Scholarly article on topic 'The imbalance of masticatory muscle activity affects the asymmetric growth of condylar cartilage and subchondral bone in rats'

The imbalance of masticatory muscle activity affects the asymmetric growth of condylar cartilage and subchondral bone in rats Academic research paper on "Biological sciences"

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{"Mandibular asymmetry" / "Condylar cartilage" / "Subchondral bone" / Asporin / TGF- / β1}

Abstract of research paper on Biological sciences, author of scientific article — Mutsumi Miyazaki, Ikuo Yonemitsu, Maki Takei, Ikuko Kure-Hattori, Takashi Ono

Abstract Objective To examine the effects of imbalance of masticatory muscle activity of the rat mandible on the condylar cartilage and subchondral bone during the growth period. Design Forty 5-week-old male Wistar rats were randomly divided into experimental (n =20) and control (n =20) groups. In the experimental group, the left masseter muscles were resected. The rats were sacrificed at 7 or 9 weeks of age in both groups. Microcomputed tomography was used to determine the three-dimensional morphology and cancellous bone structure. For histological and histochemical examination, 5-μm-thick serial frontal sections of the condyle were stained with toluidine blue and immunostained with asporin and TGF-β1 to evaluate the promotion and inhibition of chondrogenesis. Results In the experimental group, microcomputed tomography analysis showed asymmetric growth; the resected side condyles showed degenerative changes. Histological analysis showed that the total cartilage in the central region of the resected side was significantly thinner than in the non-resected side in the experimental group, as well as in the control group. Compared with the control group, the expression of asporin was significantly higher in the resected side, and significantly lower in the non-resected side. In contrast, the expression of TGF-β1-immunopositive cells in the non-resected side was significantly higher than in the resected side and the control group. Conclusions These findings imply that lateral imbalance of masseter muscle activity lead to inhibition of chondrogenesis and induce asymmetric formation of the condyle during the growth period.

Academic research paper on topic "The imbalance of masticatory muscle activity affects the asymmetric growth of condylar cartilage and subchondral bone in rats"

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Archives of Oral Biology

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The imbalance of masticatory muscle activity affects the asymmetric growth of condylar cartilage and subchondral bone in rats w

Mutsumi Miyazaki, Ikuo Yonemitsu*, Maki Takei, Ikuko Kure-Hattori, Takashi Ono

Orthodontic Science, Department of Orofacial Development and Function, Division of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan

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ARTICLE INFO

ABSTRACT

Article history:

Received 15 June 2015

Received in revised form 13 November 2015

Accepted 23 November 2015

Keywords:

Mandibular asymmetry Condylar cartilage Subchondral bone Asporin TGF-b1

Objective: To examine the effects of imbalance of masticatory muscle activity of the rat mandible on the condylar cartilage and subchondral bone during the growth period.

Design: Forty 5-week-old male Wistar rats were randomly divided into experimental (n = 20) and control (n = 20) groups. In the experimental group, the left masseter muscles were resected. The rats were sacrificed at 7 or 9 weeks of age in both groups. Microcomputed tomography was used to determine the three-dimensional morphology and cancellous bone structure. For histological and histochemical examination, 5-|xm-thick serial frontal sections of the condyle were stained with toluidine blue and immunostained with asporin and TGF-p1 to evaluate the promotion and inhibition of chondrogenesis. Results: In the experimental group, microcomputed tomography analysis showed asymmetric growth; the resected side condyles showed degenerative changes. Histological analysis showed that the total cartilage in the central region of the resected side was significantly thinner than in the non-resected side in the experimental group, as well as in the control group. Compared with the control group, the expression of asporin was significantly higher in the resected side, and significantly lower in the non-resected side. In contrast, the expression of TGF-p1-immunopositive cells in the non-resected side was significantly higher than in the resected side and the control group.

