Temporomandibular joint in miniature pigsAnatomy cell replication and relation to loading.код для вставкиСкачать
THE ANATOMICAL RECORD 266:152–166 (2002) DOI 10.1002/ar.10049 Temporomandibular Joint in Miniature Pigs: Anatomy, Cell Replication, and Relation to Loading SUSAN W. HERRING,* JAY D. DECKER, ZI-JUN LIU, AND TSUN MA Department of Orthodontics, University of Washington, Seattle, Washington ABSTRACT The mechanical environment is a regulator of growth and adaptation of the musculoskeletal system, including joints. Although pigs (Sus scrofa) are used frequently as models for temporomandibular joint (TMJ) dysfunction, no systematic description of microanatomy exists for this species. We injected the thymidine analog 5-bromo-2⬘-deoxyuridine (BrdU) into 10- to 11-month-old miniature pigs that were undergoing measurements of TMJ bone strain. Ten hr later, the animals were sacrificed and their heads were perfused. Histological sections were used to map the distribution of replicating cells. Additional observations were made on gross dissections of jaw joints obtained from an abattoir. The pig TMJ is better supported than that of humans laterally and medially, but more vulnerable posteriorly. The posterior attachment area of the intra-articular disc is fibro-fatty rather than vascular, as in humans. Cartilage lines the articular eminence as well as the condylar surface. At the posterosuperior region of the condyle, the cartilage ends abruptly and is replaced by an invaginating, actively replicating periosteum. Almost all of the BrdU-labeled cells resided in the prechondroblastic zones. The condyle had more replicating cells than did the eminence (P ⬍ 0.02), but lateral and medial locations did not differ in either element. In sagittal sections, the condyle had more replicating cells posteriorly (P ⬍ 0.001), but no A-P differences were seen in the eminence. Comparisons of these data with data on bone strain indicate that increased loading is negatively associated with cell replication. Anat Rec 266: 152–166, 2002. © 2002 Wiley-Liss, Inc. Key words: bone strain; BrdU; cell replication; pig; TMJ Dissonance in the clinical literature relative to treatment modalities for temporomandibular joint (TMJ) dysfunction (Greene et al., 1999) makes it obvious that animal models are needed to define and control experimental variables. Although no animal model can exactly duplicate the human condition, a number of authors have argued that despite its prognathism the pig, Sus scrofa, is the best nonprimate analog for human TMJ form and function (Meister et al., 1973; Ström et al., 1986; Bermejo et al., 1993; Herring, 1995). Like higher primates, pigs have bilateral occlusion at full closure, a symphysis that fuses after infancy, and sliding joint mechanics. The pig TMJ has already been used to study discal mechanical properties (Nickel and McLachlan, 1994; Kuboki et al., 1997; Nickel et al., 2000), synovial fluid pressure (Roth et al., 1984), reactions to occlusal disturbances (Ulrici et al., 1988; Dannhauer, 1992; Sindelar et al., 2000), and as a mechanical (Liu and Herring, 2000b) or surgical (Timmel © 2002 WILEY-LISS, INC. and Grundschober, 1982) model. Proteoglycan content has been examined in the disc (Sindelar et al., 2000) and the condylar soft tissues (Müller et al., 1996; Roth et al., 1997), and Moroco et al. (1997) have looked at age changes of collagen II and TGF-␤ in the condylar cartilage. Despite this burgeoning popularity, only very sketchy information about pig TMJ morphology has been pub- Grant sponsor: U.S. Public Health Service, National Institute for Dental and Craniofacial Research; Grant numbers: DE08513 and DE11962. *Correspondence to: Susan W. Herring, University of Washington, Dept. of Orthodontics, Box 357446, Seattle, WA 98195-7446. Fax: (206) 685-8163. E-mail: email@example.com Received 11 July 2001; Accepted 26 November 2001 Published online 14 February 2002 TMJ OF PIGS lished, and the coverage in veterinary texts is even more cursory. The best description of the pig TMJ (Meister et al., 1973) is unfortunately not readily accessible to English-speaking workers. The osseous elements of the joint have been described grossly (Herring and Scapino, 1973; Meister et al., 1973; Ström et al., 1986) and in terms of trabecular architecture (Teng and Herring, 1995; Teng et al., 1997). The disc has also been the subject of both macroscopic (Meister et al., 1973; Bermejo et al., 1993) and histologic (Berg and Meister, 1974; Christensen, 1975; Albu et al., 1979) investigation. There are no available