Engineering, 2013, 5, 108-113 Published Online October 2013 (
Copyright © 2013 SciRes. ENG
Bone Regeneration Enhanced by Antigen-Extracted
Xenogeneic Cancellous Bone Graft with rhBMP-2 in
Rabbits Mandibular Defect Repair*
Renfa Lai#†, Zejian Li, Ye Zhang, Zhiying Zhou
The Medical Centre of Stomatology, The 1st Affiliated Hospital of Jinan University, Guangzhou, China
Received December 2012
The effects of large piece xeno geneic bone w hich was separated from healthy pig s as a scaf fold on repair of mandibula r
defect was investigated and the applicability of antigen-extracted xenogeneic cancellous bone (AXCB) soaked with
rhBMP-2 in bone defect repair was assessed. Mandibular defects were created in 48 New Zealand Rabbits, and then
randomly divided into 4 groups, which was grafted in the mandibular defects with AXCB, AXCB soaked with
rhBMP-2, autograft bone, or blank. Equal number of animals from each group was classified into three time points (4, 8,
and 12 weeks) af ter oper ation for gr oss pa thological observation, hematoxylin and eosin (H & E) staining, radiographic
examination, and bone density measurement. H & E staining revealed that the area percentage of bone regeneration in
the group of AXCB/rhBMP-2 graft was 27.72 ± 4.68, 53.90 ± 21.92, and 77.35 ± 9.83 when at 4, 8, and 12 weeks,
which was better than that of auto bone graft, prompting that the group of AXCB/rhBMP-2 graft had commendable
osteogenic effect. And comparing with the AXCB withou t rhBMP -2, of which the area percentage of bone regene rat ion
was only 14.03 ± 5.02, 28.49 ± 11.35, and 53.90 ± 21.92, the osteogenic effect of AXCB/rhBMP-2 graft was demon-
strated to be much better. In the group of AXCB/rhBMP-2 graft, the area percentage of bone regeneration increased,
and the implanted materials were gradually degraded and replaced by autogenous bone regeneration over time. We con-
cluded that antigen-extracted xenogeneic cancellous bone (AXCB) graft soaked with rhBMP-2 had shown excellent
osteogenic effect in repair of bone defects, wit h good bioc ompabil i ty.
Keywords: Recombinant Human Bone Morphogenetic Protein-2(rhBMP-2); Antigen-Extracted Xenogeneic
Cancellous Bone (AXCB); Defect Repair; Bone Regeneration; Mandible Defect
1. Introduction
Large bone defect in oral and maxillofacial region is
frequently seen in human patients, and its proper repair is
a big challenge due to the anatomical complexity of this
region and the cosmetic issue. The main method to repair
the bone defect so far is bone transplantation, which in-
cludes autologous bone graft, bone allograft and xeno-
graft. Autologous bone graft provides not only a scaffold
but also a certain number of osteoblasts, and it has the
best osteogenic effect. Therefore, it is considered the
gold standard for bone defect repair [1]. The autologous
cancellous bone are usually taken from the iliac cancell-
ous bone, the distal femur, greater trochanter or proximal
tibia [2]. However, autologous bone graft has limitated
bone sources, and needs a second operation area, which
will increase extra trauma to the p atients and incr ease the
duration of operation. Bone allograft is another way of
providing a scaffold for bone regeneration, but it may
have a high risk of disease transmission. In addition,
some medical ethics issues may also limit the clinical
application of allogeneic bone graft [3]. Xenograft is a
good source of scaffold for bone regeneration, but it also
has a potential risk of disease transmission, for example,
the bovine spongiform encephalopathy (BSE) from bo-
vine bone xenograft, which is currently the most com-
monly used one across the world. Nevertheless, as the
development of the advanced specific antigen extraction
technology, the risk of disease transmission from xeno-
graft is no longer a health concern [4]. Recently, the
source of heterogeneous bone from pigs, cattle, sheep,
and dogs has become the focus of study for development
of biomaterials for bone regeneration. In this study, we
investigate the biocompatibility of antigen-extracted xe-
nogeneic cancellous bone (AXCB) as a scaffold, and its
This work was financially supported by a grant from Guangdong
Science and Technology Foundation (No:2011B080701053)
#Corres ponding author.
