Banats Journal of Biotechnology
Contact: web: http://www.bjbabe.ro, e-mail: [email protected]
EVALUATION OF BONE REGENERATION BY OSTRICH EGG WHITE
SUBSTITUTE IMPLANTED WITH BONE IN TIBIA BONE DEFECT IN ANIMAL
MODEL
DOI: 10.7904/2068–4738–VIII(15)–50
Mehrdad YADEGARI
Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord Branch, Islamic Azad
University, Shahrekord, IRAN
Corresponding author: e–mail: [email protected]; Phone: +989131813028; Fax:
+983833361045
Abstract. The present study was conducted to investigate the in vivo to assess the effect of
ostrich egg white (OEW)–derived graft substitutes on bone healing and comparative with self–
regeneration of control site in dogs. The eight male domestic dogs (3–4 years old, weighing 20–30
kg) were used. Bone formation was assessed by using of OEW as kind of biomaterial that has
flexibility and jelly shape with high compatibility. The implant was harvested after 4 and 8 weeks,
and the area of new bone formation was quantified by radiology and densitometry studies. The
highest bone formation was achieved with the OEW treatment, and it was significantly higher than
that achieved with the empty treatment. The quantity of new bone at end of 8 weeks was greater
than at 4 weeks in treated group. The researcher also show that Using OEW, the bone
regeneration significantly increased within the implanted site in the biomaterial group, in the second
month of the experiment, when compared to the control group. Furthermore, bone regeneration
was significantly higher in the implant treatment biomaterial site of individual OEW, after
implantation times, it’s showed little effect in enhancing bone regeneration 4 weeks post–
implantation. Overall, it can be concluded that OEW (safe, cheap, and easily available) is a potent
osteoidinductive component that plays critical role during bone regeneration and repairing in animal
model.
Keyword: Animal model, Bone regeneration, Implant, Ostrich egg, Tibia bone.
Implants that serve simultaneously
as an osteoidconductive matrix and as a
device for local growth factor delivery may
be required for optimal bone regeneration
in some applications [HING, 2005].
The osteoidinductive properties of
synthetic ceramics, including biphasic
calcium phosphate, are insufficient for the
healing of extensive bone defects.
However, to overcome the problem,
several growth factors, such as bone
morphogenetic
proteins
(BMPs),
fibroblast
growth
factors,
vascular
endothelial growth factors and natural
materials have been used for bone tissue
engineering [ARAVAMUDHAN et al., 2013].
Although many growth factors have
shown potential for use in bone
regeneration and repair, during previous
decades avian component had been
introduced as a candidate for bone
substitute in the field of maxillofacial
surgery.
Introduction
Bone is a dynamic tissue that
continuously regenerates and has the
ability to heal following fractures [TAYLOR et
al., 2007].
However, sufficiently large defects
resulting from traumatic bone loss, tumor
resections, or infection cannot heal
without intervention, and thus the
treatment of these skeletal complications
presents difficult challenges inorthopedic
medicine [GIANNOUDIS et al., 2005].
Bone grafting is widely used in
clinical procedures to promote healing of
non–unions and large defects in human
and animal.
Bone repairing is a complex
cascade of biological events controlled by
numerous cytokines and growth factors
that provide signals at local injury sites,
allowing progenitors and inflammatory
cells to migrate and trigger healing
processes [LIEBERMAN et al., 2002].
50
Available on-line at http://www.bjbabe.ro
Banats Journal of Biotechnology
2017, VIII(15)
Ostrich egg white (OEW) is rich in
ovalbumin, overrunsferine, ovummucoid,
and lysozyme [MINE, 2002].
Ovalbumin constitutes over half of
the total egg white proteins, it is a
monomeric phosphoglycoprotein, and has
been used extensively as a model for
studying
the
structure–function
relationships of proteins.
Functionally, ovalbumin is important
for the gelling, foaming, and emulsifying
properties of egg white [MINE, 2002].
Immunogenic ovalbumin peptides
have been used to enhance immune
responses for cancer immunotherapy
[GOLDBERG et al., 2003; HE et al., 2003].
