Fetal Jaw Movement Affects Condylar Cartilage Development

Abstract

Using a mouse exo utero system to examine the effects of fetal jaw movement on the development of condylar cartilage, we assessed the effects of restraint of the animals’ mouths from opening, by suture, at embryonic day (E)15.5. We hypothesized that pre-natal jaw movement is an important mechanical factor in endochondral bone formation of the mandibular condyle. Condylar cartilage was reduced in size, and the bone-cartilage margin was ill-defined in the sutured group at E18.5. Volume, total number of cells, and number of 5-bromo-2′-deoxyuridine-positive cells in the mesenchymal zone were lower in the sutured group than in the non-sutured group at E16.5 and E18.5. Hypertrophic chondrocytes were larger, whereas fewer apoptotic chondrocytes and osteoclasts were observed in the hypertrophic zone in the sutured group at E18.5. Analysis of our data revealed that restricted fetal TMJ movement influences the process of endochondral bone formation of condylar cartilage.

INTRODUCTION

The mandibular condyle, an integral part of the temporomandibular joint (TMJ), is actively involved in endochondral bone formation (Silbermann and Frommer, 1972) and contributes to the elongation of the mandibular ramus (Sarnat, 1966). The main events in the formation of this joint occur within a short period, and are completed by the end of the pre-natal period (Moffett, 1966). Previous studies have shown that chondrocyte proliferation and the thickness of condylar cartilage are modulated by mechanical factors associated with TMJ movement in the post-natal period (Simon, 1977; Hinton and Carlson, 1986; Kantomaa et al., 1994; Wang and Mao, 2002). While intra-uterine mandibular movements have been observed in mammals ([mice] Narayanan et al., 1971; [rats] Copray et al., 1988; [humans] Petrikovsky et al., 1999), the critical effects of fetal jaw movement on the development of the condylar cartilage of the TMJ remain unknown. Since the development of other joints has been suggested to be modified by intra-uterine movements (Mital and Millington, 1971; Persson, 1983; Kihara et al., 1998), we hypothesized that pre-natal jaw movement is an important mechanical factor in endochondral bone formation of the mandibular condyle.

To examine this hypothesis, we used a mouse exo utero method (Hatta et al., 2004) to manipulate fetal jaw movement. This system allowed us to examine the development of the mandibular condyle from the initial appearance of jaw movement, and to investigate the effects of mechanical factors on the development of joint cartilage without the involvement of nutritional factors.

MATERIALS & METHODS

Animals

Female Jcl:ICR mice aged 8–20 wks (CLEA, Tokyo, Japan) were used. The experimental procedures were performed according to the guidelines of the Institute of Experimental Animals of Shimane University.

Exo utero Surgery

Embryonic day (E) 0.0 was defined as midnight on the day when a vaginal plug was observed. Exo utero surgery was performed as described previously (Hatta et al., 1994, 2004). Briefly, at E15.5, pregnant dams were anesthetized with 50 mg/kg body weight (BW) pentobarbital. The embryos’ mandible and maxilla were fixed by an 8-0 nylon suture (Figs. 1A, 1B , 1C ), and these embryos were defined as the sutured group. Sham-operated embryos were defined as the non-sutured group. After operation, the embryos were allowed to develop exo utero in the abdominal cavity of the dams.

Histological Examination

At E16.5 (number of dams [Nd] = 3, number of embryos [Ne] =3) and E18.5 (Nd = 4, Ne = 7, 1 or 2 embryos from each dam), BW and crown rump length of embryos were measured. The condyles were fixed with Bouin’s solution for 48–72 hrs at room temperature, then embedded in paraffin. Five-μm serial sagittal sections were stained with hematoxylin and eosin (HE). Double-staining for bone and cartilage was performed with alizarin red and alcian blue (E18.5, Ne = 11, for each group). Osteoclasts were detected by histochemistry for tartaric acid alkaline phosphatase (TRAPase) activity (Nd = 4, Ne = 4), by the azo-dye method (Burstone and Weisburger, 1961).

