Osteogenic Alveolar Distraction

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Received May 12, 2009; revision accepted for publication November 23, 2009. doi:10.1620/tjem.220.67
Correspondence: Sawako Takeuchi, Department of Oral and Maxillofacial Surgery, Tokyo Medical University, 6-7-1 Nishi-shinjuku
Shinjuku-ku, Tokyo 160-0023, Japan.
e-mail: sawakotakeuchi2008@jcom.home.ne.jp
Tohoku J. Exp. Med., 2010, 220, 67-75
Benefcial Role of Periosteum in Distraction Osteogenesis of
Mandible: Its Preservation Prevents the External Bone Resorption

Sawako Takeuchi
1
, Akira Matsuo
1
and Hiroshige Chiba
1
1
Department of Oral and Maxillofacial Surgery, Tokyo Medical University, Tokyo, Japan
Distraction osteogenesis (DO) is a surgical process of new bone generation through the gradual extension
of two segments of existing bone. DO is applied for maxillofacial surgeries to manage defects in
mandibular continuity. Vertical DO with an oral device is often employed to augment the alveolar bone
height for better implant anchorage for esthetic purposes or functional prosthetic requirements. To
determine how the periosteum affects the vertical DO in mandibular reconstruction, we extracted the teeth
and resected the alveolar parts of the mandible on both sides of dogs, along with removal of the
surrounding periosteum in the right, but not left side. Three months later, box-shaped bone segments
(vectors) were prepared from the resected alveolar part, and the segments were vertically elongated using
a distraction device on both sides at 0.9 mm/day for one week. The extent of bone formation after
distraction was determined with micro-focused computed tomography and by measuring incorporation of
tetracycline and calcein with confocal laser scanning microscopy. During the initial two months after
distraction, new bone formation was observed more prominently in the left side than in the right side of
mandible with the periosteum. However, this difference was less clear during the bone-remodeling period.
One notable change was the reduced height of the alveolar part of the right-side mandible, a sign of
external bone resorption, observed in two out of three dogs at 6-month post-consolidation. These fndings
suggest that preservation of periosteum prevents the external bone resorption during the vertical DO of
mandible.
Keywords: vertical distraction osteogenesis/periosteum/vector/micro-focused computed tomography/confocal laser
scanning microscopy
Tohoku J. Exp. Med., 2010, 220 (1), 67-75. 2010 Tohoku University Medical Press
Distraction osteogenesis (DO) is a method of bone
generation using stimulation with stress created by gradual
traction on the bone after osteotomy (Rachmiel et al. 2001).
It has been mainly used for lengthening of the leg in the
orthopedic feld because it eliminates the need for bone
grafting (Kojimoto et al. 1988). It has also been used in
maxillofacial surgery to advance the mid and lower face
(McCarthy et al. 1992), for mandibular reconstruction
(Gaggl et al. 1999), and for alveolar augmentation
(Mazzonetto et al. 2005). Vertical DO has mainly been
used for alveolar augmentation in which new bone forms in
the gap between the osteotomized bone segment (vector)
and the base bone (Block et al. 1996). Blood supply to the
vector is sometimes insuffcient because blood is supplied
only from the attached mucosa, and the vector size tends to
be small (Rachmiel et al. 2001). Therefore, reduction of
alveolar bone height and total necrosis of the vector have
been reported in vertical DO (Amir et al. 2006).
On the other hand, we often use vertical DO after
malignant tumor resection, a procedure in which the perios-
teum is completely removed (Hirota et al. 2008). Although
the main blood supply to the vector is decreased by remov-
ing the periosteum, the effect of the periosteum removal of
the mandible in vertical DO has not been evaluated. To
focus on this important point, we made a vertical DO model
of the dog mandible that simulates tumor resection. Three-
dimensional analysis of bone microstructures was attempted
using micro-focused computed tomography (micro-CT),
and dynamic bone labeling was observed using confocal
laser scanning microscopy (CLSM).
Materials and methods
Animal model
Eleven 1- to 2-year-old beagles (8 female and 3 male), weighing
around 10 kg, were used for this study. Use and care of animals in
this study conformed to Association for Assessment and Accreditation
of Laboratory Animal Care standards and was approved by the Tokyo
Medical University Subcommittee on Research Animal Care.
