ORIGINAL ARTICLE

Mastication markedly affects mandibular

condylar cartilage growth, gene expression,
and morphology
Akiko Enomoto,a Junichi Watahiki,b Tomoki Nampo,a Tarou Irie,c Yuuta Ichikawa,d Tetsuhiko Tachikawa,e
and Koutaro Makif
Tokyo, Japan

Introduction: Mandibular growth is believed to be strongly related to mastication. Furthermore, mandibular

condylar cartilage is known to be derived from neural crest cells. We examined whether the degree of chewing
affects condylar cartilage growth of the mandible. Methods: Mice were fed diets with varying hardness. Genes
specific to neural crest–derived cells were measured by real-time polymerase chain reaction to compare the
expression changes between the mandibular and tibia cartilages. The mandibular condylar cartilage was then
evaluated histologically, and proliferation was evaluated using proliferating cell nuclear antigen.
Immunostaining was conducted for osteopontin, type X collagen, and Musashi1, and real-time polymerase
chain reaction was used to assess the expression levels of osteopontin and type X collagen. Results: Markers
including P75, Wnt-1, Musashi1, and Nestin were upregulated in the mandibular condylar cartilage as compared
with the tibial cartilage. Histologic assessment of the mandibular cartilage showed that the hypertrophic
chondrocyte zone was statistically significantly thicker in mice fed a hard diet. Chondrocyte proliferation and
Musashi1 expression were lower in mice fed a hard diet. After 4 weeks, numerous osteopontin and type X
collagen-positive cells were observed in mice fed a mixed diet. Conclusions: Mastication affects the balance
between differentiation and proliferation in the mandibular condylar cartilage. This phenomenon might be attrib-
uted to the presence of neural crest–derived cells. (Am J Orthod Dentofacial Orthop 2014;146:355-63)

