Effects of restriction of fetal jaw movement on prenataldevelopment of the temporalis muscle

Esrat Jahan a,b, Akihiro Matsumoto a, Jun Udagawa a, Ashiq Mahmood Rafiq a,b,Ryuju Hashimoto a, Omar Ibna Faizur Rahman c, Hasina Habib b,c,Joji Sekine b, Hiroki Otani a,*aDepartment of Developmental Biology, Faculty of Medicine, Shimane University, 89-1 Enya-cho, Izumoshi, Shimane 693-8501, JapanbDepartment of Oral & Maxillofacial Surgery, Faculty of Medicine, Shimane University, 89-1 Enya-cho, Izumoshi, Shimane 693-8501, JapancYamagata Dhaka Friendship Hospital, Lalmatia, Dhaka, Bangladesh

a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 9 1 9 – 9 2 7

a r t i c l e i n f o

Article history:

Accepted 27 July 2010

Keywords:

Temporalis muscle

exo utero

Mechanical stress

Fetal jaw movement

Mouse

a b s t r a c t

Jaw movement affects masticatory muscles during the postnatal period. Prenatal jaw

movement has also been implicated in the development of the temporomandibular joint;

however, its effect on prenatal development of the masticatory muscles has not been

extensively analysed. In the present study, we examined the effects of the restriction of

fetal jaw movement on the temporalis muscle, a major masticatory muscle, in mice by

suturing the maxilla and mandible (sutured group) using an exo utero development system.

We compared the morphology of the temporalis muscle between sutured, sham-operated

and normal in utero groups. At embryonic day (E) 18.5, the volume of muscle fibres, but not

that of connective tissue, in the temporalis muscle was decreased in the sutured group. The

E18.5 temporalis muscle in the sutured group appeared morphologically similar to that of

the E17.5 in utero group, except for frequent muscle fibre irregularities. By transmission

electron microscopy, in the sutured group, the myofibrils were immature and scattered, the

nuclei appeared comparatively immature, the mitochondria were expanded in volume with

fewer cristae, and cytoplasmic inclusion bodies were frequently observed. Expression of

Myf-6, a late myogenic transcription factor, by real-time RT-PCR was not significantly

different between the sutured and sham-operated groups. These findings demonstrated

approximately 1-day delay in the morphological development of the temporalis muscle in

the sutured group, and some abnormalities were observed, although Myf-6 level was not

affected in the sutured group. The present study revealed that the prenatal jaw movement

influences the development of the temporalis muscle.

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1. Introduction

The temporalis muscle is one of the three main jaw-closing

muscles (masseter, temporalis, medial pterygoid muscles).1

The combination of its broad origin on the temporal fossa and

the inner surface of the temporal fascia and its limited

insertion on the anterior border and medial surface of the

* Corresponding author. Tel.: +81 853 20 2102; fax: +81 853 20 2100.E-mail address: hotani@med.shimane-u.ac.jp (H. Otani).

0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reservedoi:10.1016/j.archoralbio.2010.07.010

coronoid process allows differential activation and variation

in the line of action.2,3 The influence of masticatory functions

of the masticatory muscles on craniofacial and dentofacial

growth has been investigated widely.3–5 Mechanical strain

regulates cell proliferation, differentiation and maturation,6

all of which are related to growth and development, therefore,

masticatory activity and jaw movement is thought to be

d.

a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 9 1 9 – 9 2 7920

related to the morphology of the jaw and jaw-related muscles.

It has been reported in adults that the jaw movement is

responsible for changes in both the volume of masticatory

muscles (masseter, medial and lateral pterygoid)7 as well as

length (masseter),8 as determined by direct measurements of

muscle and jaw movements. We previously reported that

prenatal jaw movement is an important mechanical factor in

the development of the mandibular condyle and articular

disc.9,10 Myogenesis begins when cells acquire myogenic

potential prior migration to muscle-forming regions. There

are four myogenic regulatory factors (MRFs)—namely, MyoD1,

Myf-5, myogenin, and MRF4/herculin/Myf-6, all of which have

been certified to convert non-muscle cells into myoblasts.11–14

It has also been reported that the expression of MyoD1 (palatal

muscle),15 MyoD, myogenin, and Myf-5 (masseter and tongue

muscle)16,17 mRNAs increased with development up to birth

(from embryonic day (E) 10 to 11 to birth).

