effects of lateral pterygoid muscle hyperactivity on differentiation of mandibular condyles in rats

THE ANATOMICAL RECORD 241:328-336 (1995)

Effects of Lateral Pterygoid Muscle Hyperactivity on Differentiation of Mandibular Condyles in Rats

ICHIRO TAKAHASHI, ITARU MIZOGUCHI, MASANORI NAKAMURA, MANABU KAGAYAMA, AND HIDE0 MITANI

Department of Orthodontics (I.T., I.M., H.M.) and 2nd Department of Oral Anatomy (M.N., M.K.), School of Dentistry, Tohoku University, Aoba-ku, Sendai, Japan

ABSTRACT Background: The effects of biomechanical stress on the growth and development of the mandibular condyle have been studied by many investigators. However, the role of the lateral pterygoid muscle in this development is not clear.

Methods: Hyperfunction of the lateral pterygoid muscles of male 3-week- old Sprague-Dawley rats was induced by electrical stimulation, and the responses of the mandibular condyles were compared to control tissues by a double-fluorescent staining technique using polyclonal antibodies against type I and type I1 collagen. Electrical stimulation consisted of repeated application (5 seconds od5 seconds off) of a 5 Hz current for up to 7 days.

Results: In the first 2 days, cartilaginous tissues rich in type I1 collagen disappeared in the anterior and posterior areas, which were loaded by tensional force due to direct and indirect attachment of the lateral pterygoid muscles. Tissues in these areas were replaced by intramembranous bone that was reactive for type I collagen at 7 days. By the end of the experiment, the trabecula of the condyle was remodeled more perpendic- ularly, thus resisting the compressive force due to hyperfunction of the lateral pterygoid muscles.

Conclusions: These results suggest that the activity of the lateral pterygoid muscle might play a significant role in the differentiation of progenitor cells and in the maturation and calcification of chondrocytes in mandibular condyles. o 1995 WiIey-Liss, Inc.

Key words: Mandibular condylar cartilage, Lateral pterygoid muscle, Electrical stimulation, Biomechanical force, Type I collagen, Type I1 collagen

Mandibular condylar cartilage is known to be secondary cartilage which differs from primary cartilaginous tissues, such as articular and growth plate cartilages of long bone, in terms of morphological architecture and modes of proliferation and calcification (Durkin et al., 1973; Petrovic, 1974; Hall, 1979). Histologically, condylar cartilage is covered by a fibrous connective tissue (referred to here as the fibrous layer), just beneath which are mesenchyme-like cells (progenitor cells). Progenitor cells have high proliferative activities and unique abilities to differentiate into either chondrocytes or osteoblasts (Koski, 1974; Hall, 1979; Stutzmann and Petrovic, 1982; Silbermann et al., 1987; Strauss et al., 1990), both of which depend largely on their biomechanical environments.

The biomechanical stresses exerted on condylar cartilage are largely due to the contractile forces of vari- ous masticatory muscles. Some investigators have reported that the lateral pterygoid muscle is one of the most important factors in regulating the growth and development of mandibular condyles (McNamara and

0 1995 WILEY-LISS. INC

Carlson, 1979; Kantomaa, 1982; Stutzmann and Petro- vic, 1982; Hinton, 1990). These studies primarily focused on the rate and amount of the growth of condylar cartilage; for example, the studies examined the mi- totic activity of progenitor cells, the thickness of cell layers, or the amount of bone and matrix formation. On the other hand, Petrovic (1974) examined the differentiation processes in condylar cartilage and demonstrated that resection of the lateral pterygoid muscle induces osteogenic differentiation of progenitor cells.

In a previous study (Takahashi, 19911, when the lateral pterygoid muscles were electrically stimulated to produce hyperactivity, several morphological changes were observed: 1) cartilaginous cell layers disappeared in the anterior and posterior areas, which was followed

Received March 11, 1994; accepted September 23, 1994. Address reprint requests to I. Takahashi, Department of Orthodon-

tics, School of Dentistry, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai, 980-77 Japan.

DEVELOPMENT OF THE MANDIBULAR CONDYLE IN RATS 329

by new bone formation and 2) a transient increase in the thickness of condylar cartilage in the central area. These observations suggest that activities of the lateral pterygoid muscles may influence not only the proliferation, but also the differentiation of condylar cartilage cells.

