ORIGINAL ARTICLE

Functional appliance therapy accelerates and

enhances condylar growth
A. B. M. Rabie, MS, CertOrtho, PhD,a T. T. She, BDS (Hons),b and Urban Hägg, DDS, Odont Drc
Hong Kong SAR, China

The present study was designed to quantitatively assess the temporal pattern of expression of Sox 9, the
regulator of chondrocyte differentiation and type II collagen, the major component of the cartilage matrix
during forward mandibular positioning, and compare it with the expression during natural growth. Female
Sprague-Dawley rats, 5 weeks old, were used. Results showed that the expression of Sox 9 and type II
collagen are accelerated and enhanced when the mandible is positioned forward. Furthermore, we monitored
the amount of new bone formation during mandibular advancement and after the removal of bite-jumping
appliances. A substantial increase was observed in the amount of newly formed bone when the mandible
was positioned forward. No significant difference in new bone formation could be found after the appliance
was removed when compared with natural growth. Thus, functional appliance therapy accelerates and
enhances condylar growth by accelerating the differentiation of mesenchymal cells into chondrocytes,
leading to an earlier formation and increase in amount of cartilage matrix. This enhancement of growth did
not result in a subsequent pattern of subnormal growth for most of the growth period; this indicates that
functional appliance therapy can truly enhance condylar growth. (Am J Orthod Dentofacial Orthop 2003;123:
40-8)