Furthermore, keeping in view the
cited benefits of OEW, in the work
reported here, we have investigated thein
vivo to assess the effect of OEW–derived
graft substitutes on bone healing and
comparative with self–regeneration of
control site in dogs.
Table 1.
Characteristics of ostrich egg parts
Egg weight
% of egg mass
(g)
Albumen Yolk Shell
1580
59.8
20.4
9.8
Ostrich Egg white Collection and
Preparation
OEW was obtained from Strathy
Cadmus eggs by breaking the eggshell
opposite the air chamber, discarding the
albumen and yolk.
Then, the egg white was manually
extracted by syringe. After extracting, the
syringe was individually packed and
sterilized with ethylene oxide for 1 hour at
room temperature (AX–400, Axis, and
Izmir, Turkey).
Experimental
Animals
and
Surgical Technique
In order to assess the effect of
ostrich egg white–derived graft substitutes
on bone healing, the present study was
conducted on eight adult male domestic
dogs (3–4 years old, weighing 20–30 kg).
The isoflurane anaesthetized dogs
underwent
surgery
under
sterile
conditions.
The caudomedial approach to the
medial part of the right tibia was
performed via 1–1.5 cm incisions (Figure
1).
Material and methods
Location and Research Ethics
This study was performed at
College
of
Veterinary
Medicine,
Shahrekord, Islamic Azad University,
located in Shahrekord province; Iran
(50°49ʹN and 53º44ʹE) from June to
December 2015.
The animals were kept in a
restricted access room under controlled
temperature conditions (22°C) and
light/dark cycles (12h/12h), with free
access to food and fresh water.
The
research
protocol
was
approved by the Research Ethics
Committee of the College of Veterinary
Medicine, Shahrekord Branch, Islamic
Azad University. All the experiments were
carried out in accordance with the rules of
the Institutional Animal Care and Use
Committee.
The study design and the
parameters for tissue reaction evaluation
were in accordance with the ISO 10993–6
(1994) standard.
Ostrich egg parts:
The mean ostrich egg weight is
about 1580 g (Table 1) and about 60% of
the mass of the ostrich egg is comprised
by the albumen.
Figure 1. Make two holes on the tibia by
surgical drills
After removing the surrounding
musculature to isolate the tibia, an 8–mm
wide osteotomy was performed by using
oscillating saw.
Further, 1 cc of ostrich egg white
was implanted to the site of osteotomy.
The fixation of the implant was
accomplished by using two simple
discontinuous periosteal sutures (2–0
nylon) of the cranial and caudal face of
the proximal fragment.
51
Banats Journal of Biotechnology
Contact: web: http://www.bjbabe.ro, e-mail: [email protected]
The other site of the tibia was only
subjected to osteotomy and served as
negative control for natural healing.
Densitometry was performed by
using the LC NDT FV–2009 Viewer.
Wound closure was accomplished
by the opposition of the subcutaneous
tissue in a simple continuous pattern (2–0
polyglactin 910) and by a simple
discontinuous skin pattern (2–0 nylon),
keto profen (1 mg/kg every 24 h for 3
days) being administered as an
analgesic/anti–inflammatory drug.
The animal’s radiographs were
obtained after, 0 and 4 and 8weeks
(Trophy CCX Digital, Croissy–Beaubourg,
France) at an exposure of 50 kV, 7 mA for
0.5 seconds.
Statistics and data analysis
Results
were
expressed
as
means±SD. ANOVA was performed and
mean comparisons were carried out by
Duncan's
multiple
range
tests.
Differences were considered significant
for P < 0.05. Statistical analysis was
performed using SPSS 19.1 for Windows
(SPSS Inc., Chicago, IL).
progression from the entire circumference
of the defect. In the treatment groups,
small particles were absorbed more
excessively than the larger ones.
Bone
regeneration
was
considerable in both groups, when
compared to the first group in the first
month of the experiment.