Conclusions: These findings imply that lateral imbalance of masseter muscle activity lead to inhibition of chondrogenesis and induce asymmetric formation of the condyle during the growth period. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

The temporomandibular joint (TMJ) is a bilateral synovial articulation, the most common and most movable type of joint in the body, and is subjected to a variety of loads during jaw movements (Beek, Koolstra, van Ruijven, & van Eijden, 2001). Functionally, the TMJ performs not only hinge movements but also sliding movements; therefore, it is considered that compressive, shearing, and other complex forces are exerted on the mandibular condyle during masticatory function.

Condylar cartilage acts as a part of the articulatory joint and as a major growth site for the mandible. Growth and development of the mandibular condyle substantially influence morphogenesis of the craniofacial skeleton and function of the TMJ (Hinton, 1991).

Abbreviations: Micro-CT, microcomputed tomography; OA, osteoarthritis; PBS, phosphate buffered saline; PLAP-1, periodontal ligament-associated protein-1; SLRPs, small leucine-rich proteoglycans; TGF-b, transforming growth factor-b; TMJ, temporomandibular joint.

* Corresponding author. E-mail address: yoneman.orts@tmd.ac.jp (I. Yonemitsu).

Designated as secondary cartilage, condylar cartilage differs from other cartilaginous tissues in its histological organization, its response to biomechanical stress and humoral factors, and its mechanisms of proliferation, differentiation and calcification (Berraquero, Palacios, Gamallo, de la Rosa, & Rodriguez, 1995).

Articular cartilage requires motion and joint loading to maintain its proper physical and biochemical properties (Leroux et al., 2001). In response to injury or extraordinary mechanical stress, cartilage cells decrease in number (Griffin & Guilak, 2005). Because articular cartilage lacks nerves and blood vessels, as well as lacking in self-healing ability, growth factors allow chondro-cytes to differentiate to compensate for this decrease.

The transforming growth factor-b (TGF-b) family consists of over 35 factors, including TGF-b, activins and bone morphogenetic proteins (de Caestecker, 2004). These factors play vital roles in the development and homeostasis of various tissues; regulation of cell proliferation, differentiation, apoptosis and migration; and control of extracellular matrix synthesis and degradation (Blaney Davidson, van der Kraan, & van den Berg, 2007; Redini et al., 1991). Moreover, these factors mediate cell and tissue responses to injury and modulate immune functions (Javelaud & Mauviel, 2004).

http://dx.doi.org/10.1016/j.archoralbio.2015.11.020

0003-9969/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

However, the unlimited action of the growth factor causes new problems such as abnormal cartilaginous increase, ossification, and tumor formation, so that it is necessary to regulate the system of chondrogenesis (Kizawa et al., 2005). Asporin is expressed in various tissues, including articular cartilage, aorta, uterus, heart and liver, with especially high expression in cartilage (Lorenzo et al., 2001; Henry et al., 2001). Also known as periodontal ligament-associated protein-1 (PLAP-1), asporin has been investigated as a regulator of TGF-b1, and is a new member of the family of small leucine-rich proteoglycans (SLRPs) (Lorenzo et al., 2001; Yamada et al., 2007; Iozzo & Murdoch, 1996). Asporin has been reported to be involved in the development and progression of knee osteoarthritis (OA) by suppressing the anabolic action of TGF-b1 (Kizawa et al., 2005; Lorenzo et al., 2001 ; Jiang et al., 2006), but few studies have revealed any relationship between asporin and TMJ OA.

Many studies have been conducted investigating the association between facial morphology and muscle function (Odman, Mavropoulos, & Kiliaridis, 2008; Kim et al., 2008; Navarro, Delgado, & Monje, 1995; Ingervall & Thilander, 1974). Optimal masticatory muscle force during growth is necessary for normal mandibular growth (Monje, Delgado, Navarro, Miralles, & Alonso del Hoyo, 1994), and masticatory muscle function is a determinant of bone quality in the growing mandible (Bresin, Kiliaridis, & Strid, 1999; Poikela, Kantomaa, Tuominen, & Pirttiniemi, 1995).

Mandibular asymmetry has been related to the experimental unilateral removal of the masseter muscle in animal models (Rodrigues, Traina, Nakamai, & Luz, 2009; Avis, 1961). Similar deformities or asymmetries have been produced by resecting either the muscles (Sarnat, 1988) or the nerves of mastication (Kitagawa et al., 2002).