These authors contribute equally to this work.
Copyright © 2013 SciRes. ENG
osteogenic efficiency, in combination with bone morpho-
genetic protein ( AXCB-BMP), in repairing defects of the
mandibular bone in rabbits, aiming to identify a new and
better approach for bone defect repair in the oral and
maxillofacial region using allogeneic bone as a scaffold.
2. Materials and Methods
2.1. Materials
The iliac bones are separated from healthy pigs, and cut
into 15 mm × 6 mm × 4 mm pieces for preparation of
xenogeneic antigen-extracted cancellous bone (AXCB).
The bone pieces were soaked in acetone for 48 hours to
remove the fatty composition, demineralized in 0.6 M
HCl, completely washed with water, treated with enzyme,
washed with water again according to patented technol-
ogy by the Guangdong Guan-Hao Technology Co. (Fig-
ure 1A), and then freeze-dried for pre pa ration of rhBMP-
2 incorporation. The rhBMP-2 was produced by recom-
binant expression in Escherichia coli at the Institute of
Biomedical Engineering, Jinan University (Guangzhou,
China), and purified to more than 98% purity, which was
then dissolved in gelatin solution with 0.1% acetic acid
(10 mg/m l). Each piece of AXCB was soaked with 1 ml of
gelatin solution containing rhBMP-2 (2.0 mg/ml) for 24
hours, sterilized by γ-ray irradiation with a radiation do-
sage of 25 k Gy, then freeze-dr ie d, and sto r ed fr oze n unt il
use (Figure 1B).
Figure 1. Implantation of AXCB incorporated with BMP-4
(AXCB/ rhBMP-2) in mandib ular defect in rabbit s. (A) The
prepared AXCB; (B) AXCB scaffold incorporated with
rhBMP-2; (C) Creation of a 15mm × 6mm × 6mm mandi-
bular defect in rabbit. (D) Implantation of AXCB/rhBMP-2
into the created ma ndibular defect.
2.2. Animal Experiment
Forty-eight adult New Zealand White rabbits weighing
3.0 - 3.5 kg (Experimental Animal Center of Guangdong
Province) were used for the experiment, and the protocol
was approved by the Institutional Animal Care and Use
Committee (IACUC) at Jinan University Health Science
Center. The an imals we re randomly divi de d into 4 groups
(AXCB graft with rhBMP-2, AXCB graft only, autolog-
ous bone graft, and non-graft control), with 3 subgro ups (4,
8 and 12 weeks) for e ach group (4 animals f or each con-
dition). Prior to operation, the animals were anesthetized
by intravenous injection of Nembutol (pentobarbital so-
dium) (3 0 mg/kg). In t he a ut ol ogou s b one g ra ft gr o up, the
animals were first subjected to bilateral abdominal inci-
sions parallel to the iliac crest; a 15 mm × 6 mm × 4 mm
bone piece was excised from the iliac bone on each side,
and the n pl ac e d i n sa li ne s ol ut ion u nti l use . Pre par at io n o f
the AXCB, and those soaking with rhBMP-2 were as
described above. Then a bone defect with a size of 15 mm
× 6 mm × 4 mm was created on both mandibles in each
animal for all 4 groups (Figure 1C). In the graft groups,
the created bone defect was implanted with the prepared
AXCB soaked with rhBMP-2, AXCB only, or autologous
bone (Figure 1D); while in the control (non-graft) group,
the skin incision was directly closed with sutures after
creation of the bone defect. All animals were then housed
in the same condition and monitored for postoperative
activities, emotional response, and wound healing. The
animals were sacrificed at 4, 8, or 12 weeks after opera-
tion, and the whole mandible was harvested from each
side for investigation.