Bone regeneration significantly
increased within the implanted site in the
biomaterial group, in the second month of
the experiment, when compared to the
control group. Both part of defect rated
from 0 to 4 and after that analyzed by
SPSS software, we can see P value of
them on the table 1.
Chart 1 refers to treatment
biomaterial site and chart 2 refers to
positive control site that shown all of
changes.
On the other hand, density of
previous surgery is indicator and the other
site of defect should be goes to near this
chart.
The radiographs obtained in this
study were used in the laboratory for
densitometry analysis of bone healing.
Figure 2A shows part positive controls
and part treatment biomaterial on the X–
ray images acquired after surgery. Figure
2B shows the X–ray image of the tibia 4
weeks after surgery in the first group and
Figure 2C shows the X–ray image of the
tibia 8 weeks after surgery in the second
group.
Results and discussion
Radiological
examination
was
shown that minimal bone regeneration at
the periphery of the bone defect site in the
positive control part, one month after
surgery. Therefore, the finding indicated
that bone formation displayed centripetal
Figure 2A. Part PC (Positive
controls) and part TB (Treatment
Biomaterial) on the X–ray images
acquired after surgery
Figure 2B. Part PC (Positive controls) and part
TB (Treatment Biomaterial) on the X–ray
images of the tibia 4 weeks after surgery in the
first group
52
Figure 2C. the X–ray
image of the tibia 8
weeks after surgery
Available on-line at http://www.bjbabe.ro
Banats Journal of Biotechnology
2017, VIII(15)
All radiographs consisted of both
lateral and craniocaudally views (figure 3
and 4/ table 2).
Figure 3. Before, after, 4 and 8 week of treatment biomaterial site
Results
of
radiography
and
densitometry rating effect of ostrich egg
white derived graft substitutes on bone
healing in positive and biomaterial groups.
Table 2.
Results of radiography and densitometry rating effect of ostrich egg white derived graft
substitutes on bone healing in positive and biomaterial groups
Defect
Week
Dog NO1
Dog NO2
Dog NO3
Dog NO4
Dog NO5
Dog NO6
Dog NO7
Dog NO8
Positive control (Cd/m2)
0
4
8
3.90
3.80
3.70
3.90
3.90
3.70
3.80
3.80
3.60
3.85
3.35
3.70
3.90
3.90
3.70
3.85
3.90
3.70
3.80
3.80
3.60
3.85
3.85
3.70
Biomaterial group(Cd/m2)
0
4
3.90
3.20
3.90
3.20
3.80
3.10
3.85
3.00
3.90
3.00
3.85
3.10
3.80
3.00
3.85
3.11
According to the densitometer,
number of radiographic density in terms of
Cd /m2 is lesser represents an increase
radioopacity or an increase bone density
due to X–ray pass of radiography film
8
2.50
2.50
2.50
2.50
2.50
2.50
2.50
2.50
(According to score when high opacity
has the score in the radiograph goes to 0
and when low opacity has the score in
radiograph, goes to 4 rates).
Figure 4. Before, after, 4 and 8 week of positive control site
The present study was conducted to
investigate the in vivo to assess the effect
of ostrich egg white (OEW) derived graft
substitutes on bone healing and
comparative with self–regeneration of
control site in dogs.
In recent years, this tendency has
been well documented inliterature and
53
Banats Journal of Biotechnology
Contact: web: http://www.bjbabe.ro, e-mail: [email protected]
demonstrated great promise of the
nanofibers–based and natural additive for
bone regeneration.
In the setting of bone tissue
engineering, it is critical to develop a
biomaterial that can chemically and
structurally mimic the native extracellular
matrix of the bone [HUANHUAN et al., 2013].
Thus, during previous decades in
the bone tissue engineering researches it
is rational strategy to develop composites
scaffolds with Nano fibers ultra–structure
to recapitalize the extracellular matrix of
bone.
In this study, OEW was adapted to
create composite bone graft substitutes
for tissue engineering applications and
preclinical studies of bone healing in small
animal models. While other studies have
investigated parameters related to the
powder, such as chemical composition
[KLAMMERT et al., 2010; JASON et al., 2014], particle
size [GOLDBERG et al., 2003] and a commercial
3D printer [JASON et al., 2014].