According to previous clinical studies, patients with facial asymmetry due to severe unilateral hypoplasia of the masticatory muscles show a tendency toward chewing on the normal side. The radiographic findings of a shortened ramus, smaller condyle and underdeveloped gonion on the hypoplastic side were consistent with previous studies (Sakamoto & Yoda, 2004; Parmar, Watkin-son, & Fieldhouse, 1996).

Although it is known that changes in mechanical stress affect cartilage metabolism, the mechanism is still unclear. There is little evidence demonstrating the presence and immunolocalization of asporin within the area of the TMJ. Therefore, the aim of this study was to examine the effects of masticatory muscle imbalance on the morphogenesis of the condyle and localization of asporin and TGF-b1 in the condylar cartilage.

2. Materials and methods

2.1. Animals and experimental design

The experimental protocol involving animals was approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (#015177A), and the University's Guidelines for Animal Experimentation were followed throughout the study. Forty 5-week-old male Wistar rats were randomly divided into two groups. In the experimental group (n = 20), the left masseter muscles were resected, using a method reported previously (Yonemitsu, Muramoto, & Soma, 2007). The rats were fed pellets only. Briefly, the rats were deeply anesthetized with diethyl ether and an intraperitoneal injection of 8% chloral hydrate using 1 ml/200 g of body weight. After shaving a wide area in the left gonial region, an incision was made and the masticatory muscles were exposed. All superficial and deep portions of the left masseter muscles were excised at each end and removed without damaging any major blood vessels or nerves around the muscles. The wounds were sutured, and amoxicillin (ICN Biomedicals Inc., Ohio, USA) (9 mg/60 g of body weight) was injected to prevent infection. The other group of rats (n = 20) served as control.

Morphological changes were evaluated 2 and 4 weeks after surgery (at age 7 and 9 weeks), while the rats were in the pubertal growth stage.

2.2. Microcomputed tomography analysis

The animals were perfused intracardially with 4% paraformal-dehyde in 100 mM phosphate buffer (pH 7.4). To detect the three-dimensional morphology and cancellous bone structure of the condyles, we used microcomputed tomography (micro-CT) (SMX-100CT, Shimadzu, Kyoto, Japan) with an output of 75 kV and 140 mA. Frontal cross-sections were taken initially followed by three-dimensional reconstruction to provide a histomorphometric view. The region of interest for the micro-CT analysis was determined as the midpoint of the maximum width between the lateral and medial poles of the condyle. A software package (TRI/3D-BON, Ratoc, Tokyo, Japan) was used to analyze the results (Kuroda et al., 2011). Three cubic frames (each 0.3 mm x 0.3 mm x 0.3 mm) under the osteochondral interface were located at the middle of the lateral, central and medial aspects of the mandibular condyle. Within the selected frames, we calculated bone volume/ trabecular volume (BV/TV), bone mineral density (BMD), bone surface/bone volume (BS/BV), Tb.Th (trabecular thickness), trabecular number (Tb.N) and trabecular separation (Tb. Sp).

(A) (B)

Fig. 1. (A) Frontal section of the mandibular condyle. The upper portion of the condylar head was divided into three regions of 60° each: the lateral, central, and medial regions. Bar = 200 mm. (B) Four layers of cartilage are shown: fibrous layer (F); proliferative cell layer (P); mature cell layer (M) and hypertrophic cell layer (H). The number of asporin and TGF-ß 1 immunopositive cells was calculated in an area 200 mm x 140 mm in the proliferative and mature layers of the superior region of the condylar cartilage. Bar = 50 mm.

2.3. Histological tissue preparation

After micro-CT scanning, the TMJs were dissected and immersed in the same fixative overnight, decalcified in 4.13% EDTA (pH 7.4) at 4 °C for 6 weeks, and embedded in paraffin (Yonemitsu et al., 2007). Tissue blocks were cut frontally at a 5-mm thickness with a microtome (RM2155; Leica Co., Ltd., Nussloch, Germany) (Kure-Hattori et al., 2012). Sections were stained with toluidine blue for general histological observation and the total thickness of the cartilage stained with toluidine blue was measured.