Immunohistochemistry for Cell Proliferation and Apoptosis

To examine the effects of the experimental treatment on the proliferation of condylar cartilage cells, we injected 5-bromo-2′-deoxyuridine (BrdU) (Sigma, St. Louis, MO, USA) intraperitoneally into the dams (50 mg/kg BW in distilled water) 2 hrs before their death at E16.5 (Nd = 3, Ne = 3) and E18.5 (Nd = 4, Ne = 4), 1 and 3 days, respectively, after the operation. Sections were prepared as described previously (Hatta et al., 2002). Apoptotic cells were detected with polyclonal rabbit anti-single-stranded DNA (ssDNA) antibody (DAKO Japan, Kyoto, Japan) in the condylar cartilage at E18.5 (Nd = 4, Ne = 4) (Tsukahara et al., 2004).

Quantitative Study

The condylar cartilage was divided into 3 zones according to the criteria established by Keith et al.(1982) (Table, Figs. 2E–2G ).

The condylar cartilage samples at E16.5 and E18.5 were quantitatively analyzed by a random and systematic means of sampling (Hatta et al., 2002). From an entire series of sequentially numbered sections, a number between 1 and 5 was selected randomly as the first section, and thereafter every third section (d = 3) was chosen. Total volume, total cell number, the number of BrdU-positive cells, the BrdU labeling index, and the number of apoptotic cells in each zone were analyzed (see below). The number of cross-points (P) of a grid on the microscope inside the region of interest (ROI) was counted, and the area of the ROI was calculated as P•a(p), where a(p) is the area of the square between grid points according to the Cavalieri principle (Michel and Cruz-Orive, 1988). Volume was determined according to the following formula: V = d•ΣP•a(p)•t, where t is the average thickness of the section.

The grid was superimposed on the ROI with the use of appropriate software (Adobe PhotoShop, ver.5.0). The counting box located in the top left corner inside the ROI was numbered as 1, then every third box was selected (approximately 40 boxes per section). The mean density of nuclei was multiplied by the area of the ROI in each section, so that we could obtain the estimated cell number in each ROI (n). Abercrombie’s correction (Abercrombie, 1946) was used to estimate the total number of cells (N) according to the following formula: N = n•t/(t+ϕ), where (ϕ) indicates the mean diameter of the nuclei.

Statistical Analysis

The procedures in the present study were performed in a blinded manner. Data are presented as mean ± standard deviation. We applied repeated-measures ANOVA for comparisons of groups, one-way ANOVA in the morphometric study, and Fisher’s exact probability test in the histological study. P < 0.05 was considered as significant.

RESULTS

Macroscopic and Histological Findings

Using an exo utero surgery approach, we obtained a reliable restriction of jaw movement in the mouse embryos (Figs. 1A and 1B for E15.5, 1C for E18.5). The surgical procedures did not induce general growth retardation, as was also the case in our previous study (Hatta et al., 1994). However, double-staining for bone and cartilage revealed a deformity in the mandibular condyle of the sutured group at E18.5. The total condylar cartilage was significantly reduced in size, and the margin between cartilage and bone was ill-defined in the sutured group (81.8%, 9 out of 11 embryos, P = 0.001) (Fig. 2B), in comparison with the non-sutured group (9.1%, 1 out of 11 embryos) (Fig. 2A ).

On HE-stained sections, the transitory region between the condylar cartilage and bone was clearly defined in the non-sutured group at E18.5 (Fig. 2C ), corresponding to the erosion zone; however, there was a round and broad border between the bone and cartilage in the sutured group (85.7%, 6 out of 7 embryos, P = 0.03) (Fig. 2D ), which was suggestive of the non-resorbed bone collar.