The main aim of this study attempt
to reconstruct hard injures like bone
defect. According to previous study that
performed by other scientist, we need to
improve hard tissue immediately because
skeletal system has important role
between the other organ. We could help
them by new biomaterial that is natural
and it’s not expensive.
OEW have several key attributes
that make them an attractive system for
regeneration, including biocompatibility,
degradability, and as well as porous
structure facilitate intergrowth through
newly formed bone.
Based on the results on the present
study, OEW are primary focuses for
synthetic bone graft substitutes because
they are osteoidconductive and provide
sufficient mechanical strength.
Results of the present study
showed that 8 mm critical sized defects
were created in the tibia bone of dogs
because that the bony lesions above this
critical size become scarred rather than
regenerated, leading to remain a cavity
[LIND et al., 1993; BAUER and MUTSCHLER, 2000].
Such large defects have been
traditionally treated with bone graft
substitutes.
When using a bone graft, it is
expected that the wound will heal and that
some form of new bone formation will
occur, and then support [BODNER, 1996].
It is replacement of the bone defect,
although autographs and allograft meet
most of the expectations, they also have
disadvantages such as limited availability
of donor tissue, donor site morbidity, and
unfavorable immunogenic response from
the host, potential disease transmission,
and difficulty in providing the desired
shape [BODNER, 1996, FERENCZ, et al., 2012].
From the results of the present
experiment, bone regeneration increased
significantly in both groups of defects with
an increase in implantation time from 4 to
8 weeks.
Bone regeneration was significantly
higher in the implant treatment biomaterial
site of individual OEW, after implantation
times. It’s showed that little effect in
enhancing bone regeneration 4 weeks
post–implantation.
Mann and collab. [MANN et al. 2004]
suggested that the harvesting and
sterilization methods of the graft materials
were highly effective on the osteogenic
capabilities of the materials.
A graft material would allow the
formation of bone, safe biodegradation,
and allow the functional load to native
bone [COELHO et al., 2009; LeGEROS, 1991].
The first signs of remodeling are
seen with osteoid and a vascular network
in an intra–membranous–like healing
pattern that undergoes remodeling over
the course of several months/years
[GUARNERI et al., 2004; ARAUJO et al., 2010].
Calcium plays a central role in any
discussion of bone health.
A low intake and bioavailability of
calcium may affect peak bone mass
negatively [CASHMAN, 2002].
However, most Chinese have an
insufficient intake of milk products, and
some of the green vegetables have a low
bioavailability of calcium [DAENGPROK et al.,
2003, BUTU, et al., 2014, BUTNARIU, et al., 2015].
Our previous study has confirmed
that the peptides prepared from
component of egg white can bind calcium
and prevent calcium from precipitating in
vitro [ZHAO et al., 2013].
54
Available on-line at http://www.bjbabe.ro
Banats Journal of Biotechnology
2017, VIII(15)
science. Clinical Orthopaedics and
Related Research, 2000, 371, 10–27.
4. Bodner, L. Effect of decalcified freeze–dried
bone allograft on the healing of jaw
defects after cyst enucleation. Journal
of oral and maxillofacial surgery,
1996, 54, 1282–1286.
5. Butnariu, M.; Negrea, P.; Lupa, L.; Ciopec,
M.; Negrea, A.; Pentea, M.; Sarac, I.;
Samfira, I. Remediation of Rare Earth
Element Pollutants by Sorption
Process Using Organic Natural
Sorbents. International journal of
environmental research and public
health, 2015, 12(9), 11278–11287.
6. Butu, M.; Rodino, S.; Pentea, M.; Negrea,
A.; Petrache, P.; Butnariu, M. IR
spectroscopy of the flour from bones
of European hare, Digest journal of
nanomaterials
and
biostructures.
2014, 9(4), 1317–1322.
7. Cashman, K.D. Calcium intake, calcium
bioavailability and bone health. The
British journal of nutrition Nutrition
Society, 2002, 87, 169–177.
