolis (n = 12)
1.0 (0–2)e 1.36 (0–4)f 2.27 (2–4) 0.000
1.81 (0–4)j 3.09 (1–5) 4.00 (3–5) 0.000
3.54 (1–5) 4.54 (2–6) 4.90 (3–7) 0.003
A. Meimandi-Parizi et al. International Journal of Surgery 56 (2018) 94–101

Fig. 1. Macroscopic and radiographic findings of the radial defects at the 56th day after injury. The untreated defects were mainly filled with soft fibrous connective
tissues. The defects in the chitosan-propolis and DBM-propolis groups were filled by remnants of the scaffolds associated with bone, cartilage and dense fibrous
connective tissue. At the 28th, 42nd and 56th days after injury, the autograft group had significantly better radiographic scores than the chitosan, DBM and defect
groups (p < 0.05). Bone healing on radiographs in the chitosan-propolis and DBM-propolis groups were superior to the defect group at the 56th day after injury
(p=0.003 and p=0.004). There was no significant difference between the scores of autograft and DBM-propolis group at the 56th day after injury.

The fibrous connective tissue became cartilaginous in some areas
mostly near the ulnar periosteum and ultimately become osseous in
some limited extent near the old radial bone edges. There was no
bridging bone tissue between the radial bone edges. The scaffold rem-
nants in the defect area, in the chitosan-propolis group, were enveloped
by fibrous tissue containing many blood vessels. Fibrous connective
tissue included the majority of regenerated tissue in the lesions of the
animals in this group. Woven bone could be seen near the cut edges of
the radius and beneath the scaffold while hyaline cartilage filled the
distance among the woven bones in this group. The defects treated with
DBM-propolis showed formation of fresh bone tissue and parts of the
scaffold were replaced with woven bone and cartilage tissue, in such a
way that only a small amount of hyaline cartilage were still present
between the newly formed woven bone tissues. Early stage of bone
marrow formation was observed, in the lesions of the animals of this
group. Marrow and primary osteon formation indicate initiation of the
remodeling phase of bone healing. Histopathologic results are shown in
Figs. 2 and 3 and Table 3.
The least regeneration was observed in the untreated group. We
mostly encountered with fibrous connective tissue (fibroblasts + fi-
brocytes), collagen and hyaline cartilage, in the lesions of the animals of
this group. Histomorphometric evaluation at the 56th post-operative
day demonstrated the highest density of fibrous tissue (DFT) in the
defect group (p < 0.05). The number of chondrocytes + chondroblasts
in the autograft group was significantly higher than the defect group
(p=0.027). The number of osteocytes + osteoblasts and the density of
osseous tissue (DOT) was significantly lower in the defect group than
the other groups (p < 0.05). The number of primary osteons in the
autograft, chitosan-propolis and DBM-propolis groups were sig-
nificantly more than the defect group (p < 0.05). There were no sta-
tistically significant differences in the number of osteo-
blasts + osteocytes between the autograft and DBM-propolis groups.
Histomorphometric assessment revealed that the DBM-propolis group
had significantly higher DOT compared to other groups (p < 0.05)

Fig. 2. Longitudinal sections of the radial bone defects at the 56th day after injury. Cartilage and woven bone from the cut edges advanced toward the defect and the
autograft which is still visible, in the autograft group. The radial bone is connected to the autograft with woven bone, cartilage, and dense fibrous connective tissue.
Fibrous tissue, loose areolar connective tissue, and blood vessels are the major components of healing tissue, in the defect group. Residual of chitosan and DBM are
still present in the defect site in the lesions of the chitosan and DBM groups and the scaffolds' remnants are encapsulated by fibrous connective tissue which is
infiltrated by mononuclear inflammatory cells. Fibrous connective and cartilage tissues are dominant, in the chitosan group, while in the DBM group more cartilage is
visible. In DBM-propolis group a remarkable superiority in bone formation is evident compared to the untreated and DBM groups. In the DBM-propolis group after 56
days of injury, DBM has been engaged in healing and have been replaced by woven bone and hyaline cartilage in some areas. The porous chitosan scaffold, in the
chitosan-propolis group was filled by dense fibrous connective tissue in the middle part of the defect. In DBM-propolis group primary osteons encountered very often
in the newly formed osseous tissue which indicates early stages of remodeling which was confirmed by evident of early stages of bone marrow formation. Few
mononuclear cells were diffusely distributed in the healed area in the chitosan-propolis and DBM-propolis groups. H & E staining. Abbreviations: LACT: Loose areolar
connective tissue; FCT: Fibrous connective tissue; RBE: Radial bone edge; WB: Woven bone; BV: Blood vessel; CCT: Calcified cartilaginous tissue; HC: Hyaline
cartilage; DCT: Dense connective tissue; BM: Bone marrow; R: Remnants of the scaffold; IR: Inflammatory reaction; UB: Ulnar bone; PO: Primary osteon.