E
ndochondral ossification of the mandibular
condylar cartilage is thought to occur at the
growth center site of the mandible,1-3 and the
mandibular condyle has a high ability to adapt. It is
considered that mandibular condylar cartilage is more
sensitive to changes in mechanical conditions than
other types of cartilage,4 and that muscular function
around the jaw has a marked influence on maxillofacial
growth.5,6 The mandibular cartilage is thought to consist
From the School of Dentistry, Showa University, Tokyo, Japan.
a
Postgraduate researcher, Department of Orthodontics.
b
Lecturer, Department of Orthodontics.
c
Lecturer, Dapartment of Oral Pathology.
d
Assistant lecturer, Department of Orthodontics.
e
Professor, Department of Oral Pathology.
f
Professor, Department of Orthodontics.
All authors have completed and submitted the ICMJE Form for Disclosure of
Potential Conflicts of Interest, and none were reported.
Supported by JSPS KAKENHI (23792444).
Address correspondence to: Junichi Watahiki, Department of Orthodontics,
School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta-ku, Tokyo
145-8515, Japan; e-mail, junwata2000@ybb.ne.jp.
Submitted, August 2013; revised and accepted, May 2014.
0889-5406/$36.00
Copyright Ó 2014 by the American Association of Orthodontists.
http://dx.doi.org/10.1016/j.ajodo.2014.05.028
of cells originating from the neural crest that are
multipotential with a high proliferative potency.7 How-
ever, it is unknown whether these characteristics are
observed in vivo.
We previously reported that changes in mastication
markedly influenced the expression of several genes.8 Us-
ing laser microdissection, along with cDNA array and real-
time polymerase chain reaction (PCR) techniques, we
showed that it was possible to accurately quantify gene
expression levels in vivo by selectively obtaining cells
from the mandibular condylar cartilage, and identified
the expression levels of several genes that were signifi-
cantly altered between the lactation and mastication pe-
riods. Therefore, we hypothesized that mastication would
influence condylar cartilage responses and subsequent
growth of the mandible. It is known that maxillofacial
structures change with different diets, with the ramus
height reported to be higher in rats fed a variable diet.6
The mandibular condylar cartilage also undergoes
morphologic changes with different diets, since it was
found to be thicker9 and highly proliferative10 in rats
fed a soft diet. Altered functional loading caused a
decrease in the expression of chondrocyte differentiation
markers and a decrease in proteoglycan staining in the
355
356
mandibular condylar cartilage.11 Total RNA expression
analyses of condyles from mice fed soft food showed a
significant decrease in the osteopontin levels.12 Further-
more, accelerated degeneration of the condylar cartilage
led to its enhanced replacement with bone, resulting in
thinner cartilage.8 Compressive forces have also been
reported to affect the mitotic activity of chondrocytes,
and thereby further affect the cell layer thickness.13
Although a number of studies have investigated the
effects of mastication on the growth of the mandible,
there have been no cohesive reports investigating the
adaptive ability of the mandibular condylar cartilage
and the expression levels of genes related to mandibular
condylar cartilage growth, and the 3-dimensional shape
of the mandible in relation to mastication. In our previ-
ous study, we investigated the effects of mastication on
mandibular morphology as assessed by microcomputed
tomography, and found significantly thicker condylar
widths in mice fed a hard-pellet diet (HD) for 1 week
than in mice fed a soft diet (SD).14 Interestingly, mandib-
ular length was greatest in mice fed a combined hard and
soft diet (HSD; 2 weeks of each) and smallest in the HD
group. Ramus height was greatest in the HSD group and
lowest in the SD group, and bone volume was also lower
in the SD group than in the HSD and HD groups. How-
ever, the adaptive mechanism of the mandibular
condylar cartilage has remained unclear.
In this study, we first used real-time PCR to examine
the mandibular cartilage gene expression differences
from that of other regions, using tibial cartilage as a
comparison. Next, we investigated the causes of regen-
eration in the composition of the mandibular condylar
cartilage and assessed whether changes in the level of
mastication influenced mandibular growth using histo-
logic and molecular analyses. Furthermore, we examined
the protein and mRNA expression levels of osteopontin
and type X collagen as well as the protein expression
of Musashi1 in the mandibular condylar cartilage by
immunostaining.
MATERIAL AND METHODS
Forty-eight 21-day-old male CD-1 mice (Saitama
Experimental Animals Supply, Saitama, Japan) were
used. The mice were randomly divided into 6 groups to
receive (ad libitum) diets of varying hardness and dura-
tions: control (3 animals), HD/1 week (9 animals), HD/
4 weeks (9 animals), SD/1 week (9 animals), SD/4 weeks
(9 animals), and HSD/4 weeks (9 animals). The 3 control
mice were used to generate Figure 1, whereas 3 sets of 3
mice from each of the remaining 5 groups were used to
generate Figures 2 to 5. At the end of the experimental
period, the mice were killed by cervical dislocation
September 2014  Vol 146  Issue 3 American Journal of Orthodontics and Dentofacial Orthopedics
Enomoto et al
after inhalation anesthesia with diethyl ether
(Funakoshi, Tokyo, Japan), which was applied to
cotton wool and placed with the mouse in an enclosed
space. All animal procedures, including care and
handling, conformed to the guidelines of our
Institutional Animal Care and Use Committee, and the
study protocol was approved by the Animal Research
Committee of Showa University.
Three mice were killed at 21 days of age. The remain-
ing mice were randomly divided into 5 groups of 9 when
weaned: 18 mice were fed the SD, with 9 killed at 4 weeks
of age and 9 killed at 7 weeks of age; 18 mice were fed
the HD, with 9 killed at 4 weeks of age and 9 killed at
7 weeks of age; and 9 mice were fed the HSD every other
week, and then killed at 7 weeks of age. The HD group
received an ordinary laboratory diet for mice in a hard
pellet form. The SD group received the ordinary diet after
it was ground and mixed with water in standardized pro-
portions (2 parts food to 5 parts water). No significant
differences in weight were identified among the mice
in the any of the groups either at randomization or
when they were killed.
Tissue processing for hematoxylin and eosin and
immunohistochemical staining were carried out. The
dissected cartilage specimens from the mandibular con-
dyles were fixed in 4% paraformaldehyde solution
(pH 7.4) overnight at 4
C and then decalcified in 10%
Na2EDTA (pH 7.2) for 2 weeks. Ten specimens from
each group were embedded in paraffin and sectioned
(7-mm thickness). The slices were coronal sections, which
were carefully selected at the center of the mandibular
condylar cartilage.
Five condylar sections from each group were stained
with hematoxylin and eosin. The condylar cartilage was
divided into 2 parts, comprising the articular, prolifera-
tive, and chondroblastic zones in one part, and the hy-
pertrophic zone in another.7 The representative
thicknesses were composed of the mean measurements
of these zones.
Immune complexes were formed by mixing a mouse
antibody against proliferating cell nuclear antigen
(1:200 dilution; Oncogene, Boston, Mass) and EnVi-
sionHRP antimouse detection reagent (Dako, Copenha-
gen, Denmark) for 1 hour at room temperature. After
60 minutes at room temperature, normal mouse serum
(Dako code X0910) was added to the immune complex.
Five sections from each group were incubated with
polymer immune complexes for 1 hour at room temper-
ature. Deparaffinized paraffin sections were incubated
with HRP-labeled primary antibodies using diaminoben-
zidine as the substrate. Equivalent dilutions and concen-
trations of mouse IgG2a (1:200 X0943; Dako) were used
as negative controls.
Enomoto et al 357

