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A Repetitive, Steady Mouth Opening Induced an Osteoarthritis-like Lesion in the Rabbit Temporomandibular Joint
1 Oral and Maxillofacial Rehabilitation and Correspondence: * corresponding author, kuboki{at}md.okayama-u.ac.jp
Although excessive mechanical stress is assumed to be one of the factors contributing to pathogenesis of temporomandibular joint (TMJ) osteoarthritis (OA), no pure mechanical-stress-induced OA model has been developed without surgical manipulation or puncture of the joint cavity. The purpose of this study was to establish a genuine mechanical-stress-induced OA model of the rabbit TMJ. In the experimental rabbits, repetitive, forced jaw-opening, 3 hrs/day for 5 days, was applied with the use of a general anesthesia protocol. By histological assessment of the TMJ articular tissues, partial eburnation of the articular cartilage, reactive marginal proliferation of the articular cartilage chondrocytes, and nested proliferation of chondrocytes in the subchondral bone area were observed at 7 days after the repetitive, forced-jaw-opening period. These results suggest that the repetitive, forced-jaw-opening protocol without surgical intervention can induce evident OA-like lesions in the rabbit TMJ, and this OA model may greatly contribute to the elucidation of the cartilage degradation mechanism in TMJ OA.
Key Words: articular cartilage • temporomandibular joint • mechanical stress • osteoarthritis
Osteoarthritis (OA) is a degenerative joint disease that is characterized by articular cartilage degradation and concomitant reparative/adaptive osteogenesis. Excessive mechanical stress has been recognized as one of the major implicating factors in OA. However, the exact role of the excessive joint loading on the degradation process is still unclear. Several experimental temporomandibular joint (TMJ) OA models have been developed by some researchers to elucidate the relationship between adverse mechanical stress and OA pathology. In some of those OA models, surgical manipulation of the joint structures was performed to alter intracapsular mechanical circumstances, e.g., discectomy (Takatsuka et al., 1996; Bjørnland and Haanaes, 1999), surgical induction of disk displacement (Silbermann, 1976; Ali and Sharawy, 1994), and disk perforation (Axelsson et al., 1992; Sato et al., 1998). However, these kinds of surgical procedures induced not only mechanical alteration, but also artificial surgical damage to the joint structures. Therefore, these OA models cannot be regarded as a real mechanical-stress-induced OA model that is comparable with a clinical disorder of spontaneous TMJ OA. Recently, a forced-jaw-opening protocol has been shown to be effective in inducing articular synovitis in the TMJ (Chiang and Kakudo, 1990; Muto et al., 1995; Shiga, 2001). These three studies are important since they clearly demonstrate that adverse joint loading without any surgical manipulation of the joint tissues can induce joint inflammation in vivo. Unfortunately, the experimental conditions used in these previous studies could not have reproduced OA-like lesions in the TMJ articular cartilage identical to the clinical TMJ OA findings, e.g., articular cartilage degradation (fibrillation and erosion in cartilage) and concomitant reparative/adaptive osteogenesis (sclerosis in subchondral bone and marginal proliferation in the articular cartilage). Since we have established a 3-D mathematical model of the human stomatognathic system with the mouth opened, and recognize that there is a compressive force between the articular eminence and the mandibular condyle during jaw opening (Kuboki et al., 2000), we thought that modification of the forced-jaw-opening protocol would enable us to produce OA-like lesions in the TMJ. We then selected a repetitive, steady mouth-opening protocol to produce continuous compression onto the articular cartilage, since we know that continuous compression does induce higher cartilage deformation than intermittent compression in an in vitro experimental indentation model of the pig TMJ articular cartilage (Kuboki et al., 1997) and an in vivo radiographic joint space measurement (Takenami et al., 1999). The purpose of this study was to establish a mechanical-stress-induced OA model in the rabbit TMJ, which would be compatible with clinical TMJ OA findings. Moreover, our goal was to investigate the histopathological changes in the rabbit TMJ induced by this OA model. To accomplish this, we utilized a unique repetitive, steady mouth-opening protocol that finally produced an OA-like lesion in the articular cartilage of the rabbit TMJ condyle.