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A. Meimandi-Parizi et al. International Journal of Surgery 56 (2018) 94–101

Fig. 3. The density of cartilage, fibrous and osseous tissue in regenerated tissues after 56 days of injury. Autograft and DBM groups have the highest DCT,
while the defect has the highest DFT. DBM-Propolis has the highest DOT (p < 0.05). The least amount of DOT is related to the Defect group while the lowest DFT
contributed to the DBM-Propolis groups (p < 0.05).

Table 3
Histomorphometrical findings in the bone defects of different experimental groups after 56 days of injury.
volume (number) Autograft (1) Defect (2) Chitosan (3) DBM (4) Chitosan-propolis (5) DBM- propolis (6) pa

Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

Inflammatory cells 7.0 ± 2.05b 10.86 ± 4.48c 9.71 ± 3.35d 12.4 ± 4.03e 6.5 ± 2.2 5.2 ± 1.64 0.000
Fibroblast + fibrocyte 18.6 ± 7.35f 65.8 ± 15.57g 54.14 ± 5.49h 34.4 ± 8.05i 32.63 ± 9.03j 9.2 ± 3.56 0.024
Chondroblast + chondrocyte 52.4 ± 10.54k 26.14 ± 6.98l 41.14 ± 10.12m 73.2 ± 12.79n 30.38 ± 8.7° 43.8 ± 5.49 0.000
Osteoblast + osteocyte 46.9 ± 7.78p 7.86 ± 2.26q 34.57 ± 7.85r 23.2 ± 6.61s 29.3 ± 5.26t 50.0 ± 7.90 0.000
Osteoclast 4.2 ± 1.54u 0v 1.29 ± 0.75 2.2 ± 1.3 2.0 ± 1.06 1.0 ± 1.0 0.000
Blood vessels 3.9 ± 1.72w 10.0 ± 2.30x 2.57 ± 1.27y 7.6 ± 2.4z 5.38 ± 1.99 3.6 ± 1.14 0.000
Osteons 2.1 ± 1.37 ab 0.86 ± 0.69ac 2.43 ± 1.07ad 4.2 ± 1.3 4.38 ± 1.68 5.2 ± 1.64 0.000
Density of fibrous connective tissue (%) 13.50 ± 4.76ae 54.14 ± 9.77af 37.71 ± 4.23ag 22.4 ± 2.3ah 30.5 ± 6.8ai 8.2 ± 3.49 0.321
Density of cartilage tissue (%) 39.5 ± 6.60aj 22.0 ± 6.75ak 28.71 ± 6.52al 48.2 ± 5.93am 28.6 ± 7.09an 39.0 ± 4.89 0.000
Density of osseous tissue (%) 35.6 ± 6.96ao 6.57 ± 2.14ap 24.14 ± 4.56aq 15.4 ± 4.03ar 27.63 ± 4.3as 44.0 ± 3.74 0.000