To further understand the biomolecular basis of these changes, we examined immunohistochemical localization of type I and type I1 collagens in mandibular condyle that had been subjected to hyperactivity of the lateral pterygoid muscles induced by direct electrical stimulation. Type I and type I1 collagens were used as well-established markers for the stages of differentiation in chondrogenesis.

MATERIALS AND METHODS Electrical Stimulation

Bilateral lateral pterygoid muscles were electrically stimulated in 3-week-old male Sprague-Dawley rats using monopoler needle electrodes. Electrical stimulations were applied as described previously (Takahashi, 1991). Briefly, teflon-coated electrodes 170 pm in di- ameter were inserted from the skin between the eye and the ear, through the temporal fossa, and into the lateral pterygoid muscle under pentobarbital anaesthesia (0.1 m1/100 g of body weight). The electrical stimulation was based on that described by Kantomaa (1982) and consisted of repeated applications (5 seconds od5 seconds off) of a 5 Hz current, 10 msec in duration and -1 to -3 V in intensity. This pattern of stimulation was applied to the experimental animals continu- ously for 1,2,4, or 7 days. Five animals were sacrificed to comprise each experimental group, and five 3-week- old Sprague-Dawley rats were used as controls. The final positions of the electrodes were confirmed upon resection of the temporomandibular joints (TMJ).

Control Experiments Sham operations were carried out in a previous study

(Takahashi, 1991). In these sham operations, either 1) the electrodes were inserted into the lateral pterygoids without stimulation or 2) electrical stimulation was applied to the fibrous tissue running out of the lateral pterygoid muscle. 1) The electrodes were inserted into the lateral pterygoids as described above in the section on electrical stimulation. However, to determine the effects of the mere presence of the electrodes, the electrical stimulations were not applied to the lateral pterygoids. Three animals were sacrificed on days 1,4, and 7, and the final positions of the erectrodes were verified a t resection of the TMJ. 2) To determine the effects of the electric current alone, the electrical stimulations described above were applied to the fibrous tissue running out of the lateral pterygoid muscles. The electrodes were inserted from the temporal fossa through masseter muscles and reached to the fibrous tissue just beyond the oral mucosa. Electrical stimulations of the same pattern as in the experimental groups were applied to fibrous tissue, but not the lateral pterygoids, for 7 days. The final positions of the electrodes were verified a t resection of the TMJ. At the beginning of the experiment, mandibular movement was confirmed regardless of whether or not neighbouring mas- cluture was stimulated.

Tissue Processing The mandibular condyles were taken from the con-

trol and experimental animals. Most of the experimental procedures have been described previously (Mizogu- chi et al., 1990). The animals were perfused from ascending aorta with periodate-lysine-paraformalde- hyde (PLP) in 0.1 M PBS (phosphate buffered saline) (pH 7.4) a t a rate of 3 ml/min for 25 min under pentobarbital anaesthesia. After the mandibular condyles were removed, further fixation was carried out on the specimens for 24 h at 4"C, and was followed by thorough rinsing with PBS. The specimens were decalcified with 5% ethylene diamine tetraacetic acid (EDTA) in 0.1 M PBS for 1 week a t 4°C. After dehydration in a graded series of ethanol, the specimens were passed through propylene oxide and embedded in Spurr's resin. One micrometer thick sagittal serial sections were cut with a glass knife using a Reichert ultrami- crotome and were picked up on the glass slides, placed on the slide warmer, and dried overnight.