p75 Wn t1
* 0.0018 *

Wnt1 (relative expression)

0.0018
P75 (relative expression) 0.0016
0.0016
0.0014
0.0014
0.0012 0.0012
0.001 0.001
0.0008 0.0008
0.0006 0.0006
0.0004 0.0004
0.0002 0.0002
0 0
A Mandibular Tibial B Mandibular Tibial

Musashi1 Nestin
Musashi1 (relative expression)

Nestin (relative expression)

0.008 0.02
0.007 0.018
0.006 0.016
0.014
0.005 0.012
0.004 0.01
0.003 0.008
0.006
0.002
0.004
0.001 0.002
0 0
Mandibular Tibial Mandibular Tibial
C D

Fig 1. Gene expression analysis in mandibular condylar chondrocytes and tibial cartilage chondro-
cytes using real-time PCR (n53): A, P75; B, Wnt1; C, Musashi1; and D, Nestin. Gene expression is
normalized against the expression of the glyceraldehyde-3-phosphate dehydrogenase housekeeping
gene. *P \0.05.

Five sections from each group were incubated for
2.5 hours at room temperature with an antitype-X
collagen antibody (1:100 dilution LSL-LB-0092 whole
rabbit serum; LSL, Tokyo, Japan), which was detected
using an Alexa Fluor 568-conjugated goat antirabbit
polyclonal IgG (H1L) (Molecular Probes, Eugene, Ore)
as a secondary antibody. The sections were observed
with a fluorescence microscope (Eclipse TE300; Nikon,
Tokyo, Japan) and an imaging system (AQUA COSMOS;
Nikon).
Equivalent dilutions and concentrations of rabbit
immunoglobulin fraction (1:100 X0903; Dako) were
used as the negative controls.
We performed immunohistochemistry for osteopon-
tin. Five sections from each group were incubated for
3 hours at room temperature with an antiosteopontin
antibody (1:100 dilution LSL-LB-4225 whole rabbit
serum; LSL), which was detected by incubation with
EnVision 1 HRP antirabbit detection reagent (Dako)
for 1 hour at room temperature. To prevent nonspecific
staining, we incubated the sections with a blocking
reagent containing normal mouse serum for an addi-
tional 1 hour at room temperature. Deparaffinized
paraffin sections were reacted with HRP-labeled
primary antibodies using diaminobenzidine as the sub-
strate.
Equivalent dilutions and concentrations of rabbit
immunoglobulin fraction (1:100 X0903; Dako) were
used as negative controls.
Five sections from each group were incubated for
1 hour at room temperature with an anti-Musashi1 anti-
body (1:10 dilution 1877-1 rabbit Monoclonal IgG;
Epitomics, Burlingame, Calif), which was detected by
incubation with EnVision 1 HRP antirabbit detection
reagent for 1 hour at room temperature. To prevent
nonspecific staining, we incubated the sections with a
blocking reagent containing normal mouse serum for
an additional 1 hour at room temperature. Deparaffi-
nized paraffin sections were reacted with HRP-labeled
primary antibodies using diaminobenzidine as the
substrate. In all experiments, negative controls were per-
formed in which the primary antibody was omitted.
Equivalent dilutions or concentrations of rabbit immu-
noglobulin fraction (1:10 X0903; Dako) were used as
negative controls.
For the reverse transcription and real-time PCR,
mandibular condylar cartilage was collected by dissec-
tion under a light microscope and snap-frozen in liquid
nitrogen. Total RNA from 3 mice (nonpooled) was ex-
tracted from the total cartilage layer of the mandibular
condyle and the tibial cartilage using an RNeasy Protect
Mini Kit (Qiagen, Hilden, Germany) with RNase-free
DNaseI treatment (Qiagen). Following the manufac-
turer's protocol, we were able to extract sufficient quan-
tities from 1 mouse. In addition, we confirmed by
absorption spectrometer that the mRNA obtained was
adequate and of good quality. First-strand cDNA was
synthesized from 5 mg of total RNA using Super Script