a
One-way ANOVA followed by Tukey post-hoc test; b p = 0.037(1 vs. 4).c p = 0.046, p = 0.015(2 vs. 5, 6).d p = 0.013(3 vs. 6).e p = 0.027, p = 0.013 (4 vs. 5,
6).fp < 0.05(1 vs. 2, 3, 4, 5, 6). g p < 0.05(2 vs. 4, 5, 6).h p < 0.05(3 vs. 4, 5, 6).i p = 0.001(4 vs. 6).j p = 0.000(5 vs. 6).k p < 0.05(1 vs. 2, 3, 4, 5).l p < 0.05 (2
vs. 3, 4, 6).m p = 0.002, p = 0.049(3 vs. 4, 5). n p = 0.000, p = 0.004(4 vs. 5, 6).o p = 0.006(5 vs. 6). p p < 0.05(1 vs. 2, 3, 4, 5).q p < 0.05(2 vs. 3, 4, 5, 6).r
p = 0.022, p = 0.009(3 vs. 4, 6).s p = 0.000(4 vs. 6).tp = 0.002(5 vs. 6). u p < 0.05(1 vs. 2, 3, 4, 5, 6).v p < 0.05(2 vs. 3, 4, 5).wp = 0.000, p = 0.021(1 vs. 2,
4).xp < 0.05(2 vs. 3, 5, 6).yp = 0.006, p = 0.007(3 vs. 4, 5).zp = 0.016(4 vs. 6).abp < 0.05(1 vs. 2, 4, 5, 6).ac p < 0.05(2 vs. 3, 4, 5, 6).ad p < 0.05(3 vs. 4, 5, 6).ae
p < 0.05(1 vs. 2, 3, 4, 5, 6).afp < 0.05(2 vs. 3, 4, 5, 6).ag p < 0.05(3 vs 4, 5, 6).ah p = 0.012, p = 0.000(4 vs. 5, 6).ai p = 0.000(5 vs. 6).aj p < 0.05(1 vs 2, 3, 4, 5).ak
p = 0.000, p = 0.001(2 vs. 4, 6).al p = 0.000, p = 0.011(3 vs. 4, 6).am p = 0.000, p = 0.029(4 vs. 5, 6).an p = 0.010(5 vs. 6).ao p < 0.05(1 vs. 2, 3, 4, 5, 6).ap
p < 0.05(2 vs. 3, 4, 5, 6).aq p = 0.006, p = 0.000(3 vs. 4, 6).ar p = 0.001, p = 0.000(4 vs. 5, 6).as p = 0.000(5 vs. 6).

while the chitosan-propolis group had inferior DOT compared to the
autograft and DBM-propolis groups (p=0.009 and p=0.000, respec-
tively). There were no significant differences between the chitosan and
chitosan-propolis groups in the number of inflammatory cells, osteo-
blasts + osteocytes, osteoclasts, DCT and DOT criteria. The DBM-pro-
polis group was significantly superior to the chitosan-propolis in the
DOT (p=0.000), DCT (p=0.010), number of osteoblasts + osteocytes
(p=0.002) and chondroblasts + chondrocytes (p=0.006). There was
significantly higher DOT and osteocytes + osteoblasts count in the
DBM-propolis group than the DBM group (p=0.001 and p=0.000, re-
spectively). The untreated defect and DBM groups produced sig-
nificantly higher inflammatory reaction in comparison with other
groups which could be assessed by the number of inflammatory cells
(p < 0.05).
4.3. Biomechanical performance
There was no significant difference in the assessed biomechanical fac-
tors between the autograft and the DBM-propolis group except for the
yield load criteria (p=0.047) which was higher in the DBM-propolis.
The normal group showed significantly higher maximum load, max-
imum stress and yield load and significantly lower ultimate strain and
yield strain compared with the autograft, untreated defect, chitosan,
DBM and chitosan-propolis groups (p < 0.05). However, there was no
significant biomechanical differences between the DBM–propolis and
normal groups in the maximum load (p=0.113) and yield load
(p=0.383) which indicated enhanced biomechanical performance in
the DBM-propolis treated animals. The biomechanical properties in the
chitosan-propolis group were not significantly different from those of
the chitosan group. There was a significant superiority in the DBM-
propolis over the DBM group in the measured biomechanical criteria
except for the yield strain which indicated significantly better results in
the DBM-propolis over the DBM group. The biomechanical findings are
shown in Fig. 4 and Table 4.