The masticatory muscle is known to have several unique

developmental characteristics as compared with other skeletal

muscles, such as trunk and limb muscles (reviewed in Ref. 16).

First, myoblasts of the masticatory muscles originate from non-

somitic paraxial head mesoderm,18 whereas myoblasts of the

trunk and limb muscles are derived from somatic mesoderm.19

Second, Pax3/Myf-5 homozygous mutant mice induce MyoD in

the masticatory muscles as well as in the other craniofacial

muscles, but in the trunk and limb muscles, they fail to induce

MyoD and therefore do not generate trunk and limb muscles.20

Third, adult human and murine masseter muscles contain the

neonatal isoforms of the myosin heavy chain,21,22 and delayed

MyoD family expression correlates with the delayed embryonic

development of the mouse masseter muscle.16 Despite these

unique biochemical and molecular characteristics of the

developing masticatory muscles, prenatal development of the

temporalis muscle has not been morphologically analysed in

detail, and changes in the muscle related to the prenatal jaw

movement have not yet been examined.

In the present study, to elucidate the development of the

temporalis muscle related with the jaw movement, we

examined the effects of prenatal jaw movement restriction

on the temporalis muscle development.

2. Materials and methods

2.1. Animals

Jcl:ICR female mice aged 8–20 weeks (CLEA, Tokyo, Japan) were

used and maintained at the Institute of Experimental Animals

of Shimane University, Faculty of Medicine. This study was

approved by the Ethical Committee for Animal Research of

Shimane University, and all experimental procedures were

performed according to the institutional guidelines. We use

different embryos from different dams in each and every

experiment.

2.2. Exo utero surgery

A female mouse was housed with a male overnight, and noon

of the day when the vaginal plug was found was designated as

the E0.5. For exo utero surgery, the procedure followed as

described previously.9,10,23–26 Briefly, at E15.5, the pregnant

dams were anaesthetised with 50 mg/kg body weight (BW) of

pentobarbital (Somnopentyl, Kyoritsu Seiyaku, Tokyo, Japan).

The abdominal wall was incised at the mid-line, and then a

longitudinal incision was made along the uterine wall on the

side opposite to the entrance of the uterine artery and the site

of the placenta. This exposed the fetuses covered by the

embryonic membrane. The mandible and maxilla of the

embryos were fixed using an 8–0 nylon suture (Fig. 1 in Ref. 9)

and they were designated as the sutured group. We also

performed a sham operation by passing the needle from the

mandible through the maxilla of some embryos without

creating a knot and defined them as the sham-operated group.

After the operation, the embryos were placed back in the

abdominal cavity of the dam and were allowed to develop exo

utero till E18.5.

We confirmed that the jaw movements of the embryos

were restricted. Briefly, at E15.5 we observed the embryonic

jaw movement clearly by the naked eye and more clearly by an

operation microscope. When we restricted the jaw movement

by suturing the maxilla and mandible over the amniotic

membrane, we checked very carefully whether the knot was

tight or loose. After confirming that the knot was tight, the

sutured embryos were placed back in the abdominal cavity

and left there for exo utero development. When we opened the

abdominal cavity of the dam at E18.5, we checked whether

the knot at the jaw of the embryo was tight or loose under the

operation microscope. If it was tight, we stimulated the

embryo very carefully to observe whether it moved its jaw or

not. When it did not move its jaw, we considered it a

successfully sutured embryo.

We prepared E18.5 and E17.5 in utero developed control

embryos to confirm that the exo utero experience itself did not

influence muscle development.