American Journal of Orthodontics and Dentofacial Orthopedics September 2014  Vol 146  Issue 3
358 Enomoto et al

Fig 2. Histologic analysis of condylar cartilage: A and B, histologic sections from the superior regions
of the condyles of mice at 4 weeks (W) and C-E, 7 weeks of age (n 5 3). Mice were fed the HD or SD for
1 or 4 weeks, or the HSD for 2 weeks each, respectively, over a total of 4 weeks. APC, Articular,
proliferative, and chondroblastic zones; H, hypertrophic zone. Scale bars, 20 mm.

II RT (Gibco, Grand Island, NY) and random hexamers Statistical analysis

(Gibco). For real-time PCR, we used an ABI Prism 7000
Sequence Detection System (Applied Biosystems, Darm-
stadt, Germany) with TaqMan probes. The final mixture
(20 mL) contained 4 mL of cDNA sample, 10 mL of Taq-
Man Universal PCR Master Mix (Perkin-Elmer Applied
Biosciences, Foster City, Calif), and 1 mL of primers for
osteopontin, type X collagen, P75, Wnt-1, Musashi1,
and Nestin (Mm00436767-m1Spp1, lot 455039;
Mm00487041-m1, lot 479307; Mm00446296-m1, lot
958101; Mm01300555-g1, lot 812039; Mm00485224-
m1, lot 649865; and Mm00450205-m1, lot 74463).
Gene expression levels were normalized by the corre-
sponding expression level of glyceraldehyde-3-phos-
phate dehydrogenase as a housekeeping gene. The
PCR conditions were 2 minutes at 50
C, 10 minutes at
95
C, and 40 cycles of 15 seconds at 95
C and 1 minute
at 60
C.
Measurements were performed on 3 separate tissue
sections for each anatomic area in each experimental
animal, and an average value was calculated. For gene
expression, each sample of RNA was tested on 3 inde-
pendent occasions, and the values were averaged. Aver-
aged values from 3 representative mice from each dietary
group were used for statistical analyses.
The significance of the differences between the inde-
pendent samples was assessed using the Student t test
(when comparing 2 groups) or 1-way analysis of vari-
ance (ANOVA) (when comparing 3) if the samples passed
a preliminary Shapiro Wilks test for normality. Statistical
significance was set at P \0.05. Because statistical tests
were used mainly for exploratory analysis, multiple com-
parisons were not considered in this study. Our sample
size of 3 animals for each test was not large enough to
detect statistically significant differences between or