Animals Nine adult Japanese white male rabbits (3 mos old) were used in this study. The animals were treated according to the regulations of the Animal Research Control Committee of Okayama University Dental School (approval number 2-002-024).
Application of Adverse Mechanical Stress
Radiographic Study To observe the condylar position during forced jaw-opening, we obtained a radiograph of the hemicranial specimen. One of the control rabbits was anesthetized and killed, with the forced-jaw-opening position maintained with an acrylic resin positioner. After fixation with 4% paraformaldehyde, the head was separated into hemicranial specimens in the mid-sagittal plane and subjected to x-ray exposure in a micro-FX1000 system (Fuji Film, Inc., Tokyo, Japan). This image showed that while the mandibular condyle was translated forward along the articular eminence, it did not show any evidence of dislocation (Fig. 2  ). The TMJ sample also underwent histopathological studies as described below.
Histopathological Studies Three rabbits were killed at 1 day after the mechanical stress application period (first sampling), and the other 3 rabbits were killed at 7 days after the mechanical stress application period (second sampling). One control rabbit was killed at 1 day after the mechanical stress application, and another control rabbit was killed at 7 days after the mechanical stress application period. The mandibular condyles were harvested as blocks. The condyles were fixed in 4% paraformaldehyde at 4°C for overnight, decalcified in 10% EDTA for 2 wks, and embedded in paraffin for histological study. Histologic examinations were performed on sagittal serial sections (5 microns thick), and serial sections were stained with hematoxylin and eosin (HE), and safranin O-fast green (SO) staining. All staining processes were performed at room temperature.
Macroscopic Findings of the TMJ TMJ condyles from the three control rabbits showed smooth articular surfaces without any soft-tissue damage covering the condyle. In contrast, TMJ condyles from the 3 experimental rabbits at days 1 and 7 after the mechanical stress application period showed articular surface fibrillation (roughness) and some subchondral bone exposures in the articular cartilage region of the condyle, respectively. The cartilage lesion was less severe in the first-sampled rabbits than in the second (data not shown).
Histopathological Findings
Fig. 4 shows SO-stained sagittal sections of the rabbit TMJ condyle. Control joints showed intense SO staining in the cartilaginous zone, indicating abundant proteoglycan deposition (Fig. 4A). In the experimental joints at 1 day after the mechanical stress application period, dramatic SO staining reduction, especially at the articular surface layer of the cartilage, was observed (Fig. 4B). At 7 days after the mechanical stress application period, strong SO staining was observed in the nested proliferation of chondrocytes (chondrocyte island) in the subchondral bone layer of the central and posterior aspects of the articular surface of the condyle (Figs. 4D, 4E, 4F) and marginal chondrocyte proliferation covering the anterior edge of the articular surface of the condyle (Fig. 4C). Meanwhile, significant SO staining reduction was observed at the central aspect of the articular surface (Fig. 4E), owing to complete cartilage loss with sparse nested proliferation of chondrocytes in the subchondral bone layer. In this area, invasion of synovial membrane-like-tissue was observed in some specimens.