reports on the sectional anatomy of the pig TMJ, either in terms of computed tomography (CT) or magnetic resonance imaging (MRI) or of overall histology. Most striking in its absence is any description of pig TMJ ligaments, including the attachments of the disc and ligaments to the osseous elements. Ligaments and discal attachments are likely to be critical elements in TMJ function and dysfunction. These structures are responsible for the dynamic and static stabilization of the disc (Schmolke, 1994; Sato et al., 1996; Amor et al., 1998). Disc displacements may be a consequence of weak disc attachments and clearly involve their disruption (Osborn, 1995; McNeill, 1997). Equally lacking is a comprehensive description of cell kinetics in the pig TMJ that would enable comparison with other frequently used animal models, such as macaque monkeys and the laboratory rat (Luder, 1996). Cell replication subserves growth, tissue turnover, and adaptation to new circumstances. In skeletal tissues generally and in the TMJ particularly, mechanical loading is commonly considered a potent regulator of replication. Although controversial, the general consensus is that tensile stress leads to increased replication in ligaments and their attachments to bones (Yen et al., 1990; Yousefian et al., 1992). Although hydrostatic compression is necessary to maintain articular cartilage (Frost, 1990; Carter and Beaupré, 2001), elevated compressive stress may inhibit local replication and directional growth (Ngan et al., 1992). Because the pig TMJ has been the subject of biomechanical investigation and the broad outlines of its loading are known (Liu and Herring, 2000a, 2000b), the opportunity arises to explore the connection between loading and cell replication in the joint. The purpose of this investigation was therefore threefold: 1) to provide an overall description of the pig TMJ with emphasis on histology and ligamentary attachments, 2) to define sites of cell replication, and 3) to relate these findings to probable loading patterns in the normally functioning joint. To assess replication we employed the thymidine analog 5-bromo-2⬘-deoxyuridine (BrdU) to detect S-phase cells in situ (Gratzner, 1982). In addition to the general identification of areas involved in replication, counts of BrdU-labeled cells were made from selected areas, including capsular ligament/disc attachment areas. These counts were used to test hypotheses about loading. Specifically, local replicative activity was expected to be lowest in the direction of the compressive load and, because soft tissue attachments are presumed to be tensile, higher in attachment areas than in nonattachment areas. MATERIALS AND METHODS Macroscopic observations of ligamentous anatomy were made on approximately 12 farm pig heads obtained from a local abattoir. Sex and age were unknown, but it is likely 153 that the animals were about 6 months old. Blocks containing the TMJs were removed using a band saw and individually frozen in saline until dissection. The histological and mechanical part of the study used eight male and eight female miniature pigs, Hanford strain (Charles River, Wilmington, MA), obtained at the age of 8 months. All domestic pigs, including farm animals and miniature breeds, are members of a single species, Sus scrofa. Sexual maturity occurs at 6 –7 months, but growth is not complete until animals are at least 1 year old. Thus, all animals in this study were at a stage roughly equivalent to the early teen years of humans. The diet of the miniature pigs was a commercial pelleted pig chow; that of the farm pigs was unknown. All procedures were reviewed and approved by the University of Washington Animal Care Committee. A subsample (n ⫽ 6) of the miniature pigs was imaged by MR and CT, performed on different days. For MRI, the pig was anesthetized by acepromazine/ketamine (8:1) i.m., followed by ketamine/xylazine i.v. for maintenance. The pig was placed in a prone position on a 1.5-T Signa MRI system (GE, Milwaukee, WI). Using a head coil, two initial scout sections were performed to locate the condyle sagittally and coronally. Then, circular 3-inch surface coils were placed over right and left TMJs. Sagittal and coronal T1-weighted scans produced a series of seven sagittal and eight coronal slices, 2.2 mm thick with 0.3-mm spacing, through each joint (TR ⫽ 2400, TE ⫽ 24, field of view (FOV) ⫽ 16 cm, matrix ⫽ 512 ⫻ 256). For CT, the pig was anesthetized by mask inhalation of halothane in O2/N2O and was placed prone in the bed of a GE High Advantage Tomography