The biomechanichal results showed a significant superiority in the

DBM-propolis performance compared to the chitosan-propolis group.

98
A. Meimandi-Parizi et al.
Fig. 4. Biomechanical properties of the radius and ulnar bone complex, 56 days after injury Maximum stress (a), Maximum load (b), bending stiffness (c), yield
strength (d), yield strain (e), and ultimate strain (f) included for all the untreated and treatment groups. CS (Chitosan), DBM (Demineralized bone matrix), DBM-P
(Demineralized bone matrix-Propolis), CS-P (Chitosan-Propolis).
5. Discussion
The radiographic, histopathologic and biomechanical results in-
dicated improved bone healing after using propolis along with DBM
scaffold. This is the first time that propolis along with chitosan or DBM
have been locally applied in bone healing. This study was performed to
investigate the possible role of propolis in bone healing in rat. The re-
sults of the present study confirmed the positive role of propolis in bone
healing. Quantitative and qualitative interpretation of the results of the
present study demonstrated significant differences between the propolis
treated groups and other groups.
Although the chitosan and DBM scaffolds lack osteogenic properties,
but both are osteoconductive and it has also been demonstrated that the
DBM scaffold has osteoinductive properties too. Both scaffolds have
been used, in the present study, because of their osteoconductive
properties [25]. Therefore, addition of propolis to these scaffolds
99
International Journal of Surgery 56 (2018) 94–101
provides the proper evidence needed to initiate the ability to enhance
bone healing by this material. The results of the present study indicated
that propolis along with DBM scaffold created a favorable response in
bone repair but concurrent use of propolis and chitosan scaffold did not
result in a significantly enhanced bone healing. In addition to structural
strength, DBM has the osteoinductivity property which has been de-
monstrated in in vivo studies. Chitosan also has several properties such
as antiviral, antibacterial, anticarcinogenic and cholesterol-lowering
properties that can be used to accelerate healing. Alidadi and colleagues
[5] reported that chitosan scaffold possess low biodegradability which
was also confirmed in the present study as the chitosan scaffold was
present after 56 days of implantation both in the propolis treated
chitosan or chitosan alone. They also reported high inflammatory re-
action in the chitosan treated lesions but our histopathologic results did
not agree with them. However a short period of inflammatory reaction
was noticed in the chitosan and chitosan propolis groups which
A. Meimandi-Parizi et al. International Journal of Surgery 56 (2018) 94–101

Table 4
The results of three-points bending biomechanical test on the radius-ulna complex at the 56th day after injury.
Value (number) Defect (1) Autograft (2) Normal (3) Chitosan (4) DBM (5) Chitosan-propolis (6) DBM-propolis (7) pa

Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

b c d e f
Maximum load (N) 29.9 ± 2.8 39.0 ± 3.98 47.1 ± 7.1 35.8 ± 6.3 35.3 ± 5.2 35.4 ± 2.0 41.48 ± 2.4 0.000
Maximum stress (N/mm2) 4.3 ± 0.78g 8.1 ± 1.36h 10.1 ± 1.07i 5.5 ± 2.1 4.9 ± 1.1j 5.81 ± 1.32 6.89 ± 0.81 0.000
Yield load (N) 24.4 ± 2.2k 33.2 ± 2.3l 39.7 ± 5.7m 29.3 ± 5.9n 30.4 ± 5.0° 29.71 ± 1.69p 37.22 ± 2.93 0.000
Bending stiffness (N/mm) 19.42 ± 3q 32.56 ± 5.2r 45.83 ± 7.13s 22.73 ± 6.09t 23.28 ± 5.48u 29.33 ± 1.4v 34.61 ± 2.77 0.000
Ultimate strain (%) 8.5 ± 2.1w 5.7 ± 1.0x 3.79 ± 1.01y 7.54 ± 1.9z 7.32 ± 1.8ab 7.38 ± 0.5ac 5.11 ± 0.84 0.000
Yield strain (%) 6.68 ± 1.4ad 5.04 ± 0.44ae 2.53 ± 1.3af 6.1 ± 1.5 6.1 ± 1.9 6.25 ± 0.28ag 4.69 ± 0.71 0.000