2.3. Tissue preparation for histological examination

At E18.5 and E17.5, the dams (n = 5) were sacrificed by cervical

dislocation under anaesthesia with pentobarbital. The embryos

(n = 5, one from each dam) were obtained, and their BW and

crown rump length (CRL) were measured. The heads, including

the temporomandibular joint (TMJ), were fixed in Bouin’s

solution (75 ml of saturated picric acid, 25 ml of formalin and

5 ml of glacial acetic acid) for 48–72 h at room temperature for

the histological analysis. The samples were dehydrated in a

graded series of alcohol and embedded in paraffin, and 5 mm-

thick serial sagittal sections were made and stained with

hematoxylin and eosin (HE). These sections were used for the

histological examination and morphometric analysis.

2.4. Computerised histomorphometric analysis oftemporalis muscle

For the morphometric study of the temporalis muscle in the

E18.5 in utero control (n = 5), E18.5 sham-operated (n = 5), E18.5

sutured (n = 5), and E17.5 in utero control (n = 3) groups, the

volume of the temporalis muscle was calculated from the HE

stained serial sections of the embryo’s head containing the

temporalis muscle. The initial section was determined in

which the temporalis muscle first appeared, and this section

Fig. 1 – The heads of (A) E18.5 in utero control, (B) E18.5

sham-operated and (C) E18.5 sutured embryos are shown.

The arrow indicates a tight suture fixation with 8–0 nylon

which was performed at E15.5 using an exo utero system.

a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 9 1 9 – 9 2 7 921

was designated as section 1, subsequent sections were then

numbered sequentially. Every fourth sections were chosen for

quantitative analysis (i.e., sections 1, 5, 9, . . ., n were selected).

For selected sections, micrographs containing the tempor-

alis muscle were taken; the total muscle area (containing

muscle fibres and connective tissue) and the muscle fibre area

alone were extracted separately using Adobe Photoshop

software (http://www.adobe.com/), and then the total muscle

area and the muscle fibre area were measured by using ImageJ

software (http://rsbweb.nih.gov/ij/). Using these data, we then

calculated the total muscle area, connective tissue area, and the

muscle fibre area separately (total muscle area�muscle fibre

area = connective tissue area). The total area of all selected

sections was multiplied by 20 mm (5 mm thick by 4 sections), and

the resulting value was then divided by BW for standardization

among embryos. The total volume of the temporalis muscle

(standardized) was calculated using the following formula:

standardized total muscle volume

¼ total muscle areabody weight

� 20 mm

The standardized muscle fibre volume and the connective

tissue volume were obtained similarly.

2.5. Tissue preparation for transmission electronmicroscopic (TEM) examination

The temporalis muscle in the E18.5 in utero control, E18.5

sham-operated, E18.5 sutured and E17.5 in utero control groups

(n = 5, one embryo from each dam which was different from

that used for the other experiments) was dissected under a

dissecting microscope and fixed in a solution composed of 2%

paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phos-

phate buffer, pH 7.2, at 4 8C overnight. Tissues were then post-

fixed with buffered 1% OSO4 at 4 8C for 1–2 h, dehydrated in a

graded series of ethanol, and embedded in epoxy resin (TAAB

812, Berkshire, England). Thin sections (70 nm) were created

with an ultramicrotome (RMC, Tucson, AZ, USA) and the cut

sections were stained with 2.5% uranyl acetate and lead citrate

prior to being examined under a TEM (TOPCON EM-002B,

Tokyo, Japan) at 80 kV.

We also counted muscle satellite cells from the TEM

section. Satellite cells were defined as small cells with

elongated nuclei and a relatively coarse chromatin pattern

that were located in shallow depressions on the surface of the

muscle fibres, and situated within the basal lamina that

surrounds the individual muscle fibres.27,28 Based on this

definition, we counted satellite cells in the muscle sections

from each group. We chose 3 different sections from 3

different embryos of each group. We counted more than 70

muscle fibres and the associated satellite cells, and compared

the frequency of satellite cells between the groups.

