Effect of recombinant human bone morphogenetic protein-2 on mandibular distraction osteogenesis.

Abstract

The purpose of this investigation was to study the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone formation of mandibular distraction osteogenesis. Six skeletally mature sheep underwent 10 mm of bilateral mandibular distraction osteogenesis via a custom-made distractor. Three micrograms of rhBMP-2 with a collagen carrier was implanted in the osteotomy site of one side of the mandible during the osteotomy phase. The contralateral side was used as the control group, and no material was implanted into the distracted area. At 10 days after the end of distraction, all animals were killed, and the distracted calluses were harvested for radiologic and histologic analysis. New bone was generated in the distracted zone in all groups. Histologic and radiologic examination showed that the new bone formation was greater in the rhBMP-2 group than in the control group. Quantitative computed tomography evaluation, however, did not demonstrate a significantly different mean bone density of the regenerates between the 2 groups. The results indicate that application of a rhBMP-2/collagen implant during the osteotomy phase of distraction osteogenesis increased bone formation but did not have a significant effect on bone density of the regenerates.

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Hong Kong Dental Journal 2007;4:94-100
D
HJ
ORIGINAL ARTICLE
94 Hong Kong Dent J Vol 4 No 2 December 2007
*? Oral?&?Maxillofacial?Surgery,?Faculty?of?Dentistry,?The?University?
of?Hong?Kong,?Hong?Kong
The first author won the Best Oral Presentation (Senior Category)
at the Annual Scientific Meeting of the Faculty of Dentistry, The
University?of?Hong?Kong?held?on?December?15,?2006
Correspondence to:
Prof. Lim-Kwong Cheung
Oral?&?Maxillofacial?Surgery,?Faculty?of?Dentistry,?The?University?of?
Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Hong
Kong?
Tel : (852) 2859 0267
Fax : (852) 2559 9014
e-mail : lkcheung@hkucc.hku.hk
Effect of recombinant human bone
morphogenetic protein-2 on mandibular
distraction osteogenesis
Li-Wu Zheng *,?MDS, PhD
Lim-Kwong Cheung *,?BDS, FDSRCPS, FFDRCS, FRACDS, FRACDS (OMS), FHKAM (Dental Surgery), FCDSHK (OMS), PhD
ABSTRACT Objective. This study aimed to evaluate the effect of recombinant human bone morphogenetic protein-
2 (rhBMP-2) on mandibular distraction at routine and rapid distraction rates. Methods. Eighteen New Zealand white
rabbits were randomly assigned to two groups, one treated at a routine distraction rate (0.9 mm/day) and the other
at a rapid distraction rate (2.7 mm/day). The rhBMP-2 was injected into one side of the distraction regenerate at the
end of the active distraction period, the contralateral side was used as a control. The distraction regenerates were
analyzed by plain radiography, micro-computed tomography and histological examination. Results. The rhBMP-2
appears to promote bone formation at both rapid and routine distraction rates, but no statistically significant difference
was observed between the BMP injection sides of the rapid and routine distraction groups. Conclusion.?This?study?
indicates that rhBMP-2 can enhance bone formation at both routine and rapid distraction rates. Addition of rhBMP-2
seems able to compensate for the adverse effects of rapid distraction rates on mandibular distraction osteogenesis.
Key words: Bone morphogenetic proteins; Mandible; Osteogenesis, distraction; Rabbits
Introduction
?
Distraction osteogenesis is a method of producing new
bone?directly?from?an?osteotomy?site?by?gradual?traction?
of the divided bone fragments. Since the first clinical
report on the use of distraction osteogenesis to lengthen
the human mandible in 1992 1,?it has become a widely
accepted approach to the treatment of severe craniofacial
deformities. However, one of the major disadvantages of
this?technique?is?the?lengthy?course?of?treatment?required?
for distraction and consolidation, which may result
in pin tract soft-tissue infection, bone infection, and
psychological problems. Consequently, both surgeons
and patients would welcome any technical improvement
that can speed up the treatment process.

fragments are stretched apart per day. The ideal rate for
lengthening is widely accepted to be around 1.0 mm per
day?2. Slow distraction tends to result in premature union,
whereas rapid distraction may delay bone union or even
give rise to fibrous union. In rabbit mandibular distraction
models different distraction protocols have been used: 1.0
mm?once?daily?3-5, 0.5 mm twice daily 4-6, 0.18 mm twice
daily?7,?and 0.9 mm once daily 8, all of which are capable
of leading to complete bone healing. At distraction rates
as rapid as 1.5 mm twice daily 6, 1.0 mm twice daily and
2.0 mm once daily 4, unreliable bone ossification has been
noted.
The rate of distraction is the extent to which the bone

most potent factors in bone development and healing 9-11,
and have an important role in regulating bone and
cartilage? formation? during? distraction? osteogenesis? in?
long?bones?12-14?and?in?the?mandible?15,16. The application
of recombinant human bone morphogenetic protein-2
Bone morphogenetic proteins (BMPs) are among the