Surgery
After sedation with ketamine hydrochloride (5 mg/kg), general
S. Takeuchi et al.
68
anesthesia was induced with intravenous pentobarbiturate (0.5 mg/
kg). In the frst operation, we extracted 4 premolar teeth, and per-
formed marginal resection of the mandible over a height of 5 mm
simulating tumor resection. The surrounding periosteum was com-
pletely excised over a width of 2 mm using a knife (Fig. 1A, arrow)
only on the right side, and was preserved on the left side after resec-
tion of the mandible (Fig. 1A). The excised range was over 1.5 cm of
the osteotomy line each mesially and distally, and through the lower
border of the mandible bucco-lingually. This procedure was per-
formed by a single qualifed oral and maxillofacial surgeon (A.M.).
After 3 months (Fig. 1B), under general anesthesia vectors were
produced 5 mm in height, 30 mm in length, and completely osteoto-
mized bucco-lingually (7 to 8 mm in width) on the resected area under
both conditions, and intra-oral distraction devices, TRACK 1.0 mm-
System (Gebrder Martin GmbH & Co. KG, Tuttlingen, Germany),
were fxed using screws. At frst, the vector was kept in the original
position for 1 week (latency period), then 0.9 mm of vertical distrac-
tion was performed per day, and the bone height was increased by a
total of 5.4 mm (Fig. 1C). The distraction devices were kept at the
fnal position for 2 months (consolidation period), then removed.
Observation was carried out for 6 months after completion of consoli-
dation. Tetracycline (TC, 20 mg/kg) was injected in the frst half and
calcein (Cal, 8 mg/kg) was injected in the latter half of the observa-
tion period as bone labeling agents once per week according to the
timetable (Fig. 2). The dogs were killed under general anesthesia with
perfusion fxation using 10% buffered formalin 0, 1, 2 months after
distraction or 1, 3, 6 months after consolidation. Two dogs were
killed 3 months after marginal resection without producing vectors as
controls.
3D reconstruction images using micro CT
The mandible including the whole distracted part was prepared
for micro-CT scanning, Elescan (NS-ELEX Co. Ltd., Kita-kyushu,
Japan). First, we scanned the whole image at low magnifcation under
the following conditions: slice thickness 131 m, magnifcation 1.72,
pixel size 82 82 m, and image matrix size 512 512 pixels. We
created 3D reconstruction images by volume rendering method for
morphological observations using the computer software program
TRI/3D-BON (Ratok System Engineering Co., Ltd., Tokyo, Japan).
Bone morphometrical study
Using the above images, we selected two areas of volume of in-
terest (VOI) of 2 2 1 mm in the trabecular bone for morphometri-
cal analysis, which is independently set in the mesial and distal area
of the distraction gap (Fig. 3). These areas were examined by micro-
CT analysis under the following conditions: 20 m of slice thickness,
5.56 magnifcation, 25 25 m pixel size, and 50 number of slices.
Bone volume (BV) was calculated using tetrahedrons corresponding
to the enclosed volume of the triangulated surface. Total tissue vol-
ume (TV) was the entire volume of the analysis. The bone volume
index was determined as follows: 100 BV/TV.
CLSM images
We then prepared the specimens to make the non-decalcifed
Fig. 1. Procedure of the surgery.
A. Marginal resection. Periosteum surrounding the distracted area was completely excised over a width of 2 mm using
a knife (arrow). B. 3 months after resection. C. Completion of consolidation. New bones were formed in the distrac-
tion gap macroscopically.
Osteogenic Alveolar Distraction
69
Fig. 2. Labeling schedule.
Tetracyclin (TC) injection. Calcein (Cal) injection.
The numbers inside parentheses indicate the frequency of injections.
A. Latency period. B. Distraction period. C. Consolidation period.
TC was injected at the frst half and Cal at the latter half. Both agents were injected weekly.
Fig. 3. Setting of volume of interests (VOIs) for the bone morphometries.
We selected the two areas of VOI of 2 2 1 mm in the trabecular bone, which is independently set on the mesial and
distal area of the distraction gap.
Fig. 4. Comparison of CLSM images between the original and the extracted condition.
A. The original image. In the original CLSM images, the TC labeling area was colored red, and the Cal labeling area
was green. B. The extracted image. To eliminate the background, we made CLSM extracted images drawn in the same
color using computer software.