2.4. Histomorphometry and quantitative analysis

A horizontal line was drawn through the most prominent lateral and medial points of the condylar head in the frontal plane. From the middle point of the line, the upper portion of the condyle was divided into three regions at 60° angles - lateral, central, and medial regions - by a method reported previously (Fig. 1A) (Kure-Hattori et al., 2012; Sato, Muramoto, & Soma, 2006). Fig. 1B shows

the four layers of cartilage: fibrous layer (F); proliferative cell layer (P); mature cell layer (M) and hypertrophic cell layer (H).

2.5. Immunostaining of asporin and TGF-fi1

After rinsing in PBS, the prepared sections were treated with a solution of 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. Asporin and TGF-b1 polyclonal antibody (Santa Cruz, CA, USA) were applied at a dilution of 1:200 at 4 °C overnight. After rinsing in phosphate buffered saline (PBS), the sections were incubated with biotinylated universal secondary antibody (VectastainTM Universal Quick Kit, Vector Laboratories, Burlingame, CA, USA) for 10 min at room temperature. Next, the sections were incubated with streptavidin peroxi-dase complex (VectastainTM Universal Quick Kit) for 5 min. The immunoreactive sites were visualized with 0.02% 3,3-diamino-benzidine tetrahydrochloride and 0.01% hydrogen peroxide in 0.05 M Tris-HCl buffer (pH 7.4). In each experiment, negative controls were included omitting the primary antibody. Sections were then mounted with glycerin. The number of asporin

Fig. 2. Data were taken from the rats in the control group (a and d), the non-resected side in the experimental group (b and e) and the resected side in the experimental group (c and f) at 7 weeks (a-c) and 9 weeks (d-f). (A) Horizontal micro-CT views of the condyle. (B) Three frames (each 0.3 mm x 0.3 mm x 0.3 mm) under the osteochondral interface were located at the middle of the mandibular condyle.

Fig. 3. Graphs indicating micro-CT data evaluating condylar trabecular bone in the control, non-resected side and resected side. Within the selected frames, we calculated bone mineral density (BMD) (A), bone volume/trabecular volume (BV/TV) (B), bone surface/bone volume (BS/BV) (C), trabecular number (Tb.N) (D), trabecular thickness (Tb. Th) (E) and trabecular separation (Tb.Sp) (F) Abbreviations: NRS, non-resected side in the experimental group; RS, resected side in the experimental group. Asterisks indicate significant differences (p < 0.05) between NRS and RS measured at that time.

5 " ■<-'-1 1 1-'-f"—1-

f- Control NRS RS Control NRS RS

Fig. 4. (A) Frontal section of the condylar cartilage stained with toluidine blue at 7 and 9 weeks in the control group (a and d), non-resected side (b and e) and resected side (c and f). Compared with the control group, total cartilage thickness increased in the non-resected side (b) and decreased in the lateral region of the resected side (c). Bar = 200 mm. (B) The central part of the chondroblastic layer at 7 and 9 weeks in the control group (a and d), non-resected side (b and e) and resected side (c and f). (C) Total cartilage thickness of the lateral region, central region and medial region in the three groups. Abbreviations: NRS, non-resected side in the experimental group; RS, resected side in the experimental group.

and TGF-b1 immunopositive cells was calculated in an area 200 |m x 140 |m in the proliferative and mature cell layer of the middle of the central region of the condylar cartilage, in which asporin and TGF-b1 were localized (Fig. 1B).

2.6. Statistical analysis

Statistical evaluations were performed with SPSS for Windows (IBM SPSS Statistics Version 20.0, Armonk, NY, USA). Bonferroni's multiple-comparison post hoc test was used for all statistical analysis in this study. p-values less than 0.017 (0.05/3) were considered to be statistically significant.

3. Results

3.1. Body weight

No significant body weight differences were observed between the control and experimental groups (data not shown).