2.6. mRNA extraction and quantitative real-time reversetranscriptase-polymerase chain reaction (RT-PCR)examination

After sacrifice of the mice at E18.5 the temporalis muscles of

the E18.5 sham-operated and E18.5 sutured groups were

dissected from the head of the embryos, and collected them in

five different tubes (each tube contain 6–10 whole temporalis

muscles from different dams and embryos). Total mRNA was

extracted using TRI reagent (Molecular Research Center,

Cincinnati, OH, USA), the cDNA was created by RT-PCR with

Ready-To-Go RT-PCR beads (GE Healthcare, Buckinghamshire,

UK). To validate gene expression changes, quantitative real-

time RT-PCR analysis was performed with an Applied[(Fig._1)TD$FIG]

a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 9 1 9 – 9 2 7922

Biosystems Prism 7000HT Sequence Detection System using

the TaqMan1 Gene Expression PCR master mix according to

the manufacturer’s specifications (Applied Biosystems Inc.,

Foster City, CA, USA) for the Myf-6 gene, for which validated

TaqMan Gene Expression Assays are available. The TaqMan

probes and primers for Myf-6 (assay identification number

Mm00435126_m1) and, b-actin (assay identification number

Mm00607939_s1) were assay-on-demand gene expression

products (Applied Biosystems). The mouse b-actin gene was

used as an endogenous control. The gene-specific probes were

labelled by using the reporter dye FAMTM at the 50 end, and a

nonfluorescent quencher was used at the 30 end of the probe.

The thermal cycler conditions were as follows: hold for 10 min

at 95 8C, followed by two-step PCR for 40 cycles of 95 8C for 15 s

followed by 60 8C for 1 min. All experiments were performed in[(Fig._2)TD$FIG]

Fig. 2 – Representative histological sections of the HE staining. T

and E18.5 sham-operated (B) groups were thicker, straight, cond

of the E18.5 sutured group at E18.5 were thin, curly or wavy, less

extracellular space. (D) Similar findings to those in (C) were als

magnification, the curly and wavy muscle fibres (black arrow he

(G), whereas these curly and wavy fibres were not observed in

quardruplicate. Amplification data were analysed using an

Applied Biosystems Prism Sequence Detection Software

version 1.1 (Applied Biosystems). When the efficiency of the

target gene amplification and the efficiency of b-actin

amplification were approximately equal, as verified by

determination of the absolute value (less than 0.1) of the

slope of the log input amount versus DCT, the DDCT method

recommended by the manufacturer was used to compare the

relative expression levels of the E18.5 sutured and control

groups using endogenous control b-actin as the standard.

2.7. Statistical analysis

We applied an analysis of variance (ANOVA) to compare the

groups. All data in this study are presented as mean � stan-

he temporalis muscle fibres of the E18.5 in utero control (A)

ensed and arranged in bundles, while (C) the muscle fibres

condensed and arranged in a scattered manner with more

o observed in the E17.5 in utero control group. At higher

ads) were more clearly observed in the E18.5 sutured group

the E17.5 in utero control group (H). (Scale bars = 100 mm.)

[(Fig._3)TD$FIG]

Fig. 3 – Comparison of the total muscle volume, muscle

fibre volume, and connective tissue volume of the E18.5 in

utero control, E18.5 sham-operated, E18.5 sutured and

E17.5 in utero control groups. No significant differences

were observed between the E18.5 in utero control and E18.5

sham-operated groups, but the muscle fibre volume was

significantly higher in these groups than in the E17.5 in

utero control group. On day E18.5, both the total muscle

volume and the muscle fibre volume, but not the

connective tissue volume, were significantly lower in the

E18.5 sutured group than in the E18.5 in utero control or

E18.5 sham-operated groups. *p < 0.05, **p < 0.01.

a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 9 1 9 – 9 2 7 923

standard deviation. In this study, significant variance between

groups was detected. A p-value of less than 0.05 was

considered to indicate statistical significance.