The slides were placed in a saturated solution of so- dium ethoxide in absolute ethanol for 15 min a t room temperature to remove Spurr's resin. After a thorough rinse in ethanol and rehydration in 0.01 M PBS and distilled water, the sections were dried at room temperature. The sections were subjected to enzymatic di- gestion with 0.2 mg/ml protease type XXIV (Sigma, St. Louis, MO) in 0.1 M PBS for 10 min at room temperature, followed by subsequent rinses with 0.01 M PBS.

lmmunohistochemical Double-Staining Technique The sections were first incubated with 5% BSA (bo-

vine serum albumin) (Sigma) in 0.01 M PBS containing 0.025% Triton X-100 (solution A) and 10% normal goat serum for 20 min to reduce nonspecific reactions. A rabbit anti-bovine type I1 collagen antibody (LSL Co. Ltd., Tokyo, Japan) was used as a first primary antibody, and a rabbit anti-rat type I collagen (LSL Co. Ltd.) antibody was used as a second primary antibody. A goat anti-rabbit IgG conjugated rhodamine antibody was used as a first secondary antibody, and a goat anti- rabbit IgG conjugated fluoresceinisothiocyanate (FITC) antibody (TAGO, Burlingame, CA) was used as a second secondary antibody. Each antibody was di- luted in solution A as follows: first primary antibody, 1:50; second primary antibody, 1200; first secondary antibody, 1:lO; and second secondary antibody, 1:30. The sections were incubated with each primary antibody overnight at 4°C in a moisture chamber. The control sections were incubated with normal rabbit serum or 0.1 M PBS under the same conditions. After thorough rinses with solution A, all of the sections were incubated with each secondary antibody for 2 h in a

TABLE 1. Experimental changes in the anterior area'

Morphological features TvDe 1 TvDe 11 Calcif

- Control Cartilage + + + Day 2 Cartilage and bone + + ND Day 7 Bone + + -

*Type I, type I collagen; Type 11, type I1 collagen; Calcif, calcification; ND, no data.

330 I. TAKAHASHI E T AL.

TABLE 2. Experimental changes in the posterior areal

Moruholog.ica1 features Tvve I Tvoe 11 Calcif Control

Upper Lower

Upper

Upper

Day2

Lower Day7

Lower

- - Differentiating chondroblasts + Well-differentiated cartilage + + -

ND Cartilage + + ND

- Undifferentiated polygonal cells t

Osteoblastic cells Intramembranous bone

k +

-

+ 'Upper, upper cartilaginous layers; Lower, lower noncartilaginous layers of the condylar cartilage; Type I, type I collagen; Type 11, type I1 collagen; Calcif, calcification; ND, no data.

Fig. 1. Sagittal sections of condyles from control animals (A) and after 1 day (B), 2 days (0, and 7 days (D) of stimulation. Arrowheads indicate the direct and indirect attachment of the lateral pterygoid muscle. Square a, anterior area of the condyles; square p, posterior

area of the condyles. Opposing arrows indicate the thickness of the fibrous and transitional cell layers. CC, condylar cartilage; LPM, lateral pterygoid muscle. Toluidine blue, x 20.

moisture chamber at room temperature. After several rinses with solution A and 0.01 M PBS, the specimens were mounted in polyvinyl alcohol medium (Lennette, 1978). All of the sections were examined with an Olym- pus BH2-RFK fluorescence microscope, and micropho-

tographs were taken. There is a potential problem with the immunohistochemical double-staining technique used here, since i t uses IgG from the same species of animal in cross-immunoreactions between each secondary antibody. Therefore, immunohistochemical sin-

DEVELOPMENT OF THE MANDIBULAR CONDYLE IN RATS 33 1

Fig. 2. Higher magnification of anterior (A,B) and posterior (C-E) area of condyles. A Control. B Seven days of stimulation. Note the differences in size and morphology of hypertrophic cells (arrows in A,B). C: Control. F, fibrous cell layer; H, hypertrophic cell layer; M, maturative cell layer; P, proliferative cell layer; T, transitional cell

gle-staining for anti-type I and -type 11 collagen antibodies was carried out on several sections to examine the cross-immunoreactions between primary and secondary antibodies.

Contact Microradiographs Undecalcified 50 pm thick sections were made from

7 day experimental and control specimens for contact microradiographs (CMR). Specimens from animals that had been subjected to the experimental treatment for 7 days and from 4-week-old controls were fixed as described above and embedded in Spurr’s resin after dehydration in a graded series of ethanol. CMR images were taken under soft X-ray at 60 kV, 3 mA for 60 min by a Softex CSMW-2.