S. Takeuchi et al.
70
ground sections. The specimens were dehydrated in graded alcohol
for 3 weeks, saturated with styrene monomer for 10 days, embedded
in polyester resin consisting of a 7:3 mixture of Rigorac 2004 and 70F
(Riken, Tokyo, Japan), and polymerized. The block was cut into
1-mm continuous slice sections with a band saw, and each was ground
down further to approximately 100 m using Exakt Micro Grinding
System (Exakt Apparatebau, Norderstedt, Germany). We scanned the
ground sections at 20 magnifcation using CLSM (Zeiss LSM 510,
Carl Zeiss AG, Oberkochen, Germany), and 20 images were com-
bined to make one whole slice image. In the CLSM images, the TC
labeling area representing the frst half of the observation period was
colored red fuorescence, and the Cal labeling area representing the
latter half of the observation period was colored green fuorescence
(Fig. 4A). To eliminate the background fuorescence and to distin-
guish the native bone from the original image, we made CLSM
extracted images using the computer software program Adobe
Photoshop (Adobe Systems Inc., San Jose, CA, USA, Fig. 4B)
Results
3D micro-CT reconstruction images
At 1-month post-distraction, newly generated bony
granulation tissues were observed and flled rapidly in the
distraction gap of the periosteum-preserved side of mandi-
ble (i.e., left), but not of the periosteum-removal side (i.e.,
right) in two dogs examined (Fig. 5 second column). At
3-month post-consolidation, however, this difference
became less evident and the distraction gaps in both sides
were flled with bony granulation tissues (third column). At
6-month post-consolidation, new bone formation was more
demonstrated and the height of distraction areas reached to
the original level in the left side of mandible in all three
dogs (fourth column bottom). In contrast, such tissue
regeneration features in the right side of mandible were
demonstrated in only one dog (fourth column top). The
remaining two dogs exhibited reduction of height in the
alveolar part of mandible, a sign of external bone resorption
(fourth column middle). These fndings suggest that preser-
vation of periosteum prevents external bone resorption.
CLSM images
The CLSM images were taken at the trans-section of
the central part of resected and basement bones. After com-
pletion of distraction (Fig. 6A), micropillar-like trabecular
bones were formed longitudinally toward the distraction
gap from the basement bone of both right and left sides
(green arrows). In contrast, new bone formation outside of
the resected bone fragments (vectors) was more evident in
the left side of mandible compared with the right side (pur-
ple arrows; Fig. 7A blue triangles versus black arrows; Fig.
7B yellow triangles).
At 1- to 2-month post-distraction (Figs. 6B and 6C),
new trabecular bones in the distraction gap extended from
both the basement bone and the vector and connected to
each other in both left and right sides of mandible (red
arrows). Fig. 6C shows slightly less calcein-incorporated
areas in the right side than the left side of mandible, sug-
gesting periosteum promotes remodeling in the latter half
period. The trabecular bones were apparently thicker, but
irregular in shape (Fig. 7D).
At 1-month post-consolidation (Fig. 6D), the trabecu-
lar bone structure in the distraction gap and the bone
remodeling were clearly demonstrated in both right and left
sides of mandible (see also Fig. 8A). Notably, resorption of
the native bone on the crestal area of the vector was
observed in the right, but not left, side (blue arrows).
Fig. 5. Comparison of 3D micro-CT reconstruction images between periosteum-removal and preservation.
At 6-month post-consolication, in the right (perioseum-removal) side of the mandible, only one case maintained the
alveolar height, and the remaining two cases exhibited reduction of the alveolar height, a sign of external bone resorption
(white arrows).
Osteogenic Alveolar Distraction
71
Fig. 6. Comparison of CLSM images between periosteum-removal and preservation.
A. Completion of distraction. B. 1-month post-distraction. C. 2-month post-distraction. D. 1-month post-consolida-
tion. E. One out of three cases at 6-month post-consolidation. F. The remaining two cases at 6-month post-consolida-
tion. R. Right (periosteum-removal side). L. Left (periosteum-preserved side).
At 6-month post-consolidation, one case displayed the well corporation of TC and Cal (E). However, in the remaining
two cases Cal labeling was reduced in the right side of mandible (F). These fndings are consistent with the 3D micro-
CT (Fig. 5) suggesting a protective role of periosteum from external bone resorption.
S. Takeuchi et al.
72
Fig. 7. High magnifcation images of CLSM during the new bone formation period.
A-C. The distraction gap at the completion of distraction. A. Outer border of the vector in the periosteum-preserved
side. The blue triangles indicate actively formed new bone along with the outer border of the vector. B. Outer border of
the vector in the periosteum-removal side. The yellow triangles indicate the outer border of the vector which has little
new bone formed. NB. Native bone. NEW. New bone. C. The distraction gap in the periosteum-removal side. The
green triangles indicate the top of the new trabecular bone which has not yet connected to the vector side. D. The dis-
traction gap at 1-month post-distraction. The trabecular bone had become thicker, but the structure was still irregular.
