Eur J Oral Sci 2002; 110: 144–148 Copyright # Eur J Oral Sci 2002

Printed in UK. All rights reserved European Journal of

Oral Sciences
ISSN 0909-8836

Arja Muhonen1, Timo Peltomäki2,5,

Osteoblastic activity of the rabbit Juhani Knuuti3, Olli Raitakari3,4,
Risto-Pekka Happonen1,5
temporomandibular joint during 1
Department of Oral and Maxillofacial Surgery,
University of Turku, Turku, Finland,

distraction osteogenesis assessed by

2
Department of Oral Development and
Orthodontics, University of Turku, Turku,
Finland, 3Turku PET Center, University of
[18F]fluoride positron emission Turku/ Turku University Central Hospital, Turku,
Finland, 4Department of Clinical Physiology,
Turku University Central Hospital, Turku,
tomography Finland, 5Department of Oral Diseases, Turku
University Central Hospital, Turku, Finland

Muhonen A, Peltomäki T, Knuuti J, Raitakari O, Happonen R-P. Osteoblastic

activity of the temporomandibular joint during distraction osteogenesis
assessed by [18F]fluoride positron emission tomography. Eur J Oral Sci 2002;
110: 144–148. # Eur J Oral Sci, 2002

The purpose of the study was to evaluate the effects of irradiation and hyperbaric
oxygenation (HBO) on osteoblastic activity of the temporomandibular joint
(TMJ) region during mandibular distraction osteogenesis. Unilateral distraction
was performed on 19 rabbits, which were divided into five groups. One group
served as a control group, while the others received either high- or low-dose Prof Risto-Pekka Happonen, Department of
irradiation in the TMJ region before surgery. Some of the animals were also given Oral and Maxillofacial Surgery, University of
HBO 18 times at 2.5 ATAr90 min preoperatively. Osteogenesis was assessed Turku, Lemminkäisenkatu 2, 20520 Turku,
by [18F]fluoride positron emission tomography at the end of the distraction. Finland
Osteoblastic activity was higher on the distracted side in all groups, except in the
Telefax: +358–2–2338248
high-dose irradiated group without preceding HBO. HBO increased osteogenesis E-mail: rhappone@utu.fi
on both sides after radiotherapy. It is concluded that increased osteoblastic
activity reflects increased pressure on the TMJ region of the distracted side, Key words: distraction osteogenesis;
resulting from lengthening. It seems that more remodeling is required after mandibular condyle; fluoride; positron
emission tomography; rabbit
irradiation than without preceding radiotherapy. After radiotherapy, HBO
increased osteoblastic activity. Accepted for publication January 2002

Ever since the first successful clinical cases of mandibular
distraction osteogenesis (DO) were reported (1, 2), it has
been speculated whether continuity defects resulting from
tumor resection could be treated by means of bone
lengthening. However, the most common oral malignant
tumor, squamous cell carcinoma, is usually treated with a
combination of surgery and irradiation, and it is well
known that the osteogenic capacity is reduced after
radiotherapy (3, 4). Lack of knowledge of the effects
of previous irradiation on DO has so far limited the
use of bone lengthening as a reconstructive method in
patients treated for oral malignancies. GANTOUS et al. (5)
performed bifocal mandibular DO on previously irra-
diated canine mandible and reported new bone formation
even after 50 Gy irradiation.
Our recent experimental studies have shown that DO
is feasible in previously irradiated mandibles, and that
hyperbaric oxygenation (HBO) improves bone formation
(unpublished). On the other hand, unilateral mandibular
DO was found to result in deformation and enlargement
of the irradiated condylar heads (unpublished). In
addition, severe condylar cartilage changes even after
low doses of irradiation have been noted, especially on
the condyles of the operated side.
HBO increases osteogenic capacity and is therefore
used in several hypoxic conditions, such as in the treat-
ment and prevention of osteoradionecrosis (6 –9). HBO
does not, however, seem to be able to prevent the damage
to condylar cartilage resulting from mandibular DO and
irradiation.
Positron emission tomography (PET) is a non-invasive
method used to assess tissue metabolism in vivo, and
it allows quantitative image analysis. For imagings,
positron-emitting radionuclides are used and as positron
unites with an electron, a two gamma quanta energy
annihilation (511 keV) is emitted in opposite directions.
This annihilation energy is measured by paired, opposing
detectors.
Fluorine-18 labeled fluoride [18F] is a tracer used to
define bone formation in PET imaging. [18F]fluoride
is a bone-seeking agent that is extracted from blood
circulation by bone, in proportion to blood flow and
osteoblastic activity. It is incorporated only during the
bone formation phase. The half-life of [18F]fluoride
 Osteoblastic activity in TMJ due to distraction 145