condylar growth.19 In the developing condyles, mesen-

B
iomechanical force produced by forward man-
dibular positioning solicit cellular and molecu-
lar changes in the mandibular condyles.1-4
However, the effect of functional appliances on condy-
lar growth remains a controversial issue,5-9 and the
mechanisms by which those changes are triggered are
not completely understood. Several studies have re-
ported a positive response of the condyle to mandibular
advancement.7,10-12 Some researchers believe that this
positive response is actual growth of the mandible,13-18
while others believe that functional appliances only
accelerate growth of the mandible, helping it to reach
its final size earlier, but not producing a larger size
overall.6 This controversy continues because of the
apparent lack of tissue markers to distinguish between
the 2 processes—acceleration of growth and actual
growth.
Recently, we identified some cellular and molecular
events that are responsible for key processes governing
From the Department of Orthodontics, Faculty of Dentistry, The University of
Hong Kong.
a
Associate professor.
b
Master student.
c
Chair professor.
Reprint requests to: Dr A. B. M. Rabie, Orthodontics, Faculty of Dentistry, The
University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road,
Hong Kong SAR, China; e-mail, rabie@hkusua.hku.hk.
Submitted, January 2002; revised and accepted, March 2002.
Copyright © 2003 by the American Association of Orthodontists.
0889-5406/2003/$30.00 ⫹ 0
doi:10.1067/mod.2003.45
40
chymal cells present in the proliferative layer differen-
tiate into chondrocytes. The process of differentiation is
regulated by Sox 9 transcription factor.20 Sox 9 is a
high-mobility-group–type transcription factor that con-
trols the differentiation of mesenchymal cells into
chrondrocytes by directly activating the gene expres-
sion for type II collagen.21 It is regulated by several
factors such as fibroblast growth factors, tumour necro-
sis factor-␣, and bone morphogenetic protein-2
(BMP-2) that play important roles in craniofacial de-
velopment and bone formation.22-25 Type II collagen
that is synthesized by chondrocytes is the main type of
collagen that forms the framework of the cartilage
matrix in the growing condyle.26-28 After cartilage
matrix formation, chondrocytes mature and hypertro-
phy. Hypertrophic chondrocytes secrete type X colla-
gen, which marks the onset of endochondral ossifica-
tion and the replacement of the hypertrophic cartilage
matrix with bone.12 This detailed exposition of the
cellular and molecular events participating in bone
formation in the condyle gave us the baseline data
against which changes in condylar growth in response
to functional appliance therapy could be compared. If
the effect of functional appliance therapy on condylar
growth is acceleration, then the expression of factors
regulating key processes in condylar growth should be
accelerated when compared with their expression dur-
ing natural growth. If the effect of functional appliance
American Journal of Orthodontics and Dentofacial Orthopedics
Volume 123, Number 1
therapy is actual growth, then the cartilage matrix and
the amount of bone formed during mandibular advance-
ment should be more than the amount of bone formed
during the same period of natural growth. Furthermore,
a true enhancement of growth in response to functional
appliance therapy, ie, an actual increase in growth that
would not have occurred otherwise, relies on maintain-
ing the increase in bone formation in the posttreatment
period until the end of the pubertal growth period.
The general aim of this study is to assess acceler-
ation and enhancement of condylar growth. Therefore,
this study was designed to:
1. Quantify and identify the temporal expression of
Sox 9 transcription factor in the condyles during
forward mandibular positioning and to compare it
with its level of expression during natural growth.
2. Quantify and identify the temporal pattern of ex-
pression of type II collagen and compare it with its
levels during natural growth.
3. Monitor the amount of bone formation in the con-
dyles in the posttreatment period, follow it to the end
of the pubertal growth period, and then compare it
with the amount of bone formed during natural