Fig. 8. High magnifcation images of CLSM during the bone remodeling period.
A. The distraction gap at 1-month post-consolidation. The trabecular bone structure was clearly seen. B. Comparison
of the vector between the periosteum-removal and -preserved sides at 1-month post-consolidation. Osteons at the top of
the alveolar crest were sharply cut by bone resorption from outside in the periosteum-removal side (The same fndings as
Fig. 6D).
Osteogenic Alveolar Distraction
73
Higher magnifcation images revealed that bone remodeling
in the osteons of native bone was developed in both right
and left sides of vectors (Fig. 8B green arrows). However
the labeled osteons in the native bone were sharply cut by
subsequent bone resorption from outside in the periosteum-
removal side (yellow arrow), and new bone formed in the
inner layer of the vector had already appeared on the out-
side (blue arrow).
At 6-month post-consolidation (Figs. 6E and 6F), both
right and left side of mandible in one dog displayed the well
incorporation of both tetracycline and calcein in the dis-
tracted gap and the vector (Fig. 6E). In the remaining two
dogs, however, such incorporated areas were reduced in the
right side of mandible compared with the left side (Fig. 6F).
These fndings are thus consistent with the reconstruction
images of 3D micro-CT (see Fig. 5) and suggest a protec-
tive role of periosteum from external bone resorption during
the vertical distraction osteogenesis in mandibular recon-
struction.
Bone morphometrical study
Average trabecular BV index was 28.4% and 17.8% in
the left and right side of mandible, respectively, before dis-
traction (controls, n = 2). Trabecular BV indexes of the
mesial and distal parts of the distraction gaps were 23.8%
and 21.9% in the left side and 13.1% and 20.4% in the right
side of mandible, respectively, at 6-month post-consolida-
tion (n = 3). Fig. 9 shows the changes in BV index of the
cancellous bone during mandibular distraction. BV index
of the mesial part of distraction gap was increased during
the consolidation period, but gradually decreased to approx-
imate that of the cancellous bone before surgery. On the
distal part, BV index of the distraction gap was very low,
and it was restored after the consolidation period. BV index
of the distraction gap was restored to approximate that of
the native bone at 6-month post-consolidation on both mesi-
al and distal parts, but it was delayed in the periosteum-
removal side compared with preserved side (Fig. 9).
Discussion
Since Ilizarov (1989) began using DO in limb length-
ening, it has been a procedure developed to reconstruct and
lengthen the long bones. Since McCarthy et al. (1992) clin-
ically applied this technique to correct craniofacial deformi-
ties, it has also been applied to repair segmental bone
defects in the craniofacial bone after tumor resection and
trauma. This technique has the advantage of initiating new
bone growth without bone transplantation and promoting
the growth of soft tissues (Nosaka et al. 2000). Costantino
et al. (1990) frst applied the horizontal distraction to the
mandible in a dog model, which was followed by animal
studies (Block et al. 1996) and clinical application (Chin
and Toth 1996) of vertical distraction. Several clinical
reports point out that long-term reduction of the alveolar
height is one of the main problems of this procedure, in par-
ticular post traumatic atrophic alveolar ridge (Kanno et al.
2006). In addition, there are other serious problems when
DO is performed after resection of a malignant tumor. The
surrounding soft tissues including the periosteum are usual-
ly completely removed in mandible resection to prevent the
recurrence of the tumor, and the soft tissue of the resected
part is often reconstructed with free faps or irradiated,
resulting in scars or ischemia (Nocini et al. 2004; Shao et al.
2006).
Although Ilizarov (1989) confrmed that the critical
factor of new bone formation was the preservation of the
bone marrow for limb lengthening, the effect of the perios-
teum was not clear in mandibular distraction. The most
interesting fnding of our study was that at 6-month post-
consolidation the height of the distraction area was almost
preserved in the left side, but reduced in two out of three
cases in the right (periosteum-removal) side of the mandi-
ble. What causes this difference and what role does the
periosteum play in this phenomenon? Basically, there are
two main sources of blood supply to the normal mandible:
the inferior alveolar artery and the vessels perforating the
periosteum (Miao et al. 1997). According to Elshahat et al.