2.3. X-Ray Examination
X-ray examination of the harvested mandibles was per-
formed (DR3000, Kodak, USA), and the image data were
scored by three technicians blindly based on Lane-
Sandhu scoring method [5], and analyzed using the the
Leica Image Analysis System for assessment of bone
regeneration following the mandibular defect.
2.4. Bone Mineral Density M easurement
The obtained bone samples were fixed with formalin in
posphate buffer, and bone mineral density was measured
for the repaired area using the bone density meter platform
Lunar Prodigy (GE, USA). The bone mineral content
(BMC) was presented as g/cm2.
2.5. Preparation of Bone Samples for
Pathological Staining
The obtained bone samples were decalcified, embedded in
paraffin, sectioned into 3 μm slices, and mounted onto
slides for hematoxylin and eosin (H & E) staining. The
Copyright © 2013 SciRes. ENG
stained slides were observed under optical microscope at
50× magnification for evaluation of new bone formation
and calcification, new blood vessel and fibrous tissue
generation, inflammatory cell infiltration, and implanted
scaffold degradation.
2.6. Quantitative Analysis of the New Bon e
The bone samples were fixed in 10% neutral buffered
formalin, immersed in Technovit 7200 VLC (Heraeus-
Kulzer, Germany) afte r dehydrati on , a nd sectioned into 5
thin slices of approximately 40 - 80 μm and mounted
onto slides after 24 hours’ solidification. The slides were
H & E-stained, and histomorphometry was performed
using Leica Image Analysis System.
2.7. Statistical Analysis
One-way ANOVA was used for the statistical analysis,
and the data were presented as means ± standard devia-
3. Results
3.1. Gross Observation
All animals from all groups were alive after surg ery, and
the wound healed well, though temporary postoperative
swelling was noted in all animals. Neither signs of loss,
displacement, and discharge of the implants, nor fracture
and wound infection was observed during the whole ob-
servation perio d.
3.2. Histological Observation
In the group with implantation of xenogeneic antigen-
extracted pig massive cancellous bone, at 4 weeks after
operation, there found some fibrous tissue, capillary pro-
liferation, trace of trabecular bone degradation, a small
number of osteoblasts, and a small amount of new bone
forma tion a rou nd the ed ge of the i m plant ; at 8 weeks aft er
operation, there were partial degradation of trabecular
bone, an d a large num ber of oste oblasts arou nd the edge o f
the implant; at 12 weeks after operation, there was little
mature trabecular bone tissue (Figure 2A). In the group
with implantation of xenogeneic antigen-extracted pig
massive cancellous bone soaked with rhBMP-2 , at 4
weeks after operation, the trabecular bone of the implant
was partially degraded, a large number of new bone for-
mation was observed, and around the new bone, there
were a large number of osteoblasts and m esenchymal cells,
capillary ingrowth, and osteoid formation; at 8 weeks after
operation, there were a small area of unabsorbed implant,
a large amount of trabecular bone tissue and new bone
formation, and capillary ingrowth, with a lot of osteob-
lasts and mesenchymal cells around; at 12 weeks after
operation, there were almost complete degradation of the
Figure 2. Histological images of the rabbit mandible defect
(×100). (A) A representative result at 12 weeks after surgery
in AXCB group; (B) A representative result at 12 weeks
after surgery in AXCB-rhBMP-2 group.
implant, a large amount of new bone formation with ma-
ture trabecular bone and some bone marrow . Histological
examination showed rigorous bone regeneration around
the implant. In the non-graft control group, the mandible
showed only a small amount of new bone formation, and
the created bone defect was mainly occupied by fibrous
tissue at all time points. In the autograft group, there
showed a large amount of new mature trabecular bone,
and the mandibular defect was mostly occupied by the
newly fo rmed bone (Figure 2B).