growth over the same period.
MATERIAL AND METHODS
The animal model used in this study was the same
as that described by Rabie et al.29 Bite-jumping appli-
ances fitted to the maxillary incisors of the animals in
the experimental group were worn 24 hours a day; these
produced 3.5-mm anterior and 3-mm vertical openings
of the mandible. The appliances were cemented with
PANAVIA-F (Kuraray, Osaka, Japan) resin cement.
One hundred sixty five female Sprague-Dawley
rats, 5 weeks old, were used in this study. They were
randomly divided into experimental and control groups,
which were subdivided based on type of staining and
length of experimental or observation period. Each
control group consisted of 5 rats. For the immunostain-
ing of Sox 9 and the registration of new bone forma-
tion, there were 10 rats in each experimental group.
There were 5 rats in each experimental group for the
immunostaining of type II collagen.
All rats were fed a soft diet. For the immunostain-
ing of Sox 9 and type II collagen, the rats in the
experimental groups were killed on days 1, 3, 5, 7, 9,
11, 14, 17, 30, 44, and 60 with their matched controls.
For the registration of new bone formation, the bite-
jumping appliances were removed on day 44 in the
experimental groups. The rats in the experimental
groups and the matched controls were killed on days 7,
30, 44, and 60. The animals killed on day 7 could be
Rabie, She, and Hägg 41
used for both immunostaining and the registration of
new bone formation; thus, there were 11 experimental
and control groups.
The methods of tissue preparation and sectioning
were those used by Rabie et al.29 Sections were cut
through the condyle at the parasagittal plane. The
section from the same number of cut from when the
condyle was exposed was used for the 180 specimens to
standardize the location of the condyle. The best
section of the next 5 cuts was chosen for staining.
The primary antibody for Sox 9 was kindly pro-
vided by Dr Vincent Harley of Prince Henry’s Institute
of Medical Research, Monash, Australia.
After the sections were dewaxed and rehydrated,
antigenic sites were exposed by digestion with protein-
ase K (Sigma P-6556, 10 ␮g/ml), and nonspecific
binding was reduced by treating in 3% H2O2 for 10
minutes followed by incubation in normal goat serum at
37°C (Sigma G9023, 1:9 diluted with 1xTBS).
The sections were then incubated with polyclonal
rabbit-anti-human Sox 9, dilution 1:100, and biotin-
conjugated goat-anti-rabbit IgG (Sigma B-8895), dilu-
tion 1:200, preadsorbed with normal goat serum suc-
cessively each for 1 hour at 37°C. The sections were
washed with PBS between each step.
After sections in the type II collagen groups were
dewaxed, rehydrated, and treated in 3% H2O2 and
proteinase K, they were incubated with normal rabbit
serum (Sigma G9002, 1:9 diluted with 1xTBS), poly-
clonal goat-anti-mouse collagen type II antibody (Sc-
7763, Santa Cruz Biotechnology, Santa Cruz, Calif),
dilution 1:200 and biotin-conjugated rabbit-anti-goat
IgG (Dako 0466), dilution 1:400, preadsorbed with
normal rabbit serum successively each for 1 hour at
37°C.
The sections of both Sox 9 and type II collagen
were developed in 0.05% 3,3-diaminobenzidine (Sigma
D-5637) for 1 minute and counterstained with Mayer
haematoxylin for 3 minutes.
To assess new bone formation, sections were
stained with periodic acid and Schiff’s reagent (PAS)
for identifying new bone formation. Stained with PAS,
the newly formed bone takes on a distinctive magenta
color.
Brown staining that localizes the expression of Sox
9 and type II collagen and the magenta color that
identifies new bone formation in the posterior, middle,
and anterior aspects of the condyles were measured
with a true-color RGB (red-green-blue) computer-as-
sisted image analyzing system (Leica Q550IW, Leica
Microsystems Imaging Solutions Ltd, Cambridge,
United Kingdom) with Leica QWin Pro (version 2.2)
software following the method of Rabie et al.29 This
42 Rabie, She, and Hägg
Fig 1. Photomicrograph showing anterior (A), middle
(M), and posterior (P) regions of mandibular condyle (C),
glenoid fossa (G), and articular disc (D) at 36 days of
natural growth.
system acquires high-definition digital images of the
specimen, and features from the acquired images are