September 2014  Vol 146  Issue 3 American Journal of Orthodontics and Dentofacial Orthopedics
Enomoto et al 359

Fig 3. Immunostaining and proliferation analysis of condylar chondrocytes (n 5 3): A and B, histologic
sections from the superior region of mouse condyles at 4 weeks (W) and C-E, 7 weeks. Scale bars,
100 mm. The mice were fed the HD or SD for 1 or 4 weeks, or the HSD for 2 weeks each, with measure-
ments taken at 4 or 7 weeks of age, respectively. Proliferation was measured by the number of cells
positively stained for proliferating cell nuclear antigen. **P \0.01.

among groups. However, since the variances of the data
obtained within the groups were moderately small and
well controlled, the results of the statistical tests pro-
vided useful information for our evaluation. All data
are presented as means 6 standard deviations in the fig-
ures. All analyses were performed using SPSS software
(version 12.0; IBM, Chicago, Ill).
RESULTS
We performed real-time PCR analysis of P75, Wnt-1,
Musashi1, and Nestin gene expressions in the condylar
cartilage. First, we assessed differences in gene expres-
sion in the cartilage of the mandibular condyle with
that in cartilage from another site in the body, using
tibial cartilage for the comparison. From real-time
PCR, we found a statistically significant increase (t test,
P75, P 5 0.0007; Wnt1, P 5 0.0041) in the expression
levels of the neural crest cell markers P75 and Wnt-1 in
the mandibular condylar cartilage compared with the
tibial cartilage. Similarly, the expression of Musashi1
and nestin, markers for neural stem cells, was upregu-
lated in the mandibular condylar cartilage compared
with the tibial cartilage, although this difference was
not significant (Fig 1).
We did a histologic analysis of the condylar cartilage.
At 4 weeks of age (after 1 week of feeding), the hypertro-
phic zone was significantly thicker (t test: P 5 0.0055) in
the HD group than in the SD group (Fig 2, A and B; Table).
By 7 weeks of age (after 4 weeks of feeding), the

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360 Enomoto et al

Fig 4. Immunostaining and quantitative gene expression of A and B, osteopontin, and C and D, type X
collagen in mandibular condylar chondrocytes (n 5 3). The mice were fed the HD or SD for 1 or 4 weeks
(W), or the HSD for 2 weeks each, respectively, over a total of 4 weeks. Scale bars, 20 mm.

hypertrophic zone continued to be significantly thicker C-E). However, the thickness of the entire chondrocyte
(ANOVA: P 5 0.004) in the HD group than in the SD zone was similar among the HD, SD, and HSD groups.
group, with no obvious differences noted between the In the analysis of condylar chondrocyte proliferation,
HD or SD group and the HSD group at this time (Fig 2, we found that chondrocyte proliferation was