In the condylar head of the non-sutured group, cells in the mesenchymal zone were regularly arranged in several layers (Fig. 2E ). However, in the sutured group, the mesenchymal zone had fewer layers, and the arrangement of the cells was disturbed (Fig. 2F ). In the pre-hypertrophic zone, a gradual distribution of several cell layers was maintained in the non-sutured group (Fig. 2E ), whereas the sutured group had fewer cell layers with anuclear spaces, which showed discontinuity of the cellular arrangement (Fig. 2F ). Furthermore, in the sutured group, we observed significantly more frequently acellular structures that were stained by HE in a manner similar to that of bone matrix (Figs. 2G vs. 2E , also see Figs. 2C , 2D ), and that were radially arranged to penetrate from the pre-hypertrophic to the mesenchymal zones (sutured, 85.7%, 6 out of 7 embryos; non-sutured, 14.3%, 1 out of 7 embryos, P = 0.03) (Fig. 2G ). In the hypertrophic zone of the sutured group, the chondrocytes tended to be larger and included more cytoplasmic vacuoles (Figs. 2F , 2G vs. 2E).

TRAPase reaction was detected in the osteoclasts of the erosion zone at E18.5, and significantly fewer TRAPase-positive osteoclasts (Figs. 2C , 2D insets) were observed in the sutured group (n = 115.1 ± 20.0) than in the non-sutured group (n = 176.2 ± 29.5) (F = 14.68, P = 0.005).

Morphometric Study

At E16.5, the sutured embryos differed significantly from the non-sutured embryos in terms of the volume of the condylar cartilage (F = 12.43, P = 0.02) (Fig. 3A ), and the volume of the mesenchymal zone was significantly reduced in the sutured group (F = 10.86, P = 0.03) (Fig. 3A ). The sutured embryos also differed significantly from the non-sutured embryos in terms of the total cell number in the condylar cartilage (F = 35.12, P = 0.004), and the total cell numbers were significantly lower in the mesenchymal (F = 32.11, P = 0.004) and pre-hypertrophic zones (F = 22.94, P = 0.008) (Fig. 3B ). At E18.5, we obtained results similar to those obtained at E16.5, with respect to the volume of the condylar cartilage (F = 3.67, P = 0.07), the volume of the pre-hypertrophic zone (F = 11.22, P = 0.005) (Fig. 3C ), the total cell number in the condylar cartilage (F = 16.25, P = 0.001), and total cell numbers in the mesenchymal (F = 15.27, P = 0.002) and pre-hypertrophic zones (F = 21.51, P = 0.0006) (Fig. 3D ).

Furthermore, volume/cell number, which corresponds to cell size plus extracellular matrix per single cell (cell teritory), was significantly larger in the hypertrophic zone in the sutured group (0.64 ± 0.12 μm³) than in the non-sutured group (0.51 ± 0.07 μm³) (F = 62.60, P = 0.001) at E18.5.

BrdU and ssDNA Immunostaining

At E16.5, the number of BrdU-positive cells was significantly lower in the sutured group than in the non-sutured group in the entire cartilage (F = 62.04, P = 0.001) and in the mesenchymal zone (F = 73.28, P = 0.001) (Fig. 3E ). The sutured group differed significantly from the non-sutured group in terms of the BrdU-labeling index (F = 35.54, P = 0.004), which was significantly affected in the mesenchymal zone (F = 23.68, P = 0.008) (Fig. 3F ). At E18.5, similar results were obtained, although there was no significant difference in the labeling index (not shown).

Significantly fewer apoptotic chondrocytes were observed in the hypertrophic zone in the sutured group (n = 78.6 ± 3.6) than in the non-sutured group (n = 118.6 ± 13.1) at E18.5, as observed by anti-ssDNA antibody staining (F = 13.48, P = 0.02).

DISCUSSION

Intra-uterine jaw movement in mice begins on E16 (Narayanan et al., 1971), and human intra-uterine mandibular movements have been detected by ultrasonography (Petrikovsky et al., 1999), suggesting the potential role of jaw movement in TMJ development. Disturbances in pre-natal TMJ movement could be involved in the etiology of developmental hypoplasia of the mandibular condyle (Keith, 1982).