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Hong Kong Dent J Vol 4 No 2 December 2007 95
Bone morphogenetic protein on mandibular distraction osteogenesis
(rhBMP-2) to promote the bone induction and ossification
process in distraction osteogenesis has been reported in
only a few publications. Both rhBMP-2 and rhBMP-4
were shown to enhance bone formation in the early stage
of?long?bone?and?mandible?distraction?17,18. On the other
hand, application of rhBMP-7 during distraction osteo-
genesis has been associated with conflicting results 19-21. No
study has compared the effect of rhBMPs on distraction
regenerates under routine and rapid rates of distraction.
?
rhBMP-2 on mandibular distraction at routine and rapid
distraction?rates,?and?thus?determine?the?feasibility?of?
compensating for the effects of increased distraction
rates while maintaining the quality of the regenerate.
This?study?therefore?aimed?to?evaluate?the?effect?of?
Materials and methods
Animal care
The animal experiment protocol was approved by the
Committee on the Use of Live Animals for Teaching
and Research, the University of Hong Kong. Eighteen
adult New Zealand white rabbits (3.0-3.8 kg) were used.
For this purpose, the rabbits were kept in a dedicated
animal holding facility under veterinary supervision in the
Laboratory Animal Unit of the Faculty of Medicine, the
University of Hong Kong.
Surgical procedure and postoperative care
A standardized surgical procedure used in our previous
study was performed on all the rabbits 8. Briefly, after the
administration of preoperative antibiotic and analgesic
(30 mg/kg long-acting oxytetracycline and 0.03 mg/kg
buprenorphine), each rabbit was anesthetized by
intramuscular injections of ketamine (35 mg/kg), xylazine
(5 mg/kg), and acepromazine (1 mg/kg). With the rabbit’s
head hyper-extended, the skin was incised along the
inferior border of both sides of the mandibular body. The
platysma was dissected and the periosteum was reflected
laterally. With a small bur, on both sides straight body
osteotomy cuts were performed immediately anterior to
the first premolar root. The custom-designed bone borne
distractors were adapted on both sides of the mandible
along a plane perpendicular to the osteotomy cut and
fixed by 2-mm diameter titanium screws. The periosteum,
muscle, and skin were repositioned and closed using 3-0
silk sutures.
After the operation, an antibiotic (long-acting
oxytetracycline
intramuscularly, twice per week for 2 weeks. For pain
relief, buprenorphine (0.03 mg/kg) was administered
subcutaneously twice daily for 10 days. Each animal
remained? under? close? observation? by? a? veterinary?
technician until it regained consciousness. The clinical
condition, weight and food consumption of the animals
were monitored.
30 mg/kg) was administered
Distraction procedure
Eighteen rabbits were randomly assigned to two
experimental groups, each with nine rabbits. After a 5-day
latency period, bilateral distraction was activated at 0.9
mm once daily for 12 days in the routine rate group, and
at 2.7 mm once daily for 4 days in the rapid rate group.
An 1.08 mg rhBMP-2 in phosphate buffer (Institute of
Basic Medical Sciences, Beijing, China) was injected into
one?side?of?the?distraction?regenerate?at?the?end?of?the?
active distraction period, whereas the contralateral side
was used as a control. Three rabbits in each group were
sacrificed after 1, 2, and 4 weeks of the consolidation
period, respectively.
Assessment methods
Plain radiography
Each mandibular specimen was placed on an occlusal film
with the lingual side touching the film. Plain radiography
was performed by an Orthoralix 9200 X-ray machine
(Gendex, Des Plaines, USA) under standard conditions
(50 KV, 16 mAs).
Micro-computed tomography
After plain radiographic examination, the distracted tissue
regenerate and a 2-5 mm section of the neighboring
normal bone in the distracted mandible were harvested.
The specimens were subjected to morphological and
quantitative examination by a µCT20 scan machine
(Scano Medical AG, Bassersdorf, Switzerland). Each
harvested specimen was placed into a 17-mm diameter
sample holder with the sagittal plane vertical to the X-ray
tube. For each specimen, 120-140 cross-sectional scans
with a slice increment of 100 µm were made.
?
described in our previous study was performed 8.?Briefly,
the serial scanned images for each specimen were
inspected on the computer. On each scanning image, the
total area of the distraction regenerate was outlined as the
region of interest (ROI). The bone volume fraction (the
The?standardized?method?of?quantitative?assessment?