Normal (intact radius-ulna bone complex of rat)/DBM (demineralized bone matrix) aOne-way ANOVA followed by Tukey post-hoc test. bp < 0.05(1 vs. 2, 3, 4, 5, 6,
7);cp=0.043(2 vs. 3);dp=0.047, p=0.017, p=0.009(3 vs. 4, 5, 6);ep = 0.025(5 vs. 7).fp = 0.002(6 vs. 7). gp < 0.05(1 vs. 2, 3, 6, 7);hp < 0.05(2 vs. 3, 4, 5,
6);ip < 0.05(3 vs. 4, 5, 6, 7);jp=0.006(5 vs. 7);kp < 0.05(1 vs. 2, 3, 4, 5, 6, 7).lp < 0.05(2 vs. 3, 6, 7).mp=0.039, p=0.032, p=0.007(3 vs. 4, 5, 6).np = 0.039(4 vs.
7).op = 0.016(5 vs. 7).pp = 0.002(6 vs. 7). qp < 0.05 (1 vs. 2, 3, 6, 7).rp < 0.05(2 vs. 3, 4, 5).sp < 0.05(3 vs. 4, 5, 6, 7).t p=0.009 (4 vs. 7).up=0.026, p=0.001(5 vs.
6, 7).vp = 0.009(6 vs. 7). wp < 0.05(1 vs. 2, 3, 7).xp=0.010, p=0.019(2 vs. 3, 6).yp < 0.05(3 vs. 4, 5, 6, 7).zp = 0.044(4 vs. 7).abp = 0.020(5 vs. 7).acp = 0.002(6 vs.
7). adp < 0.05(1 vs. 3, 7).aep=0.003(2 vs. 3).afp < 0.05(3 vs. 4, 5, 6, 7).agp = 0.005(6 vs. 7).