3.3. The Radiographic Evaluation
Lateral and vertical radiography was used to evaluate
bone regeneration and healing of the mandible defect
during follow-ups. New bone formation was assessed by
Lane-Sandhu scoring method. Score 0 indicated “no new
bone formation”, 1, “new bone occupied 25% of the de-
fect”, 2, “new bone occupied 50% of the defect”, and 3,
“new bone occupied 75% of the defect”. The average
scores were 1.00, 7.50, and 11.00 in the autograft group,
1.00, 5.25, and 7.50 in the AXCB/ rhBMP-2 group, and
0.20, 2.75 and 3.75 in the AXCB alone group at 4, 8, and
12 weeks after operation, respectively, indicating that
scaffold graft alone had limited effect on bone regenera-
tion and addition of rhBMP-2 greatly enhanced bone
regeneration, which is comparable to auto bone graft
(Figure 3). New bone gene ra tion increased over time.
3.4. Bone Mineral Density
Bone mineral density test revealed significant difference
in bone mineral density across groups (autogenous bone
group > AXCB/ rhBMP-2 group > AXCB only group >
control group) (P < 0.05). And the bone mineral density
was significantly increased over time (at 4, 8, and 12
weeks) within each individual group (P < 0.05).
3.5. Quantitative Assessment of Bone
Percentage of area with new bone formation was calcu-
Copyright © 2013 SciRes. ENG
Figure 3. X-ray photos of the rabbit mandible defect. (a) A
representative result at 12 weeks after surgery in AXCB
group; (b) A representative result at 12 weeks after surgery
in AXCB- rhBMP-2 group; (c) A representative result at 12
weeks after surgery in Control group; (d) A representative
result at 12 weeks after surgery in Autograft group.
lated under microscopic view of the H & E stained slides.
The area percentage of bone regeneration in the group of
AXCB/rhBMP-2 graft was 27.72 ± 4.68, 53.90 ± 21.92,
and 77.35 ± 9.83, that in the group of AXCBgraft was
14.03 ± 5.02, 28.49 ± 11.35, and 55.87 ± 10.20, and that
in the group of autograft bone graft was 30.19 ± 1.46,
49.73 ± 2.68, 68.18 ± 3.92 at 4, 8, and 12 weeks, respec -
tively. Statistical analysis result suggested that the area of
bone regeneration of the mandibular defect was signifi-
cantly greater in the group of AXCB/ rhBMP-2 (scaffold
with morphogen) than in the group of xenogeneic anti-
gen-extracted pig massive cancellous bone (scaffold only)
(P < 0.05), and there was a significant increase of bone
regeneration over time (at 4, 8, and 12 weeks after opera-
tion) within each group (P < 0.05). There was no signifi-
cant difference in the area of new bone formation between
the group grafted with autogenous bone and the group
grafted with AXCB/ rhBMP-2
4. Discussion
Tumors, especially malignant tumors, severe trauma, and
congenital malformation in the oral and maxillofacial
region often lead to a large area of bone defect. Because
of the anatomical particularity and the three-dimensional
structure complexity, the restoration of bone defects in
oral and maxillofacial region remains a challenge for
surgeons. The restoration of the original shape of the
facial skull is a prerequisite for the restitution of facial
appearance. Scientists are trying to develop new ap-
proaches aiming at the enhancement of bone regeneration
instead of using autogenous bone grafts. Autologous
bone can provide the transplant scaffolds while providing
a certain number of osteoblasts, and it has the best os-
teogenic effect and has been widely used as the gold
standard method for repair of bone defects. However,
autologous bone usually doesn’t provide an anatomically
preformed shape and meet the requirement for mechani-
cal properties, and its source and volume are very limited.
Autogenous bone graft requires a second operation area
and causes new damage for the bone-donated area, which
greatly increases the duration of operation and may result
in more complications [6]. Recent progress in regenera-
tive medicine and bone tissue engineering raises the hope
of repairing bone defects with a combination of biomate-
rials and growth factors. Application of the large can-
cellous bone (ilium) as a morphogen carrier for rhBMP-2
in skeletal repair has been extensively researched during
the past decade [7]. Bone morphogenetic proteins (BMPs)
have been successfully applied in the reconstruction of
long bones, spine and the facial skeleton in preclinical
studies [8].