Previous study of post-natal rats and rabbits has shown that mechanical forces affected not only the proliferation of chondroprogenitor cells, but also their differentiation and maturation (Kantomaa et al., 1994; Wang and Mao, 2002; Rabie et al., 2003b). Post-natal development of the condylar cartilage has been investigated in rats by the use of several different approaches, including examination of changes in the force of TMJ movement following administration of a soft or hard diet (Simon, 1977; Hinton and Carlson, 1986; Hinton, 1993), and appliance therapy (Mao et al., 1998; Rabie et al., 2003a; Fuentes et al., 2003). However, to support nutritional demands, jaw movement was not completely restricted in these post-natal studies. The present exo utero approach avoided such nutritional limitations and thus provided the opportunity for the effects of fetal jaw movement on TMJ development to be investigated.

In this study, TMJ movement restriction reduced the size of the condylar cartilage and the number of cell layers in the mesenchymal zone. Analysis of the morphometric data revealed that movement restriction significantly decreased the volume and total cell number of the condylar cartilage. Cell number was found to decrease primarily in the mesenchymal zone following 1 day of restricted movement, which became more prominent and predominant in the pre-hypertrophic zone upon extended movement restriction for 3 days. These observations suggested that this decrease in cell number was responsible for the decrease in condylar cartilage volume. Movement restriction reduced the number of BrdU-positive cells in the mesenchymal zone, thus indicating reduced cellular proliferation. At E18.5, hypertrophic chondrocytes tended to be larger and to contain prominant vacuoles in the sutured group, as compared with the non-sutured group; moreover, cell territory (i.e., cell size plus extracellular matrix) in the hypertrophic zone was larger in the sutured group than in the non-sutured group. These findings suggested that the hypertrophic chondrocytes survived longer in the sutured group, due to the inhibition of cartilage resorbtion. Whereas the erosion zone was observed in the non-sutured group, the border between bone and cartilage was rounded and broad in the sutured group, which was suggestive of a non-resorbed bone collar. Since endochondral bone formation begins with the resorption of the bone collar by multinucleated osteoclasts (Silbermann and Frommer, 1972), the significantly reduced number of TRAPase-positive osteoclasts, as well as apoptotic chondrocytes, indicated that resorption of the bone collar was inhibited in the sutured group. The present findings thus indicated that endochondral bone formation was inhibited in the sutured group. There was a disturbance of normal cellular arrangement in the mesenchymal and pre-hypertrophic zones. Previous studies of the rat hip joint have demonstrated similar abnormal morphological changes in the femoral head after pre-natal movement restriction (Kihara et al., 1998; Hashimoto et al., 2002). Taken together, these results suggest that a restriction of TMJ movement might also lead to the abnormal differentiation of chondrocytes.

Progenitor cells have high proliferative capacity, and are uniquely able to differentiate into either chondrocytes or osteoblasts, depending on their biomechanical environments (Strauss et al., 1990). Since early maturation of chondrocytes stops chondrogenesis and induces osteogenesis, maintenance of the chondroblast layer is thus a major regulatory point for the continuance of condylar growth (Kantomaa and Pirttiniemi, 1996). Simon (1977) reported observing reduced thickness of the degenerating condylar cartilage, due to accelerated differentiation in post-natal rats fed a soft diet; the results of that study were attributed to compressive force, which was deemed necessary for normal maturation of the condylar cartilage. Kantomaa et al.(1994) also observed, in rats, that the maturation of the condylar cartilage was enhanced when the compressive forces were reduced. Modified differentiation and maturation of the mesenchymal cells by reduced masticatory function are consistent with the present findings; this putative mechanism would also account for the post-natal findings in rats fed a soft diet (Bouvier and Hylander, 1984), as well as for the results obtained after rat incisors were clipped (Hinton and Carlson, 1986)—namely, that the proliferating cell layer was less thick, and that the entire condylar cartilage was diminished in size, in comparison with those of control rats.