is 110 min, and it enters bone from the circulation by

passive diffusion. Due to their small size, fluoride ions
can easily cross the capillary membrane, and the first-pass
extraction fraction of 18Fx is nearly 100% (10).
The present study was carried out to evaluate bone
formation activity in the temporomandibular joint (TMJ)
region during DO. Previous findings have shown that
after radiotherapy, unilateral DO results in alterations in
the shape of the condylar head, and severe histological
changes are seen especially on the mandibular condyle of
the lengthened side. The purpose was to evaluate the
effect of low- and high-dose irradiation and adjunctive
HBO on osteogenesis of the TMJ region during DO.
Osteoblastic activity of the TMJ region was assessed by
[18F]fluoride ion PET.

Material and methods

The Ethical Committee of the University of Turku and the
Provincial State Office of South-Western Finland approved
the study protocol. Female adult Elco rabbits (Harlan, the
Netherlands) (n=19) weighing 3500– 4000 g were used as
study subjects. There were five study groups (1): low-dose
irradiation, R1 (n=3) (2); high-dose irradiation, R2 (n=3)
(3); low-dose irradiation combined with HBO, R1-HO
(n=3) (4); high-dose irradiation combined with HBO,
R2-HO (n=5); and (5) a control group, C (n=5).
The irradiated groups received 4 MV external beam
irradiation (Clinic 4/100; Varian Medical Systems, Palo
Alto, CA, USA) bilaterally with a midplane dose of 22.4 Gy
in four 5.6 Gy fractions, which is estimated to be biologically
equivalent to 50 Gy in 25 fractions. During sessions, the
animals were under fentanyl-fluanisone sedation (Hypnorm
0.3 mL; Jansson-Cilag, Saunderton, UK), and a custom- Fig. 1. Irradiation fields used in the low-dose (A) and high-dose
made head holder was used for positioning and immobiliza- (B) irradiation groups. In the low-dose irradiation group, the
tion. In groups R1 and R1-HO, the TMJ regions were TMJ region is outside the irradiation field, whereas in the high-
shielded and received scattered irradiation of 2.2 Gy, dose group TMJs are within the radiation field. The radiation
whereas in groups R2 and R2-HO the condylar area received field is within the white rectangular area and the yellow
the total dose of irradiation (Fig. 1). shadowed area shows the lead-shielded field. Violet color
The animals in groups R1-HO and R2-HO were also given indicates TMJ regions.
hyperbaric oxygen (Hypcom monoplace chamber, Hypcom,
Tampere, Finland) preoperatively 18 times once a day at
2.5 ATA for 120 min including 15 min compression and
decompression.
The rabbits in group C were surgically treated similarly to
the animals in the other groups.