scanner. After an initial scout, a 3-mm helical scan of the pig’s head was performed (FOV ⫽ 24 cm, matrix ⫽ 512 ⫻ 512, kV ⫽ 120, mA ⫽ 180), allowing 1-mm coronal slices to be reconstructed. The entire sample of 16 animals was used for cell replication studies. At approximately 10 months of age and 45–70 kg of body weight, animals were anesthetized via mask inhalation of halothane in O2/N2O and injected i.p. or i.v. with BrdU (40 mg/kg as a 10 mg/ml solution in phosphate-buffered saline (PBS)). Stacked rosette strain gauges were bonded to the lateral surface of the left squamous temporal bone at the level of the articular eminence and to the lateral surface of the left condylar neck. Temporal and condylar bone strains were recorded during natural mastication. This methodology and the results from a larger group of pigs have been reported elsewhere (Liu and Herring, 2000a, 2000b). For the present paper, we reanalyzed the strain data from the subset of animals receiving BrdU and examined the correlations between cell replication and bone strain. After these strain measurements and 10 hr subsequent to BrdU injection, the anesthetized animals were injected i.p. with 3,000 units of heparin and sacrificed by an intracardiac injection of pentobarbital. The head was removed and both common carotid arteries were cannulated. Using a peristaltic pump, heparinized (1,000 units/l) PBS was perfused until the solution exiting the veins was clear; this was followed by perfusion with 10% neutral buffered formalin. After the tissues became stiff, the head was sagittally sectioned and blocks of tissue containing the TMJs were isolated and removed. Fixation continued for 2 days in fresh solution, followed by demineralization in Christiansen’s solution (8N formic acid, 1N sodium formate, 1:1), which required several 154 HERRING ET AL. Fig. 1. Diagram of the condyle in frontal view illustrating the division of each TMJ block into lateral, central, and medial thirds. The central third was sectioned in the parasagittal plane, whereas the lateral and medial thirds were cut in the coronal plane. months for completion. The demineralized blocks were divided into medial, lateral, and central thirds and embedded in paraffin for sectioning (Fig. 1). The medial and lateral thirds were sectioned coronally, the central third parasagittally. Sections were cut at 7– 8 m with a steel blade on a rotary microtome, floated onto a warm water bath (42°– 44° C), and mounted on polylysine-coated slides. Slides were dried on a slide warmer (32°–35° C) for 2 hr and stored at room temperature for at least 24 hr before further processing. For histology the sections were deparaffinized in a citrus-based clearing agent and hydrated in an ethanol series. For hematoxylin-eosin (H&E) sections were stained in Mayer’s hematoxylin followed by a dH2O rinse; stained in eosin; dehydrated in an ethanol series; cleared; and mounted with no. 121 coverslips. In addition to sections from the experimental animals, H&E sections of intact TMJs were available from a similarly sized animal. These had been embedded in celloidin and sectioned at about 25 m, the left side in the coronal plane and the right side parasagittally. For trichrome staining, Anton’s (1999) modification, which omits Mayer’s hematoxylin, was used. Slides were stained with acid fuchsin/orange G, rinsed, stained with phosphotungstic acid, rinsed again, stained with light green, dehydrated, cleared, and mounted. BrdU immunohistochemistry followed the procedures of Decker et al. (1996): deparaffinization; H202-methanol to inactivate endogenous peroxidases; 10% normal horse serum to minimize nonspecific binding of immunoglobulins; the primary mouse monoclonal antibody to BrdU (Becton Dickinson, Franklin Lakes, NJ) diluted 1:25 or 1:50; the secondary antibody, biotinylated horse antimouse IgG (Vector Labs, Burlingame, CA); the tertiary antibody, avidin-biotin-peroxidase (ABC Reagent, Vector Labs, Burlingame, CA); and reaction with 3,3⬘ diaminobenzidine-4HCl containing 0.02% H2O2 with nickel enhancement. Specificity controls included 10% normal horse serum substituted for primary antibody, PBS substitution alone, or omission of primary incubation entirely. The reacted sections were counterstained with 2% methyl green, washed repeatedly in N-butanol, and mounted with no. 121 coverslips. Light microscopic examination and counting of cells positive for BrdU were performed under Köhler illumination (Barer, 1953) using a Nikon E400 compound microscope with panfluor objectives. Positive cells were examined