selected by the operator. The amount of positive stain-
ing is recognized and quantified by the computer
software according to the color, shade, and contrast of
the feature selected. Sox 9 expression in the prolifera-
tive and hypertrophic zones, type II collagen expression
in the hypertrophic zone, and new bone formation in
the erosive zones in the posterior, middle, and anterior
regions (Fig 1) were quantified separately under the
fixed measurement frame on ⫻360 magnification
(Leitz Orthoplan, Wetzler, Germany).
The data was processed with SPSS for Windows
(version 10.1, SPSS Inc, Chicago, Ill) for both t test and
ANOVA.
Ten randomly drawn Sox 9, type II collagen, and
newly formed bone localized sections from the 180
specimens were quantified on 2 separate occasions
about 2 months apart to calculate the method error
(ME). ME in measuring the areas of the staining was
calculated by the formula:
ME ⫽ 冑 ⌺d2
2n
where d is the difference between the 2 registrations,
and n is the number of double registrations. Hypothesis
testing indicated no significant difference among the
registrations for Sox 9, type II collagen, and newly
formed bone.
RESULTS
Sox 9 was expressed by the cells in the proliferative
and hypertrophic zones of the mandibular condyle (Figs
2 and 3), while the expression of type II collagen was
found to be localized in the hypertrophic zone only (Fig
2). Newly formed bone was evidenced in the erosive
zones (Fig 2).
In the proliferative zone, forward mandibular posi-
American Journal of Orthodontics and Dentofacial Orthopedics
January 2003
tioning led to an earlier expression of the maximum
levels of Sox 9 on experimental day 5 in all regions
when compared with the maximum levels of expression
during natural growth, which were on day 7 (42 days of
natural growth) at the middle region and day 9 (44 days
of natural growth) at the anterior and posterior regions
(Fig 4).
When the expression was followed until experimen-
tal day 17, Sox 9 maintained a higher level of expres-
sion during forward mandibular positioning than it did
during the natural growth (Fig 4).
There was a statistically significant increase (P ⬍
.01) of 63% in the expression of Sox 9 between natural
growth and forward mandibular displacement on ex-
perimental day 5 in the posterior region.
Furthermore, the posterior region consistently pro-
duced significantly more Sox 9 when compared with
the middle and anterior regions in response to forward
mandibular positioning except on experimental days 1,
11, and 17. On these days, the expression level between
the posterior and middle regions had no statistically
significant difference (Fig 5).
In the posterior region of the hypertrophic zone, the
maximum level of expression of type II collagen was
attained on experimental day 7 (42 days of natural
growth) in response to forward mandibular positioning.
It was 4 days earlier than that of natural growth, which
was on day 46 (experimental day 11) (Fig 6).
Furthermore, there were statistically significant in-
creases (P ⬍ .05) in the expression of type II collagen
between natural growth and forward mandibular dis-
placement on experimental day 7 in both the posterior
and middle regions of 97.8% and 51.6%, respectively.
The anterior region showed no statistically significant
differences.
For the expression of Sox 9, there were no statisti-
cally significant differences between natural growth
and forward mandibular positioning in the anterior and
middle regions. However, in the posterior region, about
a 70% increase of the level of expression that was
statistically significant (P ⬍ .05) had been shown on
experimental day 7 (42 days of natural growth) during
forward mandibular positioning when compared with
natural growth (Fig 7).
Similar to the proliferative cells, during forward
mandibular advancement, the expression of Sox 9 by
the hypertrophic chondrocytes was significantly more
in the posterior than in the middle and anterior regions,
except on experimental day 17 (Fig 5).
The expression of type II collagen during forward
mandibular advancement was also significantly in-
creased in the posterior than in the middle and anterior
regions except on experimental day 3 (Fig 5), when
American Journal of Orthodontics and Dentofacial Orthopedics Rabie, She, and Hägg 43
Volume 123, Number 1