Based on the theory of creeping substitution [9], an
ideal bone graft used for bone defect repair should pro-
vide a “platform” fo r the three essential elements of bone
regeneration: osteoinduction, osteoconduction and os-
teogenesis. Osteoinduction is a process of inducing dif-
ferentiation of mesenchymal stem cells (MSCs) into os-
teoblasts and chondrocytes, presumably by some mor-
phogens. Oste oconduction is a proper ty of the bone graft
as a scaffold that ‘conduct’ the ingrowth of the osteob-
lasts (differentiation and maturation) as well as that of
the blood vessels, providing a platform for osteogenesis.
The scaffold graft is gradually replaced by creeping
substitution of the regenerated new bone [10]. So me stu -
dies have shown that the creeping substitution occurs
mainly in the facial layer and at the two ends of im-
planted bone. Large bone defects may have very limited
regeneration by using heterologous graft [11,12]. Bone
morphogenetic proteins (BMPs) are good morphogens
that induce both osteogenesis and angiogenesis [13],
which may help to overcome the above limitation of
large heterologous bone graft. Recombinant human bone
morphogenetic protein-2 (rhBMP-2) is the growth factor
most widely used for bone regeneration [14,15], but it
diffuses fast after applied. Therefore, development of
favorable carriers with slow-releasing property is critical.
The bone morphology of the oral and maxillofacial re-
gion in rabbits has similarity to that in humans. In this
study, we soaked the rhBMP-2 into a piece of xenogene-
ic antigen-extracted pig cancellous bone (AXCB), which
was then implanted in the bone defect of the same size
created in the mandibles of New Zealand White rabbits,
Copyright © 2013 SciRes. ENG
and subsequently assessed the bone regenerative effect
[16]. The results revealed that the group grafted with
AXCB soaked with BMP-4 (AXCB/ rhBMP-2) had
much better and more extensive bone regeneration than
the group grafted with AXCB only, and the bone rege-
neration increased over time from 4 weeks to 12 weeks
after operation, which indicated that rhBMP-2 has sig-
nificant bone regenerative effect over time with AXCB
as a scaffold, and AXCB is probably a good carrier for
BMP-4, which can help rhBMP-2 release slowly and
work effectively. On the other hand, the AXCB was
found to be gradually degraded over time, and at 12
weeks after operation, the implanted bone was almost
completely replaced by newly regenerated bone tissue,
which showed apparent mature trabecular structure.
There were no appreciable histological signs of inflam-
mation or immune rejection of the graft.
In conclusion, the osteogenic effect of AXCB graft
soaked with rhBMP-2 is proved much better than AXCB
graft alone (without rhBMP-2, which shows no signifi-
cant difference with the autologous bone graft). Xeno-
geneic antigen-extracted pig massive cancellous bone has
shown good biocompatibility and it may potentially re-
place aut ologo us bone graf t in re pair of l arg e bone de fect s.
This study has provided a new reference for bone rege-
neration in the oral and maxillofacial region.
5. Acknowledgements
This work was supported by a grant from the Guangdong
Science and Technology Foundation (No: 2011B0807-
01053). We thank the Department of Nuclear Medicine,
the Animal Center, and the Institute of Biomedical En-
gineering at Jinan University, and Guangdong Guan-Hao
Science and Technology Development Co. Ltd for their
generous support.
[1] C. Madrigal, R. Ortega, C. Meniz, et al., Study of Avai-
lable Bone Forinterforaminal Implant Treatment Using
Cone-Beam Computed Tomography,” Medicina Oral Pa-
tologia Oral y Cirugia Bucal, Vol. 1, No. 13, 2008, pp.
[2] W. G. De Long Jr., T. A. Einhorn, K. Koval, et al., “Bone
Grafts and Bone Graft Substitutes in Orthopaedic Trauma
Surgery,” The Journal of Bone & Joint Surgery, Vol. 89,
2007, pp. 649-658.
[3] E. N. Ebbesen, J. S. Thomsen and L. Mosekilde, “Nonde-
structive Determination of Iliac Crest Cancellous Bone
Strength by pQCT,” Bone, Vol. 21, 1997, pp. 535-540.