3.2. Micro-CT analysis of subchondral bone in the mandibular condyle

In the experimental group, micro-CT analysis showed asymmetric growth; in particular, the mandibles shifted to the resected side (data not shown). Subchondral bone increased in size with increasing age. The horizontal view showed that the condyles in the resected side were smaller than in the control group and the non-resected side of the experimental group (Fig. 2A). In addition,

the amount of trabecular bone was lower in the resected side compared with the control and the non-resected side (Fig. 2B). BMD in the lateral region of the resected side was significantly lower than in the control group and the non-resected side at 9 weeks only. In the central region, BMD in the resected side was significantly lower at both 7 and 9 weeks. However, no significant difference was seen in the medial region (Fig. 3A).

Because the most distinctive changes in BMD occurred in the central region, we focused on the central region to analyze our other measurements. Histomorphometric analysis showed that BV/TV and BS/BV (indicating the proportion of space of a cube occupied by trabecular bone) were lower in the resected side than in the non-resected side and the control group, and the same difference was noted in BMD and BV/TV. Tb.N and Tb.Sp were also lower in the resected side than in the non-resected side; in particular, the subchondral bone increased in size and thickness with increasing age. However, there was no significant difference in Tb.Th between the control and experimental groups (Fig. 3B-F).

3.3. Histomorphometry with toluidine blue staining

At 7 and 9 weeks, the cartilage layers were clearly identifiable as areas of metachromatic staining with toluidine blue (Fig. 4A and B). In the control group, the size of chondrocytes at 9 weeks was greater than at 7 weeks (Fig. 4B-a and d). However, chondrocyte size decreased in the resected side between 7 weeks and 9 weeks (Fig. 4B-c and f). Histological analysis showed that total cartilage thickness in the central region increased over the experimental

Fig. 5. (A) Immunostainingofasporin in chondrocytes at 7 and 9 weeks. Immunopositive cells in the mature cell layer are stained brown (arrows). Control: a andd; NRS: band e; RS: c and f. Bar = 50 mm. (B) Expression of asporin immunopositive cells at 7 and 9 weeks (areas shown in Fig. 2B). Significant differences (p < 0.05) are indicated by asterisks.

period in the control group and the non-resected side, while there was no marked change in the resected side. The total cartilage thickness of the central region of the resected side was significantly less than that of the control group; conversely, it was significantly thicker in the non-resected side than in the resected side. Total cartilage thickness was significantly less in the resected side condyles than in the non-resected side and control group condyles (Fig. 4C).

Histomorphometry showed that the condyle of the non-resected side became more prominent because of an increase in the thickness of the cartilage layers in the middle region at 9 weeks (Fig. 4A-e), while the resected side condyle became flattened in the lateral region (Fig. 4A-f) compared with the condyles in the control group.

3.4. Quantitative analyses of asporin and TGF-fi1 immunopositive cells

As shown in Fig. 5A, asporin immunopositive cells were found in the mature cell layer of the control group. In the non-resected side, only a few asporin immunopositive cells were found at 7 and 9 weeks. In the resected side, however, many cells showed positive cytoplasmic immunolocalization of asporin, particularly at 9 weeks. The level of asporin immunopositive cells was significantly higher in the resected side than in the control group and the non-resected side at both 7 and 9 weeks (Fig. 5B).

In the control group, TGF-b1 expression was observed particularly in the proliferative and mature layers, although as the rats grew and the chondrocytes matured, the expression of TGF-b1 decreased. Compared with the control group, the expression of TGF-b1 was higher in the non-resected side; but significantly lower in the resected side at 7 and 9 weeks (Fig. 6A and B). No immunopositive cells were observed in either the asporin or TGF-b1 negative controls. (Fig. is not shown.)