Distraction osteogenesis
Distraction surgery was done one month after the comple-
tion of radiotherapy. The mandibular bone was exposed
extraorally under general anesthesia (fentanyl-fluanisone
0.3 mL kgx1 i.m.; Hypnorm, Jansson–Cilag and midazolam
2 mg kgx1 i.m.; Dormicum, Roche, Basel, Switzerland). A
unilateral corticotomy was made with a fissured bur anterior
to the first premolar tooth, and an external custom-made
distractor was put in place with four transcutaneous,
bicortical self-tapping implants (Orthofix, Bussolengo,
Italy). An artificial fracture was made, the distractor was
fixed and the wound was closed in layers (Fig. 2). Antibiotics
and analgesics (Procaine Penicillin G 3000 I.U./kg i.m.;
Biochemie, Vienna and buprenorfine 0.01 mg kgx1 s.c.;
Temgesic, Reckitt & Colman, Hull, England) were given Fig. 2. Schematic picture of the distraction osteotomy and the
postoperatively for 4 d. distractor in place.
146 Muhonen et al.
After a 1 wk latency period, bone distraction was started
at a rate of 1 mm dayx1. Distraction was continued for
2 week, after which the PET imaging was performed. During
the distraction period, the animals were fed normal pellet
food and water ad libitum.
PET imaging
The imaging was obtained at the end of the distraction
period (Fig. 3). [18F]fluoride was produced at the Turku
PET Center by the 18O (p,n)18F reaction with a MGC-20
cyclotron. Scanning was performed with an Advance
Whole-body PET-scanner (General Electric Medical
Systems, Milwaukee, WI, USA), which acquires 35 con-
tiguous slices with an axial field of view of 15.2 cm. Slice
thickness is 4.25 mm, and the spatial resolution at FWHM
in the center of the field of view is 5 mm. All scans were
performed in 2D mode.
One pilot rabbit underwent a 60-min dynamic emission
scan starting after a 100 MBq intra-arterial injection of
[18F]fluoride. Forty min postinjection, the tracer reached the
steady state phase, and the protocol for the PET imaging
was designed according to this observed pattern of tracer
uptake. An activity of approximately 100 MBq (range
79–130 MBq) of [18F]fluoride was injected into the left ear
artery, flushed with saline, and a dynamic acquisition con-
sisting of 4r5 min frames and lasting for 20 min was started
40 min postinjection. A 10-min transmission scan for
attenuation correction was obtained after the emission
imaging using two rod sources containing germanium-68.
The rabbits were immobilized and positioned using a head
holder, and during the scanning the animals were under
sedation.
Quantitative analyzes
Data were corrected for dead time, decay and photon
attenuation, and the images were reconstructed coronally in
a 128r128 matrix employing a Hann filter with a cut-off of
4.6 mm. All frames were used to define standardized circular
regions of interest (ROIs, r=2.3 mm). One rabbit in group
C moved during the PET imaging. The scanning was
restarted, and the two last frames (57–67 min postinjection)
were used for the analyzes. ROIs were defined on the TMJ
region showing the highest osteoblastic activity. ROIs were
Fig. 3. Sagittal reconstruction of PET image at the level of the
TMJ region in a low-dose irradiated rabbit. The dark hot spot
area shows the increased activity in the TMJ region of the
distracted side. The dotted line illustrates the profile of a rabbit
skull.
also defined for the corresponding area on the preceding or
following slice, and the image having the higher activity was
chosen for the analyzes. The mean value of ROIs of these
two images was used in the analyzes. The activity was
reported as standardized uptake value (SUV), which is the
total activity in a ROI (kBq/mL) divided by the activity of
the injected dose (kBq) divided by weight (g) (11).
Statistical methods
The comparisons between multiple groups to evaluate
the effects of irradiation and HBO, and to assess the
differences in osteoblastic activity were made using analyzes
of variance (procedure Mixed in Statistical Analysis
System, SAS Institute, Cary, NC, USA). Descriptive data
are presented as meantSD, and significance inferred at
2-tailed P<0.05.
Results
The animals recovered well from radiotherapy, and there
were no problems associated with hyperbaric oxygena-
tion. Throughout the distraction period, the animals in
group C were noticeably healthier than the irradiated
animals. The irradiated rabbits recovered from surgery
better if they had received preoperative HBO. Unilateral
distraction resulted in dental crossbite, and the incisors
had to be shortened regularly as they started to over-
erupt. At the end of the distraction, the length of the
distraction generate was 14 mm.
At the end of distraction, the osteoblastic activity
was higher in the TMJ region of the distracted side
when compared to the non-distracted side (main effect
for side in ANOVA, P=0.0018) (Table 1). This effect of
distraction was evident in all other groups except in the
high-dose irradiation group (R2).
Osteoblastic activity in the TMJ region of the
distracted side was inversely related to the irradiation
dose. With higher doses, lower osteoblastic activity was
observed both with and without HBO (main effect for
irradiation in ANOVA, P=0.014).
Table 1
Osteoblastic activity reported as SUVs on the distracted (d) and
non-distracted (n-d) sides at the end of the distraction period in
control group (C), low-dose irradiation (R1), high-dose
irradiation (R2), low-dose irradiation+HBO (R1-HO), and
high-dose irradiation + HBO (R2-HO) groups
Group SUV d (n) SUV n-d (n)
C 7.5t3.88 (5) 6.4t2.17 (5)
R1 9.3t0.51 (3) 4.6t0.51 (3)
R2 3.9t1.77 (3) 4.5t1.90 (3)
R1-HO 16.6t3.56 (3) 7.2t2.60 (3)
R2-HO 7.8t3.99 (5) 6.1t2.95 (5)