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Enomoto et al
Fig 5. Immunostaining of Musashi1 in mandibular
condylar chondrocytes (n 5 3). The mice were fed the
HD or SD for 1 week (W). Histologic sections from the
superior region of the condyles at 4 weeks of age are
shown: A and B, entire chondrocytic zone. The stained
area was 100 3 100 mm. Scale bars, 20 mm. C, Graphic rep-
resentation of the number of Musashi1-positive cells in
mice fed the HD or SD for 1 week.
significantly lower (t test: P 5 0.0004) in the HD group
than in the SD group at 4 weeks of age (after 1 week of
feeding). However, the proliferative abilities of the
condylar chondrocytes did not differ significantly at
7 weeks of age (after 4 weeks of feeding) (Fig 3).
Localizations of osteopontin, type X collagen, and
Musashi1 in the mandibular condylar cartilage
At 4 weeks of age (after 1 week of feeding), there
were many osteopontin-positive cells only in the hyper-
trophic chondrocyte zone in the HD group, whereas
these cells were observed in the entire chondrocytic
zone in the SD group (Fig 4, A). Similar results were
observed at 7 weeks of age (after 4 weeks of feeding).
Compared with the other groups, there were many
osteopontin-positive cells in the entire chondrocytic
zone in the HSD group (Fig 4, A).
At 4 weeks of age (after 1 week of feeding), the num-
ber of type X collagen-positive areas in the hypertrophic
chondrocyte zone of the condylar cartilage was consider-
ably higher in the HD group than in the SD group (Fig 4,
C). At 7 weeks of age (after 4 weeks of feeding), there
were many type X collagen-positive areas throughout
the chondrocyte zone of the condylar cartilage in the
HSD group, whereas these were only present in the
American Journal of Orthodontics and Dentofacial Orthopedics
361
hypertrophic chondrocyte zones of the animals in the
HD and SD groups, and in much lower numbers (Fig 4, C).
At 4 weeks of age (after 1 week of feeding), we as-
sessed the number of Musashi-positive cells in the entire
chondrocytic zone—articular, proliferative, and chon-
droblastic zones together—and in the hypertrophic
zone in the condylar cartilage (Fig 5, A and B). Overall,
we observed more Musashi1-positive cells, but this was
not significant and was observed more often in the pro-
liferative zone of the condylar cartilage in the SD group
than in the HD group (Fig 5, C).
For the real-time PCR determination of osteopontin
and type X collagen gene expressions in the condylar
cartilage, the osteopontin and type X collagen gene
expression levels were quantitatively examined with
real-time PCR using the ratio of SD to HD group (Fig
4, B and D). For osteopontin, there were relatively large,
but not significant, differences in the expression levels
between each group at 4 weeks of age (after 1 week of
feeding) and 7 weeks of age (after 4 weeks of feeding)
(Fig 4, B). For type X collagen, similar results were ob-
tained (Fig 4, D). There was a relatively large difference,
but it was not significant in the type X collagen gene
expression levels between the HD and SD groups.
DISCUSSION
The rationale for these differences simply caused by
mastication eluded us previously, and we undertook
this study to begin to unravel the effects of mastication
on the growth and adaptive responses of mandibular
condylar cartilage.
Endochondral ossification of the mandibular condylar
cartilage is thought to be highly adaptive and occurs
through multipotent cells originating from the neural
crest that have a high proliferative capacity.15 However,
it is unknown whether these characteristics are observ-
able in vivo, and the mechanism is also unknown. There-
fore, we investigated why the mandibular condyle has a
higher adaptation ability compared with other growth
cartilages using molecular analyses.16 Our results showed
higher expression levels of the neural crest cell markers
P75 and Wnt-1 and neural stem cell markers Musashi1
and Nestin in the mandibular condylar cartilage than in
the tibial cartilage. These findings suggest that the high
adaptation ability of the mandibular condylar cartilage
might be derived from neural crest cells.
Many reports have shown a relationship between
mastication and mandibular growth: the condylar width
was thicker, and the ramus height was greater in rats
fed a hard diet than in rats fed a soft diet.9,17-20 As
expected, the force on the temporomandibular joint is
greater when masticating hard food than soft food.17
September 2014  Vol 146  Issue 3
362
Table. Histologic changes in condylar cartilage with various feeding regimens
t test
HD vs SD
HD1W SD1W (P value) HD4W HSD4W SD4W
Whole chondrocyte zone 245.0 201.8 0.537 206.2 190.0 197.6
APC zone 60.0 60.6 0.458 50.6 68.2 80.6
Hypertrophic chondrocyte 158.6 106.8 0.005y 116.2 100.4 71.8
zone
APC, Articular, proliferative, and chondroblastic zones; HD1W, hard diet for 1 week (at 4 weeks of age); SD1W, soft diet for 1 week (at 4 weeks of