4. Discussion

The present study demonstrated the influence of imbalanced masseter activity on the condylar cartilage during the growth period, which has not previously been clearly understood. This model aimed to replicate the condition of patients with unilateral mastication. The experimental period was designed to span the transition from early puberty (5 weeks) to young adulthood (9 weeks) in rats (Miki, 1972). In the experimental group, unusual jaw movement was observed during mastication; that is, grinding only on the non-resected side, with the resected side not functioning at all. Experimental rats took a long time to eat, but they ate almost the same amount as the control rats. The asymmetrical chewing trajectory was reported in the animal model of unilateral resection of the facial nerve, leading to paralysis in the masseter muscle in growing rabbits (Kitagawa et al., 2002). The study showed that the animals were not able to chew food on the denervated side, but they chewed unilaterally on

Fig. 6. Immunostaining of TGF-P1 in chondrocytes at 7 and 9 weeks. Immunopositive cells in the proliferative and mature layers are stained brown (arrows). (A) Control: a and d; NRS: b and e; RS: c and f. Bar = 50 mm. (B)The expression ofTGF-b1 immunopositive cells at 7 and 9 weeks (areas shown in Fig. 2B). Significant differences among the three groups are marked with asterisks (p < 0.05).

the surgically unmanipulated side, as determined by visual inspection. The result is similar to our model.

Resection of the unilateral masseter muscle in rats showed that the mandible was warped and smaller on the ipsilateral side and there was a severe supraeruption of the incisors, which also caused an open bite in molars (Horowitz & Shapiro, 1955). The loss of attachment of the masseter muscle function would reduce the loading on the mandible, including condyles.

It is unlikely that the surgical procedure was responsible for the changes observed, because previous studies that used a bilateral masseter resection model showed similar changes in body weight during growth (Monje et al., 1994; Kikuchi, Lu, Sebata, & Yamamoto, 1978). Many studies have observed a lateral shift in the rat mandible caused by environmental factors such as functional appliances (Kure-Hattori et al., 2012; Sato et al., 2006; Wattanachai, Yonemitsu, Kaneko, & Soma, 2009; Fuentes et al., 2003), unilateral muscle resection (Rodrigues et al., 2009), or atrophy, although the rodent TMJ performs almost exclusively antero-posterior movement rather than lateral movement (Kwon, Park, Lee, Park, & An, 2007). Therefore, it is an effective method to analyze the frontal section of the TMJ.

Our study revealed asymmetric changes in the subchondral bone and condylar cartilage.

Morphologically, the subchondral bone increased in size with increasing age in the control group. In the experimental group, the mandible shifted to the resected side and the condyles developed in unusual shapes; in particular, the resected side condyles were smaller than the control and non-resected side condyles. In the resected side, BMD, BV/TV, BS/BV and Tb.N decreased, while Tb.Sp increased, at 7 and 9 weeks. However, there was no significant difference in Tb.Th between the control and experimental groups at 7 and 9 weeks. These changes suggest that less loading affects subchondral bone degeneration characterized as decreased BMD. The resected side condyles receive less loading because of muscular depression and asymmetric masticatory activity during growth, resulting in the development of abnormal mandibular bone.

A previous study using the same experimental model evaluated by cephalograms found contour alterations in the angular process and significant differences in the length of the maxilla, the height of the mandibular body and the length of the mandible (Rodrigues et al., 2009). These results are consistent with our findings of asymmetry. In the present study using micro-CT, we revealed not only morphological alterations, but also changes in the mineralization and architectural properties of the subchondral bone. Our study provided a three-dimensional analysis of changes in the symmetry in the subchondral bone of the mandibular condyle of growing rats.

Histomorphometry showed that at 7 weeks there was no significant difference between the control and experimental groups. At 9 weeks, the condyle on the non-resected side became more prominent because of an increase in the thickness of the cartilage layer in the middle region (Fig. 4A-e), while the resected side condyle became flattened in the lateral region (Fig. 4A-f) when compared with the condyles in the control group. Additionally, the total cartilage in the central region on the resected side was significantly thinner than in the control group. A previous study showed that rats fed with a soft diet had a smaller mandibular condyle and thinner cartilage layer than rats fed a hard diet (Teramoto, Kaneko, Shibata, Yanagishita, & Soma, 2003). Another study found that bilateral resection of the masseter muscles in rats reduced the mechanical stress and loading on the condyle, resulting in a decreased number of chondrocytes (Yonemitsu et al., 2007). Consistent with these studies, our findings of degenerative changes in the resected side condylar cartilage are likely to be a result of a reduction in the mechanical stress on the condyle.