It has been difficult to develop a pure mechanical-stress-induced OA model without surgical manipulation of the joint structure. For example, it has been reported that moderate running training for a period of 25 wks led to an increase in glycosaminoglycan levels in canine knee joint articular cartilage (Kiviranta et al., 1988), and strenuous running training (40 km/day) on a treadmill for 15 wks did not produce OA change in the dog articular cartilage, but induced slightly increased thickness of the uncalcified and calcified cartilage and subchondral bone in the knee joints (Oettmeier et al., 1992). These responses of the articular cartilage and subchondral bone to long-distance running are presumably adaptive processes that provide better congruence and biomechanical stability to the articulating bone ends. In some studies, long-duration strenuous training was associated with site-dependent alterations in cartilage composition (Arokoski et al., 1996) and proteoglycan depletion (Kiviranta et al., 1992) in canine cartilage; however, the severity was not comparable with the clinical OA condition in humans. In the TMJ, experimental occlusal loss or occlusal change has been thought to cause an increased loading on the articular tissues and induce damage of the articular cartilage. However, indirect joint-loading modifications have not produced OA-like lesions in the experimental animals (Furstman, 1965; Gianelly et al., 1970; Ishimaru et al., 1994). As Huang et al. (2002) reported recently, experimental occlusal changes, e.g., unilateral removal of teeth, did not induce OA change in the rabbit TMJs. Instead, these investigators described adaptive responses, e.g., thickening of the condylar cartilage, alterations in the morphology of chondrocyte nuclei in the chondylar cartilage and disc, and increases in levels of negatively charged ions (this results in increased safranin O staining) in the hypertrophic layer of condylar cartilage, but it would not be appropriate to call these changes OA. In contrast to these earlier studies, we were able to develop an OA-like lesion in the rabbit TMJ. The repetitive, steady jaw-opening protocol used in this study was effective in developing OA-like changes compatible with the clinical findings frequently seen in TMJ OA patients. Since we could not measure the articular tissue loading in the animal, it would be difficult to identify the exact difference in the loading between the repetitive, steady jaw-opening and the occlusion change protocols; however, the biggest difference can be assumed not in the magnitude but in the nature of the joint loading. The repetitive, steady jaw-opening protocol seems to produce sustained articular tissue compression, while the occlusion change protocol would not significantly modify the mode of the TMJ loading (e.g., rhythmic). We have already reported that articular soft-tissue (cartilage) deformation of the TMJ is significantly less under intermittent compression than under sustained compression in vitro and in vivo (Kuboki et al., 1997; Takenami et al., 1999). This OA model at 7 days after the mechanical stress application period showed a clear cartilage loss (eburnation) in the central portion of the articular surface, which is a well-known OA feature. It is also extremely interesting that nested proliferation of chondrocytes (chondrocyte island formation) in the subchondral bone layer was discovered in the region with the clear articular cartilage loss. The chondrocytes in the island were hypertrophic and being replaced with mineralized tissue. We assume that this finding is closely related to a protective sclerotic change of the subchondral bone, which is also a frequently observed change in human OA joints. As far as we know, this is the first report elucidating the mechanism for generation of the sclerotic change in the subchondral bone of the TMJ. Chondrocyte proliferation covering the anterior edge of the joint surface with rich matrix deposition was also observed in this model. This reaction might also be related to osteophyte formation, which we presume to be progressive remodeling causing an enlarged articular surface area, thus reducing articular functional pressure within the physiologic limit. These site-specific differences in chondrocyte reaction might be related to biomechanical condition differences. When the animals open their lower jaws, the central and posterior aspects of the condyle are compressed against the articular eminence, while the anterior part does not receive as much compression as do the central and posterior parts. This might explain the difference in the reactions of the articular cartilage, e.g., marginal proliferation in the anterior aspect, eburnation in the central and posterior aspects. Prior to our study, production of a mechanical-stress-induced animal model for TMJ OA was difficult without surgical manipulation of the joint. Therefore, the molecular mechanisms that initiate and advance the OA change are still under study. We hope that our TMJ OA model may contribute to the elucidation of the cartilage degradation and adaptive calcification mechanism in the OA joints, leading to the development of new therapeutic strategies.
We are indebted to Dr. Glenn T. Clark, Professor, Section of Orofacial Pain and Oral Medicine, University of California Los Angeles School of Dentistry, Center for the Health Sciences, for his kind review and editing of this manuscript and for helpful suggestions. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (#12557169, #14571843), awarded to T.K. and T.F., respectively. Preliminary results of this study were presented in the AADR Annual Meeting held in Chicago, IL, USA, on March 10, 2001. Received for publication May 24, 2002. Revision received May 14, 2003. Accepted for publication May 27, 2003.
Journal of Dental Research, Vol. 82, No. 9,
731-735 (2003)
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