3. Results

Using the exo utero surgery as described in Section 2, we

obtained a reliable restriction of jaw movement in mouse

embryos (Fig. 1C), as previously reported.9,10 Also as reported

in our previous studies,9,10,29 this type of surgical procedures

caused no general growth retardation, and neither the CRL nor

the BW of the E18.5 sutured embryos was significantly

different from those of the E18.5 sham-operated animals, or

those of the E18.5 in utero control group (data not shown).

3.1. Histological observation of the temporalis muscle

The HE staining of the histological section of the temporalis

muscle at E18.5 revealed that in the in utero control (Fig. 2A)

and the E18.5 sham-operated (Fig. 2B) groups, the muscle

fibres were generally thick, straight, and homogenous, and

the muscle fibres were arranged in groups or bundles, while in

the E18.5 sutured group the muscle fibres were thin and not

straight (i.e., curly or wavy), and muscle fibres pattern

appeared scattered (Fig. 2C). Higher magnification of the

E18.5 in utero control (Fig. 2E) and E18.5 sham-operated (Fig. 2F)

groups revealed, spindle-shaped nuclei that were peripher-

ally placed, while in the E18.5 sutured group (Fig. 2G), the

nuclei were large and round, and were generally centrally

situated. When we observed the temporalis muscle of the

E17.5 in utero control group (Fig. 2D and H), the developmental

characteristics of the muscle fibres were generally similar to

those of the E18.5 sutured group (Fig. 2C and G), i.e., with a

thin, centrally situated, large and round nucleus, with more

extracellular space, and not arranged in bundles. However,

curly and/or wavy muscle fibres were frequently observed in

the E18.5 sutured muscles, whereas all muscle fibres of the

E17.5 in utero control group were straight (Fig. 2C and G vs. D

and H).

3.2. Histomorphometric analysis of the temporalis muscle

To quantitatively analyse changes in the temporalis muscle,

the muscle volume was measured. There were no significant

differences in the total muscle volume, the muscle fibre

volume and the connective tissue volume between the E18.5 in

utero control and the E18.5 sham-operated groups (Fig. 3). In

the E18.5 in utero control and E18.5 sham-operated groups, the

muscle fibre volume was significantly increased compared to

that in the E17.5 in utero control group. At 3 days after the

operation (E18.5), the volume of the temporalis muscle from

E18.5 sutured embryos was significantly different from that of

the E18.5 in utero control and the E18.5 sham-operated

embryos, as determined by repeated measures ANOVA. The

total volume of the temporalis muscle (Fig. 3, p < 0.05) and the

muscle fibre volume (Fig. 3, p < 0.01) were significantly smaller

in the E18.5 sutured group than in the E18.5 in utero control and

E18.5 sham-operated groups. On the other hand, there was no

significant difference in the volume of the connective tissue of

the E18.5 in utero control, E18.5 sham-operated and E18.5

sutured groups (Fig. 3). The reduced volume of the temporalis

muscle in the E18.5 sutured group further supported that the

jaw movement affected the development of the temporalis

muscle.

3.3. TEM analysis of the muscle fibre

The temporalis muscle was examined by TEM to evaluate the

maturity of the muscle fibres at the subcellular level. As can be

seen in the representative images shown in Fig. 4, in the cross

sections the muscle fibres exhibited a bundle-like arrange-

ment with fewer fibroblasts between them, and myofibril

density was generally high in the E18.5 in utero control and

E18.5 sham-operated groups (Fig. 4A and B). Most of the muscle

fibre characteristics were similar between the two groups;