Some studies investigating the time lag in the changes between bone and cartilage conclude that subchondral bone changes occur secondary to cartilage degradation (Chappard et al., 2006; Bobinac, Spanjol, Zoricic, & Maric, 2003; Yamada, Healey, Amiel, Lotz, & Coutts, 2002), while others report that subchondral bone changes precede cartilage degradation (Blair-Levy et al., 2008; Hayami et al., 2006). In the present study, the degenerative changes in the subchondral bone appeared significantly at 7 and 9 weeks; however, cartilage degradation appeared only at 9 weeks. It has been reported that internal changes in the subchondral bone make the cartilage more sensitive to biomechanical stimuli (Chen et al., 2014). Hypofunction of the TMJ can be induced by removing masticatory stimulation during the growing period, resulting in lower resistance of the TMJ to loading (Ikeda, Yonemitsu, Takei, Shibata, & Ono, 2014).

Our results suggest that muscular activity was weakened and jaw movement was limited on the resected side; this hypofunction then led to a decrease in chondrogenesis. On the non-resected side, however, the TMJ began to hyperfunction to compensate, leading to an increase in chondrogenesis.

TGF-b signaling stimulates proliferation and inhibits terminal differentiation of chondrocytes during chondrogenesis (Alvarez, Horton, Sohn, & Serra, 2001; Serra et al., 1997). TGF-b signaling is required for proper development of Meckel's cartilage, the condylar process and mandibular bone (Oka et al., 2007).

Asporin was expressed in the chondrocytes of the mature cell layer in the control group. Localization of asporin was seen within chondrocytes, although not in the extracellular matrix. These findings are similar to the results of a previous study which used the same anti asporin antibody in the rat humerus (Gruber et al., 2009). Our findings are also consistent with previous studies that revealed asporin expression in the meniscus of the human knee joint (Kou, Nakajima, & Ikegawa, 2007) and the intervertebral disc in an animal model (Gruber et al., 2009). In humans, asporin mRNA is expressed in many tissues. Higher asporin expression is found in OA cartilage than in normal cartilage (Kizawa et al., 2005; Lorenzo et al., 2001). An in vitro model of chondrogenesis demonstrated that asporin expression decreased during cartilage differentiation (Ikegawa, 2008). Asporin exists in tissues that are subjected to mechanical loading (Onnerfjord, Khabut, Reinholt, Svensson, & Heinegard, 2012), and may play an important role in the regulation of mechanical loading.

In our study, the level of TGF-b1 immunopositive cells was higher at 7 weeks than at 9 weeks in all the condyles. TGF-b1 is a potent factor responsible for the synthesis of extracellular matrix. It acts through the TGF-b1 type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK) and the extracellular signalregulated kinase pathway (Laping et al., 2002). Our results indicate that the decreased mechanical loading on the resected side condyle might promote asporin to bind TGF-b1 and inhibit Smad signaling in chondrocytes, and cause endochondral ossification.

The present study found significantly higher asporin expression and lower TGF-b1 expression in the resected side condyles compared with those of the non-resected side and the control group. Asporin and TGF-b1 form a functional feedback loop; thus asporin is a critical regulator of TGF-b1 in the articular cartilage (Kou et al., 2007). Because asporin binds to TGF-b1, this may suppress its ability to promote chondrogenesis. Asporin, which controls the action of TGF-b1, is important in the metabolism of the extracellular matrix and the differentiation of chondrocytes.

In conclusion, the present study showed that mandibular asymmetry in growing rats was related to asporin expression in condylar cartilage, and unilateral masseter muscle resection affected three-dimensional bone formation and chondrogenesis of condylar cartilage.

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Competing interests

The authors have no conflict of interest to disclose.

Ethical approval

The experimental procedures described here were approved by the Institutional Animal Care and Use Committee (#0150177A) and performed in accordance with the Animal Care Standards of Tokyo Medical and Dental University.

Acknowledgement

This study was financially supported by Grants-in-Aid for Scientific Research (23792422 and 25463171) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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