Fig 2. Photomicrographs showing expression of Sox 9 indicated by brown stains in proliferative (P)
and hypertrophic (H) cells. A, Natural growth day 40; B, mandibular forward positioning day 5 (40
days of natural growth). Expression of type II collagen, indicated by brown stains of hypertrophic
cells and in extracellular matrix. C, Natural growth day 42; D, mandibular forward positioning day 7
(42 days of natural growth). New bone (B) formation. E, Natural growth day 95; F, experimental day
60 with forward mandibular positioning 44 days.

there was no statistically significant difference between DISCUSSION

the middle and posterior regions.
During forward mandibular positioning, bone for-
mation was significantly higher (P ⬍ .001) than during
natural growth in the posterior condyle on experimental
day 30 (65 days of natural growth). Before and after
those periods, there was no significant difference in
bone formation in the condyle between natural growth
and forward mandibular positioning (Fig 8).
Removal of bite jumping appliances on experimen-
tal day 44 (79 days of natural growth) resulted in no
significant difference in new bone formation when
measured on experimental day 60 (95 days of natural
growth) compared with that of natural growth (Fig 9).
The current study addressed an important issue in
the field of growth modifications in orthodontics:
whether functional appliance therapy accelerates and
enhances condylar growth. To answer such a complex
question, this study was designed to address a few
aspects. How do we measure acceleration of condylar
growth? How can we put a quantitative measure to such
a process? During condylar growth, undifferentiated
mesenchymal cells proliferate, differentiate into chon-
drocytes, mature, and engage in matrix synthesis, lead-
ing to bone growth in the condyles. Therefore, if
functional appliance therapy affects condylar growth
44 Rabie, She, and Hägg
Fig 3. Photomicrograph in higher magnification show-
ing expression of Sox 9 indicated by brown stains
(arrows) in proliferative (P) and hypertrophic (H) cells
during mandibular forward positioning day 5.
through acceleration, then the progression of the differ-
ent processes leading to growth should be accelerated,
and the expression of factors regulating these processes
should be accelerated as well.
Sox 9 transcription factor is critical in the control of
the differentiation of the mesenchymal cells into chon-
drocytes.20 It is expressed in all cartilage tissues and in
knock-out gene experiments; animals without Sox 9
have defects in long bones and craniofacial areas.30 Sox
9 has been localized to cells in the proliferative layer
and hypertrophic chondrocytes in the developing man-
dibular condyles.3,19 Therefore, identifying the tempo-
ral pattern of expression of Sox 9 transcription factor
should be an excellent marker for monitoring acceler-
ation of growth. During natural growth, Sox 9 reached
a maximum level of expression on experimental day 9,
whereas during forward mandibular positioning, the
maximum level of expression was reached earlier, on
experimental day 5 (Fig 4). The earlier expression of
Sox 9 indicates earlier differentiation of mesenchymal
cells into chondrocytes and earlier entry into the chon-
drogenic route.
To further monitor the progression of chondrocytes
into the chondrogenic route, we followed their engage-
ment in the synthesis of the cartilage matrix during
forward mandibular positioning and natural growth.
We determined the temporal pattern of expression of
type II collagen, the major type of collagen in the
cartilage matrix. During natural growth, the maximum
level of type II collagen expression was reached on
experimental day 11, but the maximum level of type II
collagen was expressed on day 7 in response to forward
mandibular postioning (Fig 6). The results discussed
American Journal of Orthodontics and Dentofacial Orthopedics
January 2003
above showed that the maximum level of cartilage
matrix synthesis is reached 2 days after the maximum
level of expression of Sox 9. Forward mandibular
positioning accelerated both the expression of Sox 9
(experimental day 7) and the cartilage matrix formation
(experimental day 9). Therefore, these results clearly
demonstrate that functional appliance therapy acceler-
ates condylar growth by speeding up the entry and
progression of cells into the process of chondrogenesis.
These results lend partial support to the ideas of several
researchers.6,9,31,32
The second aspect of this study dealt with whether
functional appliance therapy enhances mandibular
growth. De Luca et al33 cultured fetal rat metatarsal
bone in the presence of recombinant human BMP-2 and
showed a concentration-dependent acceleration of lon-
gitudinal bone growth. BMP-2 is characterized by an
ability to induce ectopic cartilage and bone in soft
tissues.34 It was concluded that the rate of longitudinal
bone growth depends primarily on the rate of growth
plate chondrogenesis. Growth plate chondrogenesis is
regulated by BMP-2—a known positive regulator of
Sox 9.35 Similarly, the rate of condylar growth could
depend on the rate of chondrogenesis of the condylar
tissues. Therefore, to determine enhanced chondrogen-
esis, we carried out a quantitative analysis of chondro-
cyte differentiation, by determining the amount of
expressed Sox 9, and cartilage matrix formation, by
determining the expressed amount of type II collagen,
and correlated it to bone formation in the condyles
during mandibular advancement.
In response to forward mandibular positioning, the
expression of Sox 9 increased more than 60% in the
proliferative layer of the posterior region of the con-
dyle, indicating the increase in the differentiation of
mesenchymal cells into chondrocytes, thus possibly
increasing the population of chondrocytes potentially
available for chondrogenesis. Sox 9 also regulates the
expression of type II collagen secreted by chondro-
cytes.21 The expression of Sox 9 by chondrocytes in the
hypertrophic layer in the condyle increased by 70% in
response to mandibular advancement (Fig 6), indicating
an enhanced level of matrix synthesis by these cells. A
recent report36 showed that the lack of expression of
Sox 9 leads to campomelic displasia in humans, a
disease affecting all cartilage-derived skeletal struc-
tures, resulting in bowing and angulation of long bones,
vertebral abnormalities, small thoracic cages, craniofa-
cial defects with macrocephaly, micrognathism, and
cleft palate. This shows the important regulatory role of
Sox 9 in skeletal development. Therefore, the results of
the present study indicate that forward mandibular
positioning leads to enhanced chondrogenesis through
American Journal of Orthodontics and Dentofacial Orthopedics Rabie, She, and Hägg 45
Volume 123, Number 1

Fig 4. Temporal pattern of expression of Sox 9 of proliferative zone of posterior region of condyle
from experimental days 1 to 17 (*P ⬍ .05, **P ⬍ .01, ***P ⬍ .001).

Fig 5. Total area in square millimeters of temporal pattern of expression of Sox 9 of proliferative and
hypertrophic zones, and temporal pattern of expression of type II collagen of anterior, middle, and
posterior regions of condyle during forward mandibular positioning from experimental days 1 to 17.

Fig 6. Temporal pattern of expression of type II collagen of posterior region of condyle during
forward mandibular positioning from experimental days 1 to 17 (*P ⬍ .05).
46 Rabie, She, and Hägg American Journal of Orthodontics and Dentofacial Orthopedics
January 2003

Fig 7. Temporal pattern of expression of Sox 9 of hypertrophic zone of posterior region of condyle
during forward mandibular positioning from experimental days 1 to 17.

Fig 8. New bone formation in posterior region of condyle during natural growth and forward
mandibular positioning from experimental days 7, 30, 44, and 60 (***P ⬍ .001).