closely to confirm that the reaction was confined to the nucleus and had the pointillistic character of the tertiary antibody, peroxidase. Only sections that clearly showed both upper and lower joint cavities were examined. Typically, one to eight sections from each block met this criterion; all were counted and the results were averaged. For most subjects, only the right TMJ was available for analysis. For the few cases in which both sides were available, counts from right and left sides were averaged. Sites for quantification of replicating cells were chosen to compare areas of capsule/disc attachment with nonattachment areas, based on the idea that tensile stresses associated with attachment might increase local cell replication. All counts were performed by the same investigator (J.D.D.), who was not involved in the functional observations and was not aware of their results. Counts were made directly under the microscope. For the central third of the TMJ (parasagittal sections), counts were made using the 10⫻ objective at the anterior and posterior discal attachments (“anterior” and “posterior”) and midway between them (“middle”) for both the condyle and articular eminence, a total of six sites (Fig. 2A). The sites were defined by a circle 1 mm in diameter formed by the field diaphragm (0.8 mm2). For lateral and medial thirds (coronal sections), counts were made at the ligament/disc attachment sites (“lateral and medial poles”) and at the bearing surfaces (“lateral and medial”) of the condyle and eminence, for a total of four sites/section. Because labeled cells were less dense in the coronal sections, the 4⫻ objective was used and a larger area was examined. Each 6.6-mm2 site was defined by the ocular reticle, and each bearing surface site was 1.5 mm distant from its respective polar site (Fig. 2B). A Zeiss 5⫹100/100-mm calibration slide was used to confirm the area counted. Because of high individual variation, the results were analyzed using nonparametric rank order statistics (Friedman analysis of variance (ANOVA) followed by post hoc Wilcoxon signedrank tests, SPSS for Windows). To investigate regional variation in BrdU labeling, each articular surface was considered separately in each plane of section. Ranks were TMJ OF PIGS 155 CCD camera (Diagnostic Instruments, Sterling Heights, MI). RESULTS Bony Elements and Articular Surfaces Fig. 2. Overall histology of the TMJ, showing areas where BrdUpositive cells were counted. A: Central third of the TMJ, cut parasagittally, trichrome stain, showing the six 0.8-mm2 sites (circles): anterior and posterior disc attachments and middle area for both condyle and articular eminence. A higher magnification of the boxed region is shown in Figure 5A. B: Coronal section through the TMJ, H&E stain, showing the 6.6-mm2 areas (rectangles) at each polar discal attachment and 1.5 mm away on the lateral and medial articular surfaces. AE, articular eminence; Ant., anterior; C, condyle; Z, zygomatic bone. assigned for each individual based on the raw data (Table 1). Taking the last subject (pig 259) as an example, the anterior, middle, and posterior sites of the articular eminence were ranked 3 (most labeled cells), 1 (least labeled cells), and 2 (intermediate), respectively. Analogous sites on the condyle were ranked 1, 2, and 3, respectively. The articular eminence in the coronal sections could not be ranked because of missing data, but the condyle in the coronal sections was ranked 3 for the lateral pole, 4 for the lateral surface, 1 for the medial surface, and 2 for the medial pole. Photomicroscopy was accomplished with an attached Nikon F4 camera body equipped with a DW-21 viewfinder and Ektachrome 160T film or with a Spot RT In lateral view the TMJ is obscured by a flange of the zygomatic bone that juts beneath the zygomatic process of the squamosal part of the temporal bone (Fig. 3A). Removal of the arch reveals the presence of a distinct articular eminence oriented at 30°–50° to the occlusal plane (Fig. 3B). In contrast to the large, well-developed eminence anteriorly, the mandibular fossa consists only of a roof. Except for a small medial overlap of the mastoid area (Marks et al., 1997), there is no posterior wall and thus no postglenoid process (Fig. 3B). MR images demonstrate clearly that at rest in vivo only the anterior-superior aspect of the condyle apposes the articular eminence and that the intermediate zone of the TMJ disc is interposed between these articular surfaces (Fig. 4). As shown clearly in coronal CT scans (Fig. 3C), the eminence sits entirely