however, in the E18.5 sutured group, the muscle fibres were

arranged more sparsely, with a larger number of fibroblasts

between them, and the myofibril density was generally low

(Fig. 4G). In the E17.5 in utero control group, the muscle fibres

were arranged more sparsely, the muscle fibres were less

mature, and there were fewer myofibrils and more extracel-

lular gaps (Fig. 4F). The muscle fibre characteristics of the E17.5

in utero control group were similar to those of the E18.5 sutured

group, but in the E18.5 sutured group there were some

abnormalities. In the longitudinal sections the muscle fibres

again appeared more mature, with more myofibrils in the

E18.5 in utero control (data not shown) and E18.5 sham-

operated group (Fig. 4C), while in the E18.5 sutured group, the

muscle fibres were less mature and had fewer myofibrils

[(Fig._4)TD$FIG]

Fig. 4 – Representative electron micrographs of the temporalis muscle. In the cross sections, muscle fibres were condensed,

arranged in bundle, contained few fibroblasts in-between and there was less extracellular matrix/space in the muscles of

the E18.5 in utero control (A) and E18.5 sham-operated (B) groups. In the E17.5 in utero control (F) and E18.5 sutured (G)

groups muscle fibres density was low, a scattered arrangement was seen, and there were more fibroblasts, as well as more

extracellular matrix/space. In the longitudinal sections, myofibrils were more densely arranged in the E18.5 in utero control

(data not shown) and E18.5 sham-operated (C) groups, whereas the muscle fibres contained comparatively fewer myofibrils

in the E18.5 sutured group (H). (G and H) In the E18.5 sutured group, muscle cell nuclei were centrally situated, large in size,

a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 9 1 9 – 9 2 7924

a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 9 1 9 – 9 2 7 925

(Fig. 4H). The extracellular spaces were wider in the E18.5

sutured group than in the E18.5 in utero control (data not

shown) and E18.5 sham-operated group (Fig. 4A and B vs. G and

C vs. H) which corresponded to the light-microscopic

observation (Fig. 2). We also observed that in the E18.5 sutured

group, the nuclei of muscle cells were centrally localised, were

round in shape and large in size, and their electron density

was relatively low, and the muscle cells frequently contained

generally electron-dense inclusion bodies of unknown origin

(Fig. 4G–I). In contrast, in the E18.5 in utero control (data not

shown) and E18.5 sham-operated group, the muscle cell nuclei

were placed at the periphery of the cells, and the nuclei were

rather spindle-shaped and had a relatively high electron

density (Fig. 4A–C). Thus, the nuclei in the E18.5 sutured group

appeared comparatively immature. Very few (if any), and

much smaller inclusion bodies were found in the muscle cells

in the E18.5 in utero control (data not shown) and E18.5 sham-

operated group (Fig. 4A–E). Distances between the cell

membrane and the outermost myofibrils in the muscle cells

were generally wider in the E18.5 sutured (Fig. 4I) than in the

E18.5 in utero control (data not shown) and E18.5 sham-

operated (Fig. 4D) muscle cells. At higher magnification, the

mitochondria in the E18.5 sutured group were expanded in

volume and contained fewer cristae, and their electron density

was low (Fig. 4J), whereas in the E18.5 in utero control (data not

shown) and E18.5 sham-operated group, the mitochondria

were normal in terms of size and shape, contained more

numerous and parallel cristae, and generally exhibited higher

electron density (Fig. 4E).

We observed and counted the numbers of satellite cells to

further assess the differentiation status of the temporalis

muscle. The number of satellite cells in the E18.5 in utero

control group was significantly higher than that in the E17.5 in

utero control group, corresponding to the differentiation of the

muscle fibres. However, there were no significant differences

among the E18.5 in utero control, E18.5 sham-operated, and

E18.5 sutured groups (data not shown).