Fig 9. New bone formation in posterior region of condyle on experimental day 60 with bite-jumping
appliance removed on day 44.
American Journal of Orthodontics and Dentofacial Orthopedics
Volume 123, Number 1
increases in the expression of Sox 9 transcription
factor.
To further investigate the effect of the increased
expression of Sox 9 on cartilage matrix synthesis in
response to mandibular advancement, we quantitatively
assessed the level of expression of type II collagen, the
major component of cartilage matrix. Forward mandib-
ular positioning led to 98% and 52% increases in the
levels of type II collagen expression in the posterior and
middle regions, respectively (Fig 6), when compared
with matching untreated controls. Therefore, we con-
cluded that forward mandibular positioning enhances
chondrogenesis by enhancing the synthesis of the major
component of cartilage matrix, type II collagen.
We also found a close correlation between the
increase in the cartilage matrix (98%) and the increase
in the amount of bone formed (90%) in response to
forward positioning of the mandible (Figs 6 and 8), thus
lending support to the work of De Luca et al,33 who
showed that longitudinal bone growth primarily de-
pends on chondrogenesis. Furthermore, the design of
this study allowed continuous forward positioning of
the mandible that resembled that of the Herbst appli-
ance used in the clinic. Clinically, it seems that the
reported fixed functional appliance, ie, Herbst appli-
ance, consistently indicated an enhancement of man-
dibular growth during treatment,18,37,38 but the effect
on the mandible with removable functional appliances
remains unclear.39 Therefore, considering these results,
forward mandibular positioning solicits cellular
changes that enhance chondrogenesis and osteogenesis
in the mandibular condyles.
The third aspect of this study dealt with whether
such enhancement of growth was at the expense of
future growth in the posttreatment period or whether
such growth could be maintained at levels equal to or
higher than those expressed during natural growth
when followed to the end of the growth period. There-
fore, we designed the experiment to measure the
amount of bone formed as a result of mandibular
advancement for 44 days and then removed the appli-
ances and monitored the amount of bone formed in the
posttreatment period until day 60 of the experiment.
The animals were 35 days old at the beginning of the
experiment; this is at the decelerating phase of the
pubertal growth spurt, approaching the postpubertal
growth period.40 Therefore, we chose to measure the
amount of new bone formation on day 60 of the
experiment, or day 95 of age, because it is near the end
of the growth period. Mandibular advancement led to
an increase in bone formation on days 7 and 30 of
advancement followed by a decrease on day 44 when
the appliances were removed (Fig 8). The levels of
Rabie, She, and Hägg 47
bone formation after the removal of the appliances on
day 44 and day 60 showed no significant difference
from the amount of bone formed during natural growth
(Fig 9).
Our results support the work of Mills and McCul-
loch,32 whose use of the Twin-block appliance resulted
in an average increase in mandibular length during
active treatment. In the posttreatment period, there was
a slight reduction in the mandibular growth rate, but
most of the increase in mandibular length achieved
during treatment was still evident 3 years later.
However, the current results disagree with those of
Petrovic et al,41 who reported that, at the end of the
growth period, no increase in mandibular length was
observed when the experimental group was compared
with untreated controls. In their experiment, similar to
the current methodology, the mandible was advanced,
the appliance was removed, and growth was then
compared with untreated controls. Mandibular growth
was evaluated by measuring the distance between the
posterior edge of the condyle and the mental foramen
on enlarged photographs and radiographs. Using the
image analyzer with the computerized software in the
present study is a more accurate method to determine
the amount of newly formed bone. Therefore, we
believe that forward mandibular positioning leads to
acceleration and enhancement of condylar growth. The
issue of sustaining the amount of growth should be
followed for a longer time so that we can draw a solid
conclusion. Currently, we are following the growth rate
to the end of the growth period on day 121.
CONCLUSIONS
Forward mandibular positioning accelerates and
enhances chondrocyte differentiation and cartilage ma-
trix formation in the mandibular condyle by accelerat-
ing and enhancing the expression of Sox 9 and type II
collagen. This enhancement of growth did not result in
a subsequent pattern of subnormal growth for most of
the growth period, indicating that functional appliance
therapy could induce true enhancement of condylar
growth.
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