lateral to the braincase and appears to be cantilevered from it. The CT scans also show that at their point of maximal congruence the articular surfaces of both the eminence and the condyle slope from dorsolateral to ventromedial (73° ⫾ 2° from the sagittal plane, n ⫽ 6). The articular surfaces are also slightly inclined in the horizontal plane, particularly the eminence, with the medial border lying posterior to the lateral border (Fig. 3D). The internal architecture of the eminence and condyle differ markedly (Fig. 2), as reported elsewhere (Herring and Liu, 2001). The eminence has a robust cortex, whereas the condylar head has little or no cortical bone. The trabeculae of the eminence are large, relatively sparse, and randomly arranged. In contrast, the condylar trabeculae are fine, densely packed, and roughly perpendicular to the condylar surface both in parasagittal and coronal sections. In parasagittal sections the condylar trabeculae become gradually finer and denser from anterior to posterior. Trabeculae are particularly long and thin beneath the posterior disc/capsular attachment (Figs. 2 and 5A). The articular surfaces of the eminence and condyle are similar in that both are lined by fibrous tissue underlain by cartilage. Between the fibrous and cartilaginous layers is a zone of fibroblast-like cells. This fibroblastic layer contained the vast majority of proliferating (BrdU-positive) cells for both the eminence and the condyle (see below). The cartilage layer is not continuous on the eminence, and even where present the cartilage is much thinner than on the condyle. Specifically, the eminence lacks the hypertrophic, mineralizing, and erosive zones that characterize growth cartilages, including the pig condyle (Roth et al., 1997). The surface coverings of the articular eminence show no obvious regional variation, but those of the condyle show striking changes from anterior to posterior. The condylar cartilage is thin anteriorly and gradually thickens posteriorly. The thickening is most marked from the central part of the condyle (underlying the intermediate zone of the disc in occlusion) to the posterior retrodiscal attachment area (Fig. 2A). In the posterior and polar areas of the condyle, perichondrial connective tissue invaginates deeply into cartilage. At the retrodiscal attachment, where the elongated bony trabeculae nearly reach the fibrous surface layer, the cartilage layer almost disappears (Fig. 5A). 156 HERRING ET AL. TABLE 1. BrdU-labeled cell counts by site Parasagittal sections (0.8 mm2 areas)b Articular eminence a Coronal sections (6.6 mm2 areas)b Condyle Articular eminence Condyle Pig Ant Middle Post Ant Middle Post Lpole Lat Med Mpole Lpole Lat Med Mpole 214m 215m 216f 217f 221m 222m 223f 224f 239f 241m 245m 246f 249m 250f 258f 259m –c 48 – 90 36 12 – 18 32 130 – – – 6 4 53 – 55 – 58 28 0 – 0 0 34 18 – – 0 10 10 – 52 – 34 0 0 – 0 0 38 32 – 4 22 12 47 0 18 0 0 38 4 30 0 34 74 – 10 16 33 16 26 0 87 0 128 75 0 18 0 24 2 28 70 56 0 23 81 14 62 229 118 114 43 43 42 229 293 152 83 103 96 148 257 0 0 8 2 5 1 4 0 – 0 5 0 10 0 3 95 2 32 10 39 20 7 5 0 – 0 2 0 12 0 13 132 6 7 0 8 42 62 – 10 1 24 21 9 14 5 0 – 2 8 0 2 3 14 – 6 0 16 10 1 0 12 0 – 38 22 51 55 35 10 35 5 10 0 28 5 38 10 0 85 40 47 57 44 21 1 32 4 6 0 51 15 14 31 36 89 48 24 64 28 18 10 5 17 12 34 33 16 22 54 4 34 70 27 63 34 28 10 4 21 15 29 16 4 8 74 2 51 n Mean S.D. 10 43 40 11 19 22 12 20 20 15 20 20 16 37 40 16 127 85 15 9 24 15 18 37 14 15 18 14 5 6 16 27 24 16 30 24 16 26 17 16 28 24 a f, m, female, male. Ant, Middle and Post are the regions indicated on Figure 2A; Lpole, Lat, Med, and Mpole are indicated on Figure 2B. Dashes indicate data missing because of incomplete histological material. b c Intra-articular Disc and TMJ Capsule The pig TMJ disc and its similarities to the human disc have been described often (Meister et al., 1973; Ström et al., 1986). In brief, the disc is ovate in dorsal view and biconcave in parasagittal section (Figs. 2A and 4A). The anterior and posterior bands feature dense interwoven collagen fibers oriented predominantly mediolaterally, whereas the crimped collagen fibers of the intermediate zone run anteroposteriorly (Sindelar et al., 2002). Elastic fibers are abundant and multidirectional in the anterior and especially the posterior bands, infrequent and aligned anteroposteriorly in the intermediate zone (Christensen, 1975). The reflections of the anterior band form the anterior capsule (Fig. 2A), and the medial portion of this area receives the partial insertion of the lateral