The present morphological and morphometric findings clarified that pre-natal TMJ movement plays an essential role in all processes of endochondral bone formation, i.e., proliferation, differentiation, and apoptosis of chondrocytes, as well as in resorption of the bone collar and cartilage matrix of the condylar cartilage.

Table.

Three Different Zones of Condylar Cartilage^a

Zones	Characterization
^a See Fig. 2E.
^b The pre-hypertrophic zone includes the proliferating zone (Keith et al., 1982), since the border in between was unclear in some cases.
Mesenchymal	The outermost zone, where cells are flat, condensed, and undifferentiated.
Pre-hypertrophic^b	The intermediate zone, where cells are arranged uniformly basically in a columnar fashion, with a small nucleus, without cytoplasmic vacuoles.
Hypertrophic	The innermost zone, where cartilage cells are enlarged, with a nucleus and prominent vacuoles. The cells are haphazardly arranged with matrix in between.

Figure 1.

Exo utero surgery. (A) At E15.5, the abdominal cavity of the dam was surgically revealed, and embryos covered with the embryonic membrane were exposed; then, the mandible and maxilla were fixed by suture, while the umbilical cord and placenta were kept intact inside the amnion. (B) Schematic representation of the suture fixation in (A) performed to restrict fetal jaw movement. (C) Sutured (left) and non-sutured (right) embryos obtained at E18.5. Arrows indicate suture fixation with 8-0 nylon.

Figure 2.

Macroscopic (A,B) and histological (C,D,E,F,G) views of the mandibular condyle of non-sutured (A,C,E) and sutured (B,D,F,G) embryos at E18.5. (A) Non-sutured embryos (10 out of 11) show a clear bone-cartilage margin (arrow) between the condylar head and neck. (B) In contrast, sutured embryos (9 out of 11) show a smaller volume of cartilage with an ill-defined margin (arrow). (C,D) Photomicrographs of the condyle. (C) Non-sutured embryos exhibit a clearly defined junction (arrows) between the cartilage and bone, corresponding to the erosion zone. The boxed area is magnified in E. (D) The hypertrophic zone in the sutured embryos (6 out of 7) had a broader and rounded junction (arrows). The boxed areas are magnified in F and G. The insets in C and D show the junction stained for TRAPase. Fewer osteoclasts (arrowheads) with a strong reaction to TRAPase were observed in the sutured group (insets D vs. C). (E,F,G) Histological changes in the condylar cartilage at E18.5. (E) The condylar cartilage in the non-sutured embryos was subdivided into mesenchymal (M), pre-hypertrophic (P), and hypertrophic (H) zones. (F) In the sutured embryos, the mesenchymal and pre-hypertrophic zones showed fewer and irregularly arranged cell layers. In the pre-hypertrophic zone, the anuclear space led to a discontinuity of the cellular arrangement (arrowheads). The pre-hypertrophic zone appeared to be underdeveloped structurally and was significantly thinner than that of the non-sutured embryos (E). (G) In the sutured embryos (6 out of 7), the acellular structure in the pre-hypertrophic zone was HE-stained in a manner similar to that of bone matrix (arrows). (A,B) Double-staining with alizarin red and alcian blue. (C,D,E,F,G) HE staining. Scale bars: 100 μm.

Figure 3.

Morphometric study of the developing condylar cartilage. Comparison of cartilage volume (A,C), and total number of cells (B,D) between the non-sutured and sutured groups at E16.5 (A, B; N = 3) and E18.5 (C, D; N = 7). (E) BrdU-positive cells and (F) BrdU-labeling indices at E16.5 (N = 3). For the details, see RESULTS. M, mesenchymal zone; P, pre-hypertrophic zone; H, hypertrophic zone; □, non-sutured group; ▪, sutured group; data are represented as mean ± SD.

Footnotes

Notes

Acknowledgements

We are thankful to Ms. Yumiko Takeda for her help in histological preparation. This study was supported by a grant from the Japanese Ministry of Education, Science, Sports and Culture through the Faculty of Medicine, Shimane University.

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