(2004) and Gosain et al. (2004), the periosteum is not as
essential as the bone marrow. To the contrary, Costantino et
Fig. 9. Changes of the bone index (BV/TV) between perioste-
um removal and preservation.
A. The periosteum-removal side. B. The periosteum
preserved side.
Distraction gap (mesial).
Distraction gap (distal).
The arrow indicates the level of BV/TV before distrac-
tion. New bone formation was delayed in the periosteum-
removal side.
S. Takeuchi et al.
74
al. (1990) stressed the importance of the periosteum for
mandibular lengthening. Zimmermann et al. (2005) wrote
that in distraction of rat and rabbit long bones and sheep
mandible, new bone formation was predominantly seen in
the sub-periosteal region in every case. Cope and
Samchukov (2000) reported that in osteogenesis in alveolar
distraction, either from the bone marrow or the periosteum,
the modeling period continued almost for the initial 2
months, and after that consolidation period, secondary
remodeling began. We observed the same fndings on our
micro-CT and CLSM images in the periosteum-preserved
side. However, in the periosteum-removal side, although
remodeling of osteons inside the cortical bone was main-
tained, no bone formation but only resorption from the out-
side progressed after the consolidation period. Moreover,
there was little Cal labeling at the distraction gap and the
vector in two out of three cases exhibited reduction of
height in the alveolar part of mandible in the periosteum-
removal side at 6-month post-consolidation. This fnding
indicated that when periosteum was removed bone remodel-
ing was sparse during the latter half period. On the contrary,
in a case in which the alveolar height was maintained, both
TC and Cal were labeled in both right and left sides in all
periods. Morphometrical study confrmed the bone struc-
tures quantitatively (Parftt et al. 1987). Three-dimensional
observation is possible without destruction of the specimen
using micro CT (Odgaard 1997). Some studies have adopt-
ed two-dimensional morphometrical parameters for DO
(Zimmermann et al. 2005; Amir et al. 2006), but it has not
been confrmed three-dimensionally. Our morphometrical
observations that remodeling is delayed without periosteum
are in line with the CLSM fndings that bone remodeling
was sparse during the latter half period. To summarize our
results, only inner remodeling (remodeling in the distraction
gap or in the osteon) was continuing in the periosteum-
removal side, whereas inner and outer remodeling (remod-
eling on the outer bone surface along the periosteum) was
continuing normally in the periosteum-preserved side. In
addition, active inner remodeling maintained the bone
height even without periosteum, the condition of which is,
however, unpredictable.
If the total amount of bone formation is higher than
that of resorption during the bone remodeling period, exter-
nal bone resorption might be protected even without perios-
teum. Several methods are considered to compensate the
external bone resorption to obtain prospective vertical DO.
Firstly, excessive elongation (overcorrection) is an actual
solution as many reports point out (Saulacic et al. 2005;
Kanno et al. 2006; Ettl et al. 2009). Secondly, the distrac-
tion rate is an important condition, and it has been indicated
that frequent distraction was more effective for good bone
formation (Zimmerman et al. 2005; Amir et al. 2006).
Thirdly, combination of growth factors may be useful. Hu
et al. (2003) suggested that angiogenic factors like VEGF
and bFGF may be effective as therapeutic agents. Weight
bearing may also protect against resorption, because func-
tional pressure promotes the bone remodeling (Chiba 1976).
Many reports point out that implant placement protects
against bone resorption of the alveolar crest on the grafted
(Chiapasco et al. 2000) or distracted (Nosaka et al. 2000)
mandible. According to Radomisli et al. (2001), weight
bearing alerts the expression of collagen I and II, BMP, and
osteocalcin to affect early healing in DO.
Some limitations exist in our study. Firstly, bone turn-
over is different according to species, and the results of the
present study are limited to dogs. We selected the dog in
this study because its bone turnover is similar to that of
humans (Dannucci et al. 1987). Secondly, using young ani-
mals is also a limitation, because most malignant tumors
occur in elderly people. Therefore, we should perform the
same study in older animals in the future. Thirdly, quantita-
tive evaluation was performed in this study but statistical
evaluation was not performed because of the small number
of cases.
In conclusion, the preservation of periosteum prevents
the external bone resorption and maintains the augmented
bone height in vertical DO of mandible.
Acknowledgments
We greatly appreciate the precious advice and editing of Dr.
Hiromi Kubagawa, Professor, Division of Laboratory Medicine,
Department of Pathology, University of Alabama at Birmingham.
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