8. Coelho, P.G.; Coimbra, M.E.; Ribeiro, C.;
Fancio, E.; Higa, O.Z.; Suzuki, M.;
Marincola,
M.
Physic/chemical
characterization
and
preliminary
human histology assessment of a B–
TCP particulate material for bone
augmentation. Materials science &
engineering. C, 2009, 29, 2085–91.
9. Daengprok, W.; Garnjanagoonchorn, W.;
Naivikul,
O.;
Pornsinlpatip,
P.;
Isogenies K.; Mine, Y. Chicken
eggshell matrix protein enhance
calcium in transport the human
intestinal epithelial cells, Caco–2.
Journal of Agricultural and Food
Chemistry, 2003, 51, 6056–6061.
10.Ferencz, A.; Juhasz, R.; Butnariu, M.;
Deer, A.K.; Varga, I.S.; Nemcsok, J.
Expression analysis of heat shock
genes in the skin, spleen and blood of
common carp (Cyprinus carpio) after
cadmium exposure and hypothermia,
Acta biologica hungarica, 2012, 63(1)
15–25.
11.Giannoudis, P.V.; Dinopoulos, H.; Tsiridis
E. Bone substitutes: an update. Injury,
2005, 36, 20–27.
12.Goldberg, J.; Srikanth, P.; Melcher, M.F. In
vivo augmentation of tumor–specific
CTL responses by class I peptide
antigen complexes on microspheres
(large
multivalent
immunogen).
Journal of immunology, 2003, 170:
228–235.
Also some complex proteins in
avian egg white can help to transport
calcium into skeletal tissue [LV et al., 2013; LV et
al., 2008].
On
the
other
hand,
some
component of egg white help to
angiogenesis next to hard tissue for high
blood supply that is useful for healing and
treatment of hard and soft lesion [LIU et al.,
2010].
Conclusions
In conclusion, according to result of
this article we arrived to best quality of
bone repair in short time and the bone
doesn’t have any misshape after
treatment by OEW implantation.
This
system
promotes
large
volumes of regenerated bone and a high
probability of bridging in a dog critical
defect. Further study its osteoproductive
activity alone or in combination with other
materials.
Conflicts of interest
The authors declare that they have
no conflicts of interest.
This study it is in agreement with
the aim of the Basel Declaration (Basel
Declaration Society) is to bring the
scientific community together to further
advance the implementation of ethical
principles such as the 3Rs whenever
animals are being used and to call for
more
trust,
transparency
and
communication on the sensitive topic of
animals in research.
References
1. Araujo, M.G.; Liljenberg, B.; Lindhe, J.
Beta–tricalcium phosphate in the early
phase of socket healing: an
experimental study in the dog. Clinical
oral implants research. 2010; 21:
445–54.
2. Aravamudhan, A.; Ramos, D.M.; Nip, J.;
Subramanian, A.; James, R.; Harmon,
M.D.;
Yu.
X.;
Kumbar,
S.G.
Osteoinductive small molecules:
growth factor alternatives for bone
tissue
engineering.
Current
Pharmaceutical Design, 2013, 19,
3420–3428.
3. Bauer, T.W.; Mutschler, G.F. Bone graft
materials. An overview of the basic
55
Banats Journal of Biotechnology
Contact: web: http://www.bjbabe.ro, e-mail: [email protected]
13.Guarnieri, R.; Pecora, G.; Fini, M.; Aldini,
N.N.; Giardino, R.; Orsini, G.; Piattelli,
A. Medical grade calcium sulfate
hemihydrate in healing of human
extraction sockets: clinical and his–to
logical observations at 3 months.
Journal of periodontology, 2004, 75,
902–8.
14.He, X.; Tsang, T.C.; Luo, P.; Zhang, T.;
Harris,
D.T.
Enhanced
tumor
immunogenicity through coupling
cytokine expression with antigen
presentation. Cancer gene therapy,
2003, 10, 669–677.
15.Hing, K. Bioceramic bone graft substitutes:
influence of porosity and chemistry.