ANOVA main effects: effect of side, p=0.0018; effect of
irradiation, P=0.014; effect of HBO, P=0.0055.
 Osteoblastic activity in TMJ due to distraction
The highest osteoblastic activity was seen in the
distracted side in the group receiving HBO treatment
and low-dose irradiation. Also in the group with HBO
and high-dose irradiation, the osteoblastic activity was
comparable to the control group (main effect for HBO
in ANOVA, P=0.0055).
Discussion
[18F]fluoride-PET has proved to be an appropriate means
to study bone formation in vivo. [18F]fluoride-PET has
been used to assess the incorporation and healing of
various bone grafts used for mandibular reconstruction
(12, 13), and to investigate bone formation in metabolic
bone disorders (14, 15). In addition, whole-body
[18F]fluoride-PET has been used to investigate normal
skeletal uptake of [18F]fluoride and uptake of malignant
and benign lesions (16). To our knowledge, PET has not
been used to evaluate bone formation of the TMJ region
before. Although the imaged structures were small, the
TMJ regions were clearly identifiable. Analysis of the
whole series of contiguous PET images produced form
the head of each rabbit gave a definite picture of the bony
anatomical structures including TMJ thus enabling
proper location of the area to be measured.
The present study helps us to understand previous
morphological and histological findings. It has been
reported that increased pressure promotes bone forma-
tion and results in enlargement of a mandibular condyle
(17, 18). High osteoblastic activity on the distracted side
in rabbits in all but the high-dose irradiation group
indicates that unilateral DO creates greater pressure on
the TMJ region of the distracted than of the non-
distracted side. This is in accordance with earlier
histological and morphological findings on the effects
of DO on TMJ (19, 20). Condylar enlargement, and thus
increased osteoblastic activity, can also result from
mechanical loading caused by occlusal trauma (21). In
our study, change in occlusion resulting in crossbite
was probably also partially responsible for the bony
remodeling of the TMJ region.
In the non-irradiated control animals, the osteoblastic
activity seemed to be only slightly elevated on the
distracted side when compared to the non-distracted side.
This indicates that, despite increased loading on the
condyle during DO, healthy condylar cartilage is capable
of gradual cartilaginous adaptation, and therefore no
remarkable osseous remodeling is needed. This is in line
with histological findings that DO affects the condylar
cartilage in a reversible manner (19, 20).
Despite the limited number of experimental animals,
the effects of irradiation and HBO on osteoblastic
activity in TMJ regions seem obvious. Low dose
irradiation was associated with increased osteogenesis
on the distracted side, whereas decreased osteogenesis
was seen with high-dose irradiation. It may be assumed
that, due to previous radiotherapy and resulting cartila-
ginous damage following distraction, more osseous
remodeling and repair is required than without preceding
irradiation. Possibly, the destroyed condylar cartilage
147
layer on the distracted side is replaced by bone, and this
replacement process is seen as increased osteoblastic
activity in the TMJ region. In the high-dose irradiation
groups, the difference in osteoblastic activity between the
lengthened and non-lengthened side was less than in the
low-irradiation groups. Without HBO, the activity was
even lower on the distracted than on the non-distracted
side. An explanation for this could be that, although the
condylar damage on the distracted side was severe and
bony repair and remodeling would be needed, the
osteoblastic function would be impaired due to high-
dose radiotherapy. Therefore, new bone formation was
noticeably defective. Also in the high-dose irradiation
groups, the osteoblastic activity was increased if HBO
was used, but to a lesser extent than in the low-dose
irradiation animals. These findings indicate that previous
radiotherapy suppresses osteoblastic activity dose-
dependently, and HBO improves osteogenic capacity.
Although, histologically, HBO has not been shown to
have a beneficial effect on the condylar cartilage of the
irradiated and distracted mandible, it seems to improve
osteogenesis. In fact, on the basis of its known effects,
HBO can be expected to increase the osteogenic capacity
rather than chondroid adaptability.
In conclusion, at the end of the unilateral mandibular
distraction osteogenesis, osteoblastic activity was found
to be elevated on the distracted side compared to the non-
distracted side in all animals, except the rabbits in the
high-dose irradiation group. This elevation is presumably
partly a response to increased pressure in the TMJ region
due to distraction. After low-dose radiotherapy, osteo-
blastic activity was further increased on the distracted
side. This can be explained as a result of the reparative
process occurring in the condylar process with advanced
cartilage damage. Following HBO treatment in the low-
dose radiotherapy animals, osteoblastic activity on the
operated side reached the highest values in the experi-
ment. Probably, HBO further potentiated the healing
capacity of the bone. In the high-dose irradiation group
with HBO, a smaller increase in the osteoblastic activity
was found due to greater suppression of bone healing
capacity. Increased pressure caused by distraction,
reparative need in the condylar process due to cartilage
damage, and altered occlusion probably all had an effect
on the osteoblastic activity.
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