subsided at the third week after injury.
A wide range of pharmacological properties such as antimicrobial,
antioxidant, anti-inflammatory, immunomodulatory, anti-cancer, anti-
ulcer, hepatoprotective, cardioprotective, and neuroprotective proper-
ties have been attributed to propolis, however the definitive under-
standing of the mechanisms behinds these effects has not been known
yet [26]. The composition of raw propolis or its extract varies according
to the area of collecting the propolis which is largely attributed to di-
versity among wild plants in different geographical areas. It is likely
that as many natural compounds, the known properties of propolis are
due to synergistic effects of its different bioactive components [27].
Paper chromatography of the propolis collected in the same area of our
propolis confirmed that approximately 80% of the propolis flavonoids
were present in the aqueous extract [20]. It has been shown that caffeic
acid phenethyl ester (CAPE) enhances the wound healing process [28].
Anti-inflammatory action shown on mast cells may be due to inhibition
of cell degranulation or regulation of purinergic signaling or both,
which led to an anti-inflammatory pattern and modulating the release
of cytokine, nitric oxide and prostaglandins in the area. Many studies
focused on the effects of propolis and CAPE on osteoclasts and its effect
on bone healing and also the mechanism of action of these ingredients
in healing of bone fractures [29–31].
It is not known how propolis could affect bone metabolism, but it is
clear that activation and differentiation of osteoclasts occur by RANK-
RANKL system. Mechanisms of modulation and activation of osteoclasts
shares with some known inflammatory pathways. It has been shown
that CAPE suppresses osteoclastogenesis in the bone marrow progenitor
cells in vitro [29]. It also has been shown that CAPE significantly in-
hibits formation of osteoclasts in vivo [32]. It has been suggested that
CAPE affects RANKL/OPG signaling and confronting with osteoclasto-
genesis while protecting osteoblasts [33]. Also, the use of propolis on
mouse bone marrow medium showed that the ethanol extract of pro-
polis inhibits final stages of osteoclast maturation including fusion of
progenitor cells and forming actin ring in vitro [34]. In the present
study, there was no significant difference between the chitosan-propolis
and DBM-propolis groups with the chitosan and DBM groups in the
number of osteoclasts at the 56th day after injury, but a significantly
higher number of osteoblasts + osteocytes were observed in the DBM-
propolis group. It has also been demonstrated that oral administration
of propolis increases bone density which was supported with the results
of radiological and histopathological assessments [16].
Measurements of superoxide dismutase (SOD), glutathione and
myeloperoxidase has shown the antioxidant effect of propolis on bone
healing [11]. Local or systemic administration of CAPE in a rat calvarial
bone defect model in a 28 days period demonstrated substantially
higher new bone formation in the systemically treated group compared
with other groups [31].
It has been shown that propolis has stimulating and regulatory
effects on immune system in vivo and on macrophages in vitro [35].
These biological effects could be related to the propolis ability in up-
regulating the expression of TGF-β which is involved at earlier stages of
wound healing [36]. It has been demonstrated that local application of
propolis enhances wound healing through reducing inflammatory re-
actions [37]. Specific results that make the Chitosan-Propolis group
inferior include the insignificant radiographic differences between the
chitosan-propolis with the DBM and chitosan groups at the 56th post-
operation day so that no significant differences were found between the
chitosan and chitosan-propolis groups in the number of inflammatory
cells, osteoblasts + osteocytes, and osteoclasts, and also density of
cartilage and osseous tissues. In addition, the biomechanical properties
in the chitosan-propolis group were not significantly different from
those of the chitosan group. The chitosan-propolis group did not lead to
a significant improvement in bone healing, which could be related to
overlapping of antioxidant and anti-inflammatory properties in both
chitosan scaffold and propolis extract. The histopathological assess-
ments of the DBM-propolis group showed appropriate integration in the
bone healing process. Radiographic, histopathologic and biomechanical
findings indicated a marked improvement in quality and quantity of
new bone formation in the DBM-propolis group compared to other
groups. Although in most benchmarks significant differences was not
found between the propolis receiving groups and the autograft, the DOT
in the DBM-propolis indicated its superiority on the autograft group
(p=0.010). Significant differences between the chitosan-propolis and
DBM-propolis groups indicate superiority of the latter. This superiority
of the DBM-propolis group may be related to lower biodegradability of
chitosan and also osteoconductive properties of DBM scaffold along
with lack of antioxidant activity in the DBM scaffold which let propolis
to demonstrate its healing effect. Unfortunately, our study did not in-
clude the in vitro assays to determine possible mechanisms or com-
pounds which are responsible to improve bone healing. The DBM
scaffold along with propolis may be an appropriate option in treatment
of critical radial bone defects, but determining specific mechanisms for
improving bone regeneration using these natural materials should be
considered in future studies.
6. Conclusions
The present study well established that local propolis along with
DBM scaffold induced more bone formation in the healing of a critical
bone defect in rat. The DBM scaffold along with propolis is an appro-
priate choice in healing of the radial critical bone defects and would not
initiate a devastating inflammatory reaction. The results of this study
indicated the potential role of DBM-propolis in improving bone healing
as an effective combination in bone tissue engineering. However more
research is still needed for uncomplicated and safe application of pro-
polis in clinical bone defects.

100
A. Meimandi-Parizi et al.
Ethical approval
The study was approved by the local Ethics Committee of
“Regulations for Using Animals in Scientific Procedures” in the School
of Veterinary Medicine of Shiraz University.
Sources of funding
Shiraz University.
Conflicts of interest
The authors have declared that no conflict of interests exist.
Guarantor
All authors are responsible for the work.
Author contribution
All authors were involved in all study procedures such as study
design, data collections, data analysis and writing.
Acknowledgement
This work was supported by Veterinary School, Shiraz University,
Shiraz, Iran, and National Science Foundation (Grant number
96006039), Iran.
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