age); HD4W, hard diet for 4 weeks (at 7 weeks of age); HSD4W, hard diet for 2 weeks after soft diet for 2 weeks (at 7 weeks of age); SD4W, soft diet
for 4 weeks (at 7 weeks of age).
*P \0.05; yP \0.01.
Furthermore, the histologic changes showed a thicker hy-
pertrophic chondrocyte zone of the mandibular condylar
cartilage in rats fed a hard diet than in those fed a soft
diet.21,22 Reduced proliferative ability and matrix
production has also been reported in rats fed a hard diet
compared with rats fed a soft diet,23 and the expression
levels of several genes related to mandibular condylar
cartilage growth are significantly altered between the
stages of lactation and mastication in mice.8 Taken
together, these reports suggest that mastication markedly
affects mandibular condylar cartilage growth in rodents.
To further understand the associations between
mastication and mandibular condylar cartilage growth,
we compared the histology and proliferative ability of
the mandibular condylar cartilage among the 3 groups.
Histologic analysis showed that the hypertrophic chon-
drocyte zone in the central region of the mandibular
condylar cartilage was significantly thicker in the HD
group than in the SD group after feeding the mice the
different diets for 1 week (4 weeks of age). Similarly,
the hypertrophic chondrocyte zone was thicker in the
HD group than in the SD group after feeding the mice
the respective diets for 4 weeks (7 weeks of age), suggest-
ing that the hard diet induced terminal differentiation of
the mandibular condylar chondrocytes. This finding is
consistent with the results from a previous report.21
The proliferative ability of chondrocytes was signifi-
cantly higher in the SD group than in the HD group after
feeding the mice the different diets for 1 week (4 weeks of
age), suggesting that masticatory stimulation with the
soft diet might have promoted chondrocyte proliferation.
The expression levels of osteopontin, which is involved in
calcification, and type X collagen, which is involved in
terminal differentiation, were the highest in the entire
condylar cartilage in the HSD group after feeding the
mice the combined diets for 4 weeks (7 weeks of age).
Musashi1 is involved in the proliferation and mainte-
nance of neural stem cells.24 This represents a mechanism
by which sequence-specific translational repression oc-
curs in stem cells through the control of translation
September 2014  Vol 146  Issue 3
Enomoto et al
ANOVA HD t test t test t test
vs HSD vs SD HD vs SD HD vs HSD HSD vs SD
(P value) (P value) (P value) (P value)
0.358 0.295 0.124 0.239
0.163 0.464 0.089 0.126
0.004y 0.035* 0.171 0.207
initiation.25 This initiating factor exists in all cells and reg-
ulates protein expression levels. Therefore, it is possible
that this function is not exclusive to neural stem cells.
In our study, Musashi1 protein expression was stronger
in the SD group than in the HD group. Furthermore, the
proliferative ability of the condylar chondrocytes was
significantly lower in the HD group than in the SD group
at 4 weeks of age (after 1 week of feeding). These data
suggest that Musashi1 might be related to the prolifera-
tive ability of the condylar chondrocytes, which have
important implications in the cell cycle where prolifera-
tion and differentiation are finely balanced.
Proliferative potential was greater in the SD group
than in the HD group. We concluded that Musashi1
activity leads to proliferation in the SD animals but to
differentiation in the HD animals. Furthermore, the
stem cell-like characteristics of cells originating from
the neural crest are maintained until a growth process
is initiated in the mandibular condyle. As a result,
mandible growth is largely affected by mastication
changes, as we observed in this study.
In our statistical analysis, we used the t test to
compare 2 groups, and 1-way ANOVA to compare 3 or
more groups, with an alpha level of 0.05 for primary
analysis. When we found a statistical difference from
the 1-way ANOVA, the t test with an alpha level of
0.05 was adopted for each pair-wise comparison of 3
or more groups as an ad hoc test. Because the ad hoc
tests were used mainly for exploratory purposes, multi-
ple comparisons were not considered in this study.
CONCLUSIONS
The results of this study suggest that neural crest-
derived cells might be responsible for the high
adaptation ability of the mandibular condylar carti-
lage. Together with the results of other authors, our
findings suggest that mastication markedly affects
mandibular condylar cartilage growth in rodents, and
we concluded that food consistency influences the
American Journal of Orthodontics and Dentofacial Orthopedics
Enomoto et al
morphologic and histologic characteristics of mandib-
ular growth.
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September 2014  Vol 146  Issue 3