International Journal of Applied
Ceramic Technology, 2005, 2, 184–
99.
16.Huanhuan, L.; Hongju, P.; Yan, W.; Can,
Z.; Youzhi, C.; Guowei, X.; Qin, L.;
Xiao, C.; Junfeng, J.; Yanzhong, Z.;
Hong Wei, O.Y. The promotion of
bone regeneration by nanofibrous
hydroxyapatite/chitosan scaffolds by
effects
on
integrin–BMP/Smad
signaling
pathway
in
BMSCs.
Biomaterials, 2013, 34, 4404–4417.
17.Jason, A.; Zana, D.O.; Seth, M.F.; James,
P.K.; Olivia, A.G.; Edward, M.S.;
Stephen, L.K.; Hani, A.A. 3D printing
of composite calcium phosphate and
collagen
scaffolds
for
bone
regeneration. Biomaterials, 2014, 3,
4026–4034.
18.Klammert, U.; Vondra, E.; Reuther, T.;
Muller, F.A.; Zorn, K.; Gbureck, U.
Low temperature fabrication of
magnesium
phosphate
cement
scaffolds by 3D powder printing.
Journal
of
Materials
Science:
Materials in Medicine, 2010, 21(11),
2947–2953.
19.LeGeros, R.Z. Calcium phosphates in oral
biology and medicine. Monographs in
Oral Science, 1991, 15, 1 201.
20.Lieberman, J.R.; Daluiski, A.; Einhorn, T.A.
The role of growth factors in the repair
of bone. Journal of bone and joint
surgery. American, 2002, 84, 1032–
44.
21.Lind Holm, T.S.; Gao, T.J. Functional
carriers for bone morphogenetic
proteins. Annales chirurgiae et
gynaecologiae, 1993, 82, 3–12.
22.Liu, J.B.; Yu, Z.P.; Zhao, Z.; Lin, S.Y.;
Wang, E.L.; Zhang, Y.; Hao, H.;
Wang, Z.Z.; Chen, F. Isolation and
identification
of
angiotensin–
converting enzyme inhibitory peptides
from egg white protein hydrolysates.
Food chemistry, 2010, 122, 1159–
1163.
23.Lv, Y.; Bao, X.L.; Yang, B.C.; Ren, C.G.;
Guo, S.T. Effect of soluble soybean
protein
hydrolysate–calcium
complexes on calcium uptake by
Caco–2 cells. Journal of Food
Science, 2008, 73, 168–173.
24.Lv, Y.; Boa, X.L.; Liu, H.; Ren, J.H.; Guo,
S.T. Purification and characterization
of calcium–binding soybean protein
hydrolysates by Ca2+/Fe3+ immobilized
metal affinity chromatography (IMAC).
Food chemistry, 2013, 141, 1645–
1650.
25.Mann,
K.;
Siedler,
F.
Ostrich
(Struthiocamelus) eggshell matrix
contains two different C–type lectin–
like proteins. Isolation, amino acid
sequence,
and
posttranslational
modifications,
Biochimica
et
Biophysica Acta, 2004, 14, 1696:41–
50.
26.Mine, Y. Recent advances in egg protein
functionality in the food system.
World's poultry science journal, 2002,
58, 31–39.
27.Taylor, D.; Hazenberg, J.G.; Lee, T.C.
Living with cracks: damage and repair
in human bone. Nature Materials,
2007, 6, 263–268.
28.Vorndran, E.; Klarner, M.; Klammert, U.;
Grover, L.M.; Patel, S.; Barralet, J.E.;
Gbureck, U.; Grover, L.M.; Barralet J.
3D powder printing of beta–tricalcium
phosphate ceramics using different
strategies. Advanced Engineering
Materials, 2008, 10 (12), 67–71.
29.Zhao, N.N.; He, H.; Hu, Q.; Hou, T.; Wang
C. Preparation and deamination of
promoting
calcium
absorption
peptides from desalted duck egg
white. Food Science, 2013.
Received: October 21, 2016
Article in Press: April 26, 2017
Accepted: Last modified on: May 20, 2017
56