Histological and lmmunohistochemical Evaluations To estimate histological changes, condylar cartilage

was divided into the following five cell layers, as described by Luder et al. (1988) and in our previous study (Mizoguchi et al., 1990): a fibrous layer which involves spindle-shaped fibroblasts and is rich in type I collagen, the proliferative cell layer with polygonal-shaped progenitor cells which have proliferative ability, a

layer. D Two days of stimulation. * indicates the area occupied by the polygonal-shaped cells rather than the transitional and maturative cells. E Seven days of stimulation. Arrowheads indicate the newly formed intramembranous bone. Toluidine blue. x 40.

transitional cell layer with cells intermediate between progenitor and cartilaginous cells which biosynthesize type I1 collagen, a maturative cell layer which includes well-differentiated chondrocytes and type I1 collagen- rich extracellular matrix (ECM), and hypertrophic cell layers with hypertrophied chondrocytes. The fibrous, proliferative, and transitional cell layers are noncartilaginous, whereas the maturative and hypertrophic cell layers have cartilaginous characteristics. In the anterior of the condyle, cartilage localization is slightly different than that in other areas which lack a transitional cell layer.

RESULTS Histological Observations

In the present study, remarkable histological changes were observed primarily in two areas of the experimental condyles: 1) the anterior area which is characterized by direct attachment of the lateral pterygoid muscle and 2) the posterior area, where the lateral pterygoid muscle attaches indirectly (Tables 1 ,2 ; Figs. 1, 2). On the other hand, since the sham-operated condylar cartilages presented no changes histologically and 4-week-old Sprague-Dawley rats showed qualita-

332 I. TAKAHASHI ET AL.

Fig. 3. CMR images of the posterior area of control (A) and experimental (B) sections. Intramembranous formation of bone matrix (arrowheads) was observed in the posterior area of experimental condyles. x 40.

tively identical histological features as 3-week-old rats, the 3-week-old controls were acceptable as controls.

In the control condyles, the lateral pterygoid muscles were attached to the fibrous layer of the anterior bor- der of the cartilage in the anterior area (Fig. 1). After 2 days of experimental treatment, the fibrous and proliferative layers increased in thickness (Figs. 1,4), and activation of bone modeling was observed in the anterior area and in vital staining sections in our previous study (Takahashi, 1991). At the end of the experiment, hypertrophic chondrocytes disappeared from the anterior area (Fig. 2A,B). In the posterior area, cartilages of control sections were thick and have the five layers, as described above (Fig. 2C-E). The thickness of the cartilage decreased considerably in the transitional, maturative, and hypertrophic cell layers after 2 days of stimulation. This area was first occupied by polygonal- shaped progenitor cells which were replaced by bone matrix at the end of the experiment (Fig. 2). These changes began with the disappearance of the transitional and maturative cell layers in the early stages, followed by the formation of intramembranous bone (Fig. 2C-E). CMR observations (Fig. 3) confirmed calcification of the newly deposited bone matrix in the posterior area of the condyles.

lmmunohistochemical Observations The specificity of the antibodies used in this study

was previously confirmed by Western blotting (Mizogu- chi et al., 1990). In the immunohistochemical double- staining technique used in this study, it is possible that the secondary antibody would cross-react to both pri-

mary antibodies. Therefore, to verify the results of immunohistochemical double-staining, single-staining for type I and type I1 collagens was carried out on several sections. As a result, little or no difference in the immunolocalization of both collagens was observed between single- and double-stained sections in terms of the intensity and the distribution of reactions for each antibody (data not shown).

Anterior Area (Fig. 4; Table 1 ) In the control sections, type I1 collagen was richly

distributed in the maturative and hypertrophic cell layers, whereas type I collagen was distributed in the fibrous cell layer and in the matrix between the maturative and hypertrophic cell layers. Type I1 collagen was localized in both the maturative and hypertrophic cell layers and on the region between them.

The sections after 2 days of stimulation presented different patterns of reactivity from those of controls for both antibodies. There was almost no type I1 collagen in the thickened fibrous and transitional cell layers, where type I collagen was abundant. Reactivity for type I1 collagen was faintly observed in the maturative and hypertrophic cell layers. The type I1 collagen antibody showed intense reactivity in the territorial matrix, as compared to the interterritorial matrix, in these areas. The reactivity for type I1 collagen antibody increased gradually with depth in the maturative and hypertrophic cell layers, while that for type I collagen decreased below the maturative cell layer in both experimental and control sections.