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96 Hong Kong Dent J Vol 4 No 2 December 2007
Zheng and Cheung
ratio between bone volume and total volume, BV/TV)
within the ROI on each section was calculated individually
and a mean value of BV/TV for the total regenerate was
obtained by pooling from all the scanned sections within
the distraction gap. To determine the threshold, all the
ROIs on each specimen were subjected to an adaptive
procedure in which the bone fraction was determined for
a range of thresholds. The optimal threshold (120) was
defined at the minimum change of bone fraction.
Histology
After the micro-computed examination, the samples were
decalcified in a solution of 14.5% ethylenediaminetetra-
acetic acid buffered (pH 7.2) at room temperature. The
decalcified specimens were processed and embedded
in paraffin wax. Sections of 5 µm in thickness were cut
longitudinally in the axial plane with a microtome and
stained with hematoxylin and eosin for light microscopy.
Statistical test
The intra-individual controls were compared by paired
t test, and groups at different distraction rates were
compared by the two sample t test with the Statistical
Package for the Social Sciences for Windows (SPSS Inc.,
Chicago[IL], USA). A probability of 0.05 or less was
considered significant.
Results
Clinical examination
All 18 rabbits completed the experimental process
uneventfully. None of the animals experienced
postoperative complications, and the distractors remained
stable till the day of sacrifice. Both sides of the mandible
were lengthened symmetrically, and obvious crossbite
and overgrowth of the lower incisor developed in all the
rabbits.
Plain radiographic examination
In the routine distraction group, after 1 week of
consolidation two of the three rabbits sacrificed still
demonstrated incomplete union at the central area of
the distraction regenerate on the control side. Whereas,
confluent radio-opacity across the distraction gaps of
the BMP injection side was noted in all three cases. In
the corresponding rapid distraction group, radio-opaque
streaks were observed in both the BMP injection and
control sides, but partial union only was noted in two of
the three cases of the BMP injection side.

demonstrated bony continuity in both the BMP injection
and control sides of the routine distraction group, and the
radio-density of BMP injection side seemed to be greater.
In the rapid distraction group, complete bone union was
observed in two of the three cases on the BMP injection
side, but the control side still demonstrated obvious non-
union in the central area.
At week 2 of consolidation, the osteotomy site

injection sides of both the routine and rapid distraction
groups appeared similar. When compared with the
control sides, the radio-density in the distraction gap was
greater and clear corticalization was only observed on
the BMP injection side. On the control side, bone union
was complete in all the cases, but corticalization was not
clearly seen.
At week 4 the radiographic images of the BMP
Micro-computed tomography
Distraction regenerate in the serial computed
tomographic (CT) images demonstrated continuous
ossification with gradually increasing mineralization from
the center to the edges. Bone formation and remodeling
of the distraction regenerate on the BMP injection sides
(in both the routine and rapid distraction groups) were
more?obvious?than?on?the?control?sides?at?all?observation?
time-points.

formation was seen in all mandibular samples of the
experimental animals. More bone formation was noted
on the BMP injection side of both the normal and rapid
distraction groups than on the control side (Figures 1a-
d).
At week 1 of consolidation, obvious new bone

more obvious in all the mandibular samples (Figures
1e-h). The BMP injection side of both the normal and
rapid distraction groups displayed more new bone than
on the control side.
At week 2 of consolidation, new bone formation was

injection side of the normal distraction group. The
peripheral bone became denser and the marrow cavity
became evident, although it was not yet very clearly
defined (Figure 1f).
Early corticalization was only observed on the BMP

injection side of the normal and rapid distraction groups
were quite similar; advanced remodeling was noted, and
At week 4 of consolidation, the CT images of the BMP

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Hong Kong Dent J Vol 4 No 2 December 2007 97
Bone morphogenetic protein on mandibular distraction osteogenesis
obvious corticalization and marrow cavity formation
were clearly seen (Figures 1j and l). On the control side,
multifocal bony defects were still obvious (Figures 1i and
k).
?
showed that bone volume of the BMP injection sides was
higher?than?that?on?the?control?sides,?in?both?the?routine?
and rapid distraction groups (Figures 2a and b).
Quantitative? analysis? of? the? distraction? regenerate?