3.4. Quantitative real-time RT-PCR analysis of Myf-6

We used RT-PCR analysis to examine the relative expression of

Myf-6 in E18.5 sham-operated and E18.5 sutured groups,

expecting that the relative expression of Myf-6 might be lower

in the E18.5 sutured group than in the E18.5 sham-operated

group, based on the relatively immature morphology of the

temporalis muscle in the E18.5 sutured group. However, in this

respect, we found no significant differences between the two

groups (data not shown).

and their electron density was low, and muscle cells frequently

heads). In the E18.5 in utero control (data not shown) and E18.5

peripherally situated, spindle shaped, and exhibited greater ele

and much smaller, inclusion bodies. At higher magnification, w

membrane and the outer most myofibrils of the muscle cells we

in utero control (data not shown) and E18.5 sham-operated (D) gr

volume, contained fewer cristae, and lower electron density wa

the E18.5 in utero control (data not shown) and E18.5 sham-opera

and shape, and contained more and parallel cristae, and the elec

H), and 1 mm for (D, E, I and J).

4. Discussion

Our present study using the mouse exo utero development

system9,10,23–26 demonstrated that prenatal jaw movement

restriction affected the development of the temporalis muscle.

This restriction of jaw movement clearly led to histological

changes in the temporalis muscle in the E18.5 embryo in the

E18.5 sutured group; histomorphometric study in particular

revealed a significant decrease in the temporalis muscle

volume in the E18.5 sutured group. In addition, TEM observa-

tion further supported the relative immaturity of muscle fibres

in the E18.5 sutured group, and also revealed potential

abnormalities at the subcellular level in this group.