In sections that had been stimulated for 7 days, the anterior area had changed from endochondral bone to intramembranous bone, accompanied with an absence of reactivity for type I1 collagen antibody, whereas type I collagen was observed in the bone matrix. Through- out the experiment, the staining intensity for type I1 collagen in all of the layers was less in the experimental sections than in the controls.

Posterior Area (Fig. 5; Table 2) In the posterior area, control sections showed intense

reactivity for type I collagen in almost all of the cell layers, whereas type I1 collagen was only weakly de- tectable in the maturative and hypertrophic cell layers. In sections after 2 days of stimulation, reactivity for type I1 collagen was almost entirely absent from all of the cartilage layers. On the other hand, a fibrous reac- tion pattern for type I collagen was observed in the ECM of the cartilaginous, maturative, and hypertrophic cell layers.

In sections that had been stimulated for 4 and 7 days, the posterior area of the condyles was occupied with intramembranous bone matrix which reacted only for anti-type I collagen. Type I1 collagen was not observed in this intramembranous bone matrix. Newly formed intramembranous bone matrix showed weaker reactivity for anti-type I collagen antibody. The reactivity for type I collagen was maintained in the perichondrium of this area throughout the experimental period.

DISCUSSION The lateral pterygoid muscle originates at the exter-

nal surface of the lateral pterygoid plate and passes backward, superiorly and laterally, into the capsule


Fig. 4. Immunolocalization of type I (A,C,E) and type I1 (B,D,F) collagen in the anterior area of condyles. A,B: Control. C,D: Two days of stimulation. E ,F Seven days of stimulation. A: Control. F, fibrous cell layer; H, hypertrophic cell layer; M, maturative cell layer; T, transitional cell layer. A,C: Small arrows indicate the thickness of the

fibrous and transitional cell layers. E: Arrowheads indicate intramembranous bone that is reactive to type I collagen. D , F Large arrows and bars indicate the anterior margin of the cartilage which moved backward. x 50.

and disc of the TMJ and muscularly into the anterior margin of the condylar cartilage. Based on studies of anatomical architecture (Hiiemae, 1971; Weijs, 1973) and electromyographic activity of the lateral pterygoid muscle (Byrd and Chai, 1988), it has been suggested that this muscle forms a stabilizing system with the mandibular condyle and the articular capsule in the TMJ during mastication. Many investigators have reported the significance of mastication on craniofacial growth (Simon, 1977; Whetten and Johnston, 1985; Watanabe, 1990), especially with regard to the effects of compressive forces on proliferation or chondrogenic differentiation of progenitor cells. In mandibular condylar cartilage, such relationships have been demonstrated in in vitro and in vivo studies (Silbermann et al., 1983; Copray et al., 1985) using 3H-proline and 35S-sulphate. These studies have shown that certain functional compressive forces promote the biosynthetic

activity of collagenous matrix and cartilage-character- istic proteoglycans, as well as cellular proliferation.

In the present study, where we focused on the biomechanical aspects of the TMJ under electrical stimulation, periodic contraction of the lateral pterygoid muscle translated the mandibular condyle anteriorly and changed the biomechanical environment of the TMJ. The articular capsule in the experimental group mediated more contractile force to the mandibular condyle than that of the controls and loaded compressive force on its articular surface, while tensional force was predominant at the anterior and posterior area to which the lateral pterygoid muscle and retrodiscal pad were attached via a fibrous layer.

The histological changes we observed in the anterior area coincided with changes reported in studies of the functional protrusion of the mandible induced by functional orthopedic appliances (McNamara and Carlson,


Fig. 5. Immunolocalization of type I (A,C,E) and type I1 (B,D,F) collagen in the posterior area of condyles. A , B Control. C,D: Two days of stimulation. E ,F Seven days of stimulation. A,B: control. F, fibrous cell layer; H, hypertrophic cell layer; M, maturative cell layer;

P, proliferative cell layer; T, transitional cell layer. C,D: Arrowheads indicate the area (a) from which cartilage matrices disappeared. E,F: Type I collagen was observed in the intramembranous bone (b) indicated by arrows. x 50.