became statistically significant at 2 weeks and 4 weeks
of consolidation. When the bone volume of the BMP
injection sides was assessed, the routine distraction group
tended to have higher values than the rapid distraction
group, but the differences were not statistically significant
(Figure 2c).
Difference in bone volume between the groups
Histology
Corresponding to the findings demonstrated by the
micro-CT study, histological examination showed that
bone formation and remodeling on the BMP injection
sides was more advanced than on the control sides.

group the distraction regenerate of both the experimental
and control sides was composed of primary trabeculae
and loose fibrovascular stroma; fibrous tissue still being
obvious in the central area. On the control side, there were
still a few small cartilage islands at the central area close
to the periosteum. In the rapid distraction group, most
of the distraction gap was bridged by fibrous tissue; new
bone had generated at the edges of the distracted gap, and
also?at?the?medial?surface?of?the?distraction?regenerate?
At week 1 of consolidation, in the routine distraction
Figure 1 Micro-computed tomographic images of the middle section of the distraction regenerate in the transverse plane
respectively at week 1, 2, and 4: (a,e,i) control side under normal distraction; (b,f,j) bone morphogenetic protein injection side
under normal distraction; (c,g,k) control side under rapid distraction; and (d,h,l) bone morphogenetic protein injection side
under rapid distraction
(a)(b)(c) (d)
(e)(f)(g)(h)
(i)(j)(k)(l)
Week 1
Week 2
Week 4

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98 Hong Kong Dent J Vol 4 No 2 December 2007
Zheng and Cheung
(by periosteal reaction). At week 2, bone formation and
remodeling was more advanced in BMP injection sides
of both the routine and rapid distraction groups, and
complete bony union was observed in the central area
of the distraction regenerate. On the control sides, focal
fibrous tissue was obvious in the routine distraction
group, and even more so in the rapid distraction group.

bone remodeling was evident; increased thickness of the
new cortex and bone marrow cavity formation were clearly
seen. On the control sides, though the distraction gap
was completely united, corticalization and marrow cavity
formation were not yet evident in the routine distraction
group. On the control side in the rapid distraction group,
the presence of fibrous tissue was obvious.
At week 4, on the BMP injection sides of both groups,
Discussion
Varying the rate of distraction significantly influences
the? outcome? of? distraction? osteogenesis?2. A higher
rate of distraction may lead to poor bone quality and a
high incidence of soft tissue complications 22,23. Li et al 22
investigated the angiogenic response to different
distraction?rates?and?demonstrated?that?vascularization?
in the central fibrous zone was maximally stimulated at
distraction rates of 0.7 to 1.3 mm/day. A slow rate of
distraction at 0.3 mm/day does not maximally stimulate
angiogenesis? in? the? distraction? regenerate,? and? a? high?
rate (2.7 mm/day) appeared to impair this response. A
study in our department demonstrated that change in the
mechanical?environment?resulting?from?different?rates?of?
distraction leads to different expression of the BMP-2
and -4. The biological environment created by distraction
at a routine rate (0.9 mm/day) is superior to that ensuing
with a rapid rate (2.7 mm/day) during the early stage of
consolidation.

maxillofacial and orthopedic surgery would wish to
reduce the treatment course of distraction osteogenesis.
Reducing the consolidation period or increasing the
distraction rate are possible options. The present study
attempted to facilitate an accelerated distraction rate with
the injection of rhBMP-2, and compare the ensuing bone
ossification process at routine and rapid distraction rates.
From the clinical perspective, most surgeons in

family) has been shown to promote bone regeneration and
remodeling during the bone repair process in a variety of
animal?models?24-29. The development of the recombinant
DNA techniques enables recombinant human BMPs to
be produced reliably and in substantial quantities, which
offers promise for hard tissue engineering.
Use of rhBMP-2 (among the most potent of the BMP

department demonstrated that the osteogenic activity
of rhBMP-2 in mandibular distraction osteogenesis is
dosage-dependent, and that a single injection of rhBMP-
2 of 1.08 mg is just as effective as multiple injections 8. In
the current study, a single injection of 1.08 mg rhBMP-
2 was used at the end of the active distraction period.
In reconstructive surgery, exogenous BMPs are normally
delivered to the site of regeneration by the carrier matrix.
A baseline study for this work conducted in our
*? P<0.01
**?P<0.001
Figure 2 Quantitative analysis of bone volume/total volume (BV/TV) of the regenerates under (a) routine distraction,
(b) rapid distraction, and (c) comparison of routine and rapid distraction with recombinant human bone morphogenetic
protein 2 (rhBMP-2) injection
0
0.2
0.4
0.8
0.6
123412341234
Week Week Week
BV/TV
**
**
**
*
(a) (b)(c)
BMP injection side
Control side