In humans, the masticatory muscles (i.e., temporalis and

masseter) play an important role in jaw movement during

opening and closing of the jaw.30,31 It has been reported that the

masticatory muscle movement and function influence cranio-

facial growth, as well as mandibular4 and articular disc32

growth. Mechanical stress plays a fundamental role in regulat-

ing cellular activities during tissue morphogenesis.33,34 As

regards influences on the growth and differentiation of

masticatory muscles, it has been shown that mechanical

stimulation is an important factor in muscle development; as

such stimulation directly acts on cells.35 The development of

masticatory muscles has been observed in terms of the

relationship between masticatory function and muscle proper-

ties in postnatal mice.36 It has also been suggested that delayed

MyoD family gene expression is related to delayed develop-

ment, differentiation, and maturation of the masticatory

muscles during the prenatal period in mice.16 The temporalis

muscle has been shown to play an important role in marked

functional changes in the oral cavity from lactation to

mastication, which contributes to oral function in cooperation

with other masticatory muscles.37 Intrauterine jaw movement

starts on E14 in mice,38 on E16 in rats39 and in humans

intrauterine mandibular movements have been detected at

midterm by ultrasonography.40 These findings suggest a

potential role for fetal jaw movements in TMJ development,

aswellas that of relatedmuscles.However, no investigation has

yet been undertaken in order to elucidate the precise effects of

prenatal jaw movement on the development of the temporalis

muscle. Our previous studies demonstrated that a restriction of

fetal movement of the TMJ in mice9,10 as well as that of the hip

joint in rats41,42 caused abnormal morphological changes in the

mandibular condyle and articular disc, and the femoral head,

respectively. Several studies have predicted that mechanical

forces are initial factors for tissue morphogenesis31,32 and

morphological changes in cartilaginous structures.43,44

contained inclusion bodies of various sizes (white arrow

sham-operated groups (A–C), the nuclei (arrows) were

ctron density, the muscle cells contained very few (if any),

ider gaps (double-ended arrows) between the cell

re observed in the E18.5 sutured group (I) than in the E18.5

oups. Mitochondria (black arrow heads) were expanded in

s observed in the E18.5 sutured group (I and J), whereas in

ted (E and G) groups, the mitochondria were of regular size

tron density was higher. Scale bars = 10 mm for (A–C and F–

a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 9 1 9 – 9 2 7926

Our present histological observations suggested that the

development of the temporalis muscle was more delayed in

the E18.5 sutured group with respect of the timeline of

development than in the E18.5 in utero control and the E18.5

sham-operated groups. Developmental characteristics of the

muscle in the E18.5 sutured group were generally similar to

those of the E17.5 in utero control group. However, curly or

wavy muscle fibres were frequently observed in the E18.5

sutured group, whereas such muscle fibres were observed in

both neither E17.5, E18.5 in utero control nor E18.5 sham-

operated groups. Histomorphometric study revealed that the

muscle fibre volume of the temporalis muscle, but not that of

connective tissue, was decreased in the E18.5 sutured group. A

TEM study revealed that in the E18.5 sutured group, the

muscles tended to be less mature than those of the E18.5 in

utero control and the E18.5 sham-operated groups, although

some immature muscle fibres were also identified in those

groups. It has been reported that certain experimental

treatments of the masticatory muscles can initiate cellular

changes, particularly changes in intercellular and extracellu-

lar organelles, in muscle cells.45–47 In addition to obtaining

findings suggestive of the immaturity of muscle cells, we also

observed abnormalities in the mitochondria and inclusion

bodies of unknown origin inside the muscle cells in the E18.5

sutured group; such changes also appeared to be related to the

restriction of jaw movement. These findings, taken together,

suggested that jaw movement restriction might have delayed

muscle differentiation and progress to maturity, and might

possibly have had other abnormal effects on the E18.5 sutured

group.

We therefore examined whether or not myogenic regulatory

factors might have been affected by the jaw-restrictive

treatment. We selected Myf-6 from among a number of

candidate factors, because MRF4/Mfy-6 mRNA begins to be

expressed in the masticatory muscles of the mouse embryo

from E13 to 15 (i.e., at a later time point than that of other

factors), and its expression increases steeply until to the time of

birth,16,17 this time frame roughly corresponds to the period,

from E15.5 to 18.5, when we restricted fetal jaw movement by

the suturing technique described above. However, in the

present study, Myf-6 expression in the temporalis muscle did

not significantly differ between the E18.5 sutured and E18.5

sham-operated groups, suggesting that the genetic commit-

ment to myogenesis was not affected by the present procedure.

Satellite cells are a type of stem cells related to the

differentiation of muscle fibres from the late gestation.27,28 In

the present study, we used TEM analysis of muscle fibres to

observe and count the satellite cells. We found that the number

of satellite cells was significantly greater in the E18.5 in utero

control group than in the E17.5 in utero control group, which is

consistent with the finding that these cells started to appear as

undifferentiated stem cells at the end of the second wave of

myogenesis at E17.5.28 However, we found no significant

differences between the E18.5 in utero control, E18.5 sham-

operated andE18.5suturedgroup.Therefore, these findingsmay

correspond to the result regarding Myf-6 expression, although

our TEM observations were limited to a very small area.

Although no significant changes were observed at theMyf-6

level or in the frequency of satellite cells in the E18.5 sutured

group, the present morphological findings suggested that the

development of the temporalis muscle was delayed by

approximately 1 day. Certain abnormalities were observed

in the muscle fibres of the E18.5 sutured group, which suggests

that jaw movement restriction indeed induced developmental

differences from the E18.5 in utero control and the E18.5 sham-

operated groups.

In conclusion, the histological, histomorphometric analy-

ses and TEM findings from the present in vivo study using the

exo utero method suggested that the fetal jaw movement plays

a role in the proper development of the temporalis muscle in

the prenatal period.

Conflict on interest

None.

Acknowledgements

We are grateful to Ms. Yumiko Takeda for her help with the

histological preparation and to Mr. T. Yoneyama of the Central

Research Laboratories of Shimane Medical University, for his

technical assistance with the preparation of the TEM speci-

mens.

Funding: A grant KAKENHI from the Japanese Ministry of

Education, Science, Sports and Culture through the Faculty of

Medicine, Shimane University.

Competing interest: None declared.

Ethical approval: This study was approved by the Ethical

Committee for Animal Research of Shimane University, and

all experimental procedures were performed according to the

institutional guidelines.

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