1979). McNamara and Carlson reported that the lateral pterygoid muscles were activated by functional protrusion of the mandible. Therefore, the thickening of the fibrous layer and the increase in type I collagen observed in the anterior area were considered to be adaptive changes made possible by activation of the synthetic function of cells due to increased functional forces exerted by the lateral pterygoid muscle. Further- more, our observations in the anterior area during the early experimental stages agreed with reports indicat- ing that fibroblastic differentiation is dependent upon tensional forces (Bassette, 1961).

In the posterior area during the early experimental periods, which may primarily be subjected to tensional forces, the cartilaginous, transitional, and maturative cell layers disappeared, and type I1 collagen was almost undetectable. This area was occupied by polygonal cells, which were similar in morphology to the progenitor cells in the proliferative cell layer and which were surrounded by some ECM, type I collagen. With regard to the phenotypic changes of the collagens, spatial and temporal localization of type I and type I1 collagen expressed in the ECM of the growth-plate cartilages in developing bones has been demonstrated by Silber-


mann et al. (1987) and von der Mark (1980). In chick limb bud, a temporal phenotypic switch from type I to type I1 collagen occurs as the mesenchymal cells differentiate into chondrocytes (von der Mark et al., 1976). In studies of fracture healing under conditions of compressive force, similar patterns of expression of the ECM, including type I and type I1 collagen and some proteoglycans, have been reported (Ashhurst, 1986; Page et al., 1986). A similar phenotypic transition of the collagen components occurs spatially in condylar cartilage, which contains not only type I1 collagen, but also noncartilaginous ECM of type I collagen (Livne, 1985; Silbermann et al., 1987; Mizoguchi et al., 1990). These studies concluded that type I collagen is expressed in the ECM during the early cell condensation stages of the morphogenesis of hyaline cartilage and type I1 collagen is expressed in the later maturation stages. During the early stages of this experiment, the tensional forces generated by stimulation of the lateral pterygoid muscles may have caused the disappearance of cartilaginous ECM rich in type I1 collagen and an increase in progenitor cells. These findings suggest that the progenitor cells in this area stopped differentiating into chondrocytes, and typical cartilaginous ECM was not biosynthesized under these conditions.

After 4 and 7 days of stimulation of the lateral pterygoid muscle, reactivity for anti-type I collagen was observed in the calcified bone matrix in both the anterior and posterior areas. However, reactivity for anti-type I1 collagen, which would indicate residual calcified cartilage matrix, was completely absent in this bone matrix. These results indicate that intramembranous bone formation, rather than endochondral osteogenesis as under physiological conditions, occurred continu- ously after the disappearance of cartilaginous matrix. This finding did not coincide with those of Bassette (1961), in that bone formation did not progress under conditions of tensional force. However, Kantomaa and Hall (1988) indicated that condylar cartilage was calcified when removed from functional compressive force in vitro. Studies of the experimental expansion of intermaxillary sutures due to the displacement of buccal musculature found that intermaxillary suture cartilage responded to expansive forces by biosynthesizing the bone matrix in rats (Ghafari, 1984; Sawahata, 1988). Therefore, osteoblasts, which synthesize the intramembranous bone matrix, may be differentiated from the progenitor cells which appeared in earlier stages.

In summary, our results suggest that the differentiation pathway of condylar secondary cartilage repre- sented by the biosynthesis of type I and type I1 collagen depends considerably on the functional and biomechanical environment of the TMJ. Furthermore, the expression of specific ECM by condylar cartilage cells may depend on the direction of the applicable force vectors.

ACKNOWLEDGMENTS We are grateful to Dr. Koji Kindaichi and Dr. Ya-

suyuki Sasano, 2nd Department of Oral Anatomy, for their valuable advice. We also thank Mr. Toshihiro On- odera, Mr. Masami Eguchi, and Mr. Yasuto Mikami for their excellent technical assistance. This work was supported in part by a grant in aid (06771986) for sci-

entific research from the Ministry of Education, Sci- ence and Culture, Japan.

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effects of lateral pterygoid muscle hyperactivity on differentiation of mandibular condyles in rats

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