[4] C. G. Finkemeier, “Bone-Grafting and Bone-Graft Subs-
titutes,” The Journal of Bone & Joint Surgery, Vol. 84A,
2002, pp. 454-464.
[5] A. S. Herford and P. J. Boyne, Reconstruction of Man-
dibular Continuity Defects with Bone Morphogenetic
Protein-2 (rhBMP-2),” Journal of Oral and Maxillofacial
Surgery, Vol. 66, 2008, pp. 616-624.
[6] S. A. Jovanovic, D. R. Hunt, et al., “Bone reconstruction
Following Implantation of rhBMP-2 and Guided Bone
Regeneration in Canine Alveolar Ridge Defects,Clinical
Oral Implants Research, Vol. 18, 2007, pp. 224-230.
[7] Z. Luo, Y. Hu and Q. Wang, The Experimental Studies
of Immune Response of Antigen-Extracted Bovine Can-
cellous Bone Grafting,Zhonghua Wai Ke Za Zhi, Vol.
35, 1997, pp. 690-693.
[8] S. Oeberg, C. Johansson and J. B. Rosenquist, “Bone For-
mation after Implantation of Autoly s ed Ant ige n E xtracted
Allogeneic Bone in Ovariectomized Rabbits,” Interna-
tional Journal of Oral and Maxillofacial Surgery, Vol. 32,
2003, pp. 628-632.
[9] H. Schliephake, “Application of Bone Growth Factors:
The Potential of Different Carrier Systems,” Oral and
Maxillofacial Surgery, Vol. 14, 2010, pp. 17-22.
[10] K. H. Schuckert, S. Jopp and S. H. Teoh, “Mandibular
Defect Reconstruction Using Three-Dimensional Polyca-
prolactone Scaffold in Combination with Platelet-Rich
Plasma and Recombinant Human Bone Morphogenetic
Protein-2: De Novo Synthesis of Bone in a Single Case,”
Tissue Engineering Part A, Vol. 15, 2009, pp. 493-499.
[11] C. Shi, W. Chen, Y. Zhao, et al., “Regeneration of Full-
Thickness Abdominal Wall Defects in Rats Using Colla-
gen Scaffolds Loaded with Collagen-Binding Basic Fi-
broblast Growth Factor,” Bi omaterials, Vol. 32, 2011, pp.
[12] D. I. Spector, J. H. Keating and R. J. Boudrieau, “Imme-
diate Mandibular Reconstruction of a 5 cm Defect Using
rhBMP-2 after Partial Mandibulectomy in a Dog,” Vete-
rinary Surgery , Vol. 36, 2007, pp. 752-759.
[13] R. Visser, P. M. Arrabal, J. Becerra, et al., “The Effect of
an rhBMP-2 Absorbable Collagen Sponge-Targeted Sys-
tem on Bone Formation in Vivo,” Biomaterials, Vol. 30,
2009, pp. 2032-2037.
[14] B. Wenz, B. Oesch and M. Horst, “Analysis of the Risk
of Transmitting Bovine Spongiform Encephalopathy through
Bone Grafts Derived from Bovine Bone,” Biomaterials,
Vol. 22, 2001, pp. 1599-1606.
[15] J.-C. Xu, G.-H. Wu, H.-L. Liu, et al., “The Effect of Lep-
tin on the Osteoinductive Activity of Recombinant Hu-
man Bone Morphogenetic Protein-2 in Nude Mice,Sau-
di Medical Journal, Vol. 31, 2010, pp. 615-621.
[16] M. Zhou, X. Peng, C. Mao, et al., “Primate Mandibular
Reconstruction with Prefabricated, Vascularized Tissue-
Engineered Bone Flaps and Recombinant Human Bone
Copyright © 2013 SciRes. ENG
Morphogenetic Protein-2 Implanted in Situ,” Biomate-
rials, Vol. 31, 2010, pp. 4935-4943.