pterygoid muscle (Herring and Scapino, 1973; Ström et al., 1986). The posterior band expands into a massive retrodiscal pad of fibrous tissue with internal clusters of adipose cells (Figs. 2A and 4A). The retrodiscal tissue divides into temporal and condylar attachments and constitutes the posterior part of the TMJ capsule. Medially and laterally the disc attaches to the poles of the condyle (Figs. 2B and 4B). The medial, lateral, and particularly the posterior disc attachments are robust compared to the anterior. The TMJ capsule attaches to the borders of the articular surfaces. This attachment is greatly enlarged posteriorly, where the temporal and condylar divisions of the retrodiscal tissue insert broadly onto the expanded posterior aspects of the condyle and temporal bone (Figs. 3D and 6). The capsule is closely associated with the disc not only anteriorly and posteriorly, but also at the condylar poles, where medial and lateral capsular thickenings, referred to here as capsular ligaments, join the discal insertions (Figs. 2B and 6). The lateral capsular ligament is a collateral thickening that begins at the medial side of the zygo- matic/squamous temporal suture line at the level of the posterior half of the articular eminence. The lateral ligament courses directly medially and attaches to the lip of the condyle at its lateral pole; it is thin, ribbon-like, nearly transparent, strong, and when distended, is approximately 4 –5 mm long, 2 mm wide, and 1 mm thick. The medial capsular ligament is also a collateral thickening and courses from a small pit posteromedial to the eminence to the medial condylar pole. The medial ligament is prominent and dense, measuring approximately 4 –5 mm in length, 4 –5 mm in width, and 2 mm in thickness. In addition to these collateral ligaments, the anterior capsule has an extension that stretches inferiorly and anteriorly from the anterior edge of the eminence and condyle, eventually attaching to the border of the mandibular notch up to the tip of the coronoid process. This anterior capsular ligament is highly elastic and nearly transparent. Small to medium vessels are frequent in the loose connective tissue anterior and lateral to the TMJ capsule. The delicate anterior capsular ligament has an abundant vascular supply and occasional small deposits of adipose tissue. The retrodiscal tissue is comparatively avascular. Cell Replication Sections incubated without the primary antibody for BrdU exhibited normal histologic characteristics and cellular architecture, but no nuclear reactivity. The positive histochemical reaction for BrdU in cell nuclei is prima facie evidence of DNA synthesis and thus of cells in the S-phase of replication. The distribution of replicating cells was very specific. Most labeling was found in the fibroblast-like cells (presumably undifferentiated mesenchymal cells) between the fibrous and cartilage layers of the articular surfaces (Figs. 5B, C and 7). Replicating cells were found throughout this TMJ OF PIGS 157 Fig. 3. Bony morphology of the TMJ. A: Lateral view, perpendicular to sagittal plane. With the zygomatic arch intact, the articular surfaces of the TMJ are obscured from view. B: Lateral view, perpendicular to mandibular surface. With the arch removed and the teeth in occlusion, the posterior aspect of the articular eminence can be seen to be in relation to the articular surface on the anterior aspect of the condyle. There is no posterior wall for the mandibular fossa. C: Coronal CT scan through both TMJs, showing their dorsolateral to ventromedial inclination. D: Palatal view of the skull, showing the anterolateral to posteromedial slope of the articular eminence (outlined area); the hatched area depicts the posterior attachment area for the disc/capsule. AE, articular eminence; B, auditory bulla; C, condyle; H, hyoid bone; PT, posterior temporal attachment area; ST, squamous part of temporal bone; Z, zygomatic bone. cambial layer, although there were some quantitative differences. Strikingly, the invaginations of the perichondrium into the cartilage at the medial and lateral poles and the posterior aspect of the condyle included this layer of replicating cells (Fig. 5B and C), creating an appearance of labeled canals. There were no similar invaginations subjacent to the disc attachment sites on the eminence. BrdU labeling was also seen in presumed osteoprogenitor cells in the periostea and marrow cavities of both the eminence and the condyle. Chondroblasts near the cambial layer were occasionally labeled. However, labeled cells were never seen anywhere in the disc (Fig. 7B) or the capsule, including the capsular ligaments. Because of the locations of the quantified areas (Fig. 2), the labeled cells counted were almost all from the cambial layer between the fibrous articular covering and the underlying cartilage. Raw counts are presented in Table 1, and Table 2 summarizes and compares the total counts for 158 HERRING ET AL. the center of the TMJ, the articular eminence showed no significant variation among the anterior, middle, and posterior sites, although there was a tendency (P ⫽ 0.12) for higher cell replication anteriorly. However, regional variation in the condyle was highly significant (P ⬍ 0.001). Here the anterior and middle areas had relatively low BrdU labeling (but comparable to labeling in the eminence, Table 1), whereas the posterior condylar site had by far the highest number of replicating cells. In coronal sections through the lateral and medial thirds of the TMJ, both the eminence and the condyle showed higher BrdU labeling in the bearing surfaces of the joint than at the poles, although this trend was significant only for the articular eminence (P ⫽ 0.015). Although the medial pole and surface tended to have more replicating cells than the lateral pole and surface, respectively, these trends were not statistically significant for either the eminence or the condyle (Fig. 8). Correlations Between Replication and Bone Strain Fig. 4. MR images of the TMJ. A: Parasagittal slice through the central area. The disc itself is dark because of its dense collagenous structure, but the retrodiscal tissue can be seen to consist of fibrous temporal and condylar attachments (arrows on dark bands) sandwiching a more fatty internal area (labeled RT). B: Coronal slice through the intermediate zone of the disc. The disc appears dark, but its attachments to the lateral and medial poles of the condyle can be visualized (arrows). The interior of condyle and articular eminence are lighter because of the contained marrow. AE, articular eminence; C, condyle; D, disc; RT, retrodiscal tissue; T, temporalis muscle; Z, zygomatic bone. the eminence and condyle. Replicating cells were far more numerous in the condyle, outnumbering those in the eminence 2:1 in parasagittal sections and 3:1 in coronal sections. There was a low but significant correlation between total eminence labeling and total condylar labeling (r ⫽ 0.73, P ⫽ 0.04). Individual variability was high, with eminence totals ranging from 34 –242 and condylar totals from 78 – 623. In addition to the greater labeling in the condyle than in the eminence, every region in the central part of the TMJ showed more cell replication than every region in the lateral and medial areas. This fact is not obvious from Table 1 until it is remembered that the lateral and medial sites counted in coronal sections were approximately eight times larger than the central sites counted in parasagittal sections (6.6 mm2 vs. 0.8 mm2). The analysis of regional variation of cell replication is presented in Figure 8. In parasagittal sections through Masticatory strains for the present sample of pigs are summarized in Table 3 and illustrated in Figure 9. Working and balancing side cycles differ little (Liu and Herring, 2000a) and were combined to represent the average loading of the TMJ during chewing. The gauges recorded strain on the lateral surfaces of the bones, roughly equivalent to a parasagittal plane. The squamosal part of the temporal bone was strongly tensed in the vertical direction, a pattern we have previously attributed to cantilever bending of the articular eminence (Herring and Liu, 2001) and out-of-plane bending of the entire zygomatic process of the temporal bone (Rafferty et al., 2000). In contrast, the condyle was mainly compressed, with the compressive axis running from anterosuperior to posteroinferior. Correlations between cell replication and the magnitude of masticatory bone strain are presented in Table 4, and Figure 10 shows scatter plots for the best correlations. Strikingly, all correlations were negative. As bone strain increased, cell counts decreased. In the articular eminence, BrdU labeling was negatively associated with all measures of strain in the temporal bone, particularly shear strain (P ⬍ 0.01; Table 4, Fig. 10), which is the sum of the magnitudes of tensile and compressive strain. By contrast, articular eminence labeling was not statistically correlated with condylar strain. In the condyle, BrdU labeling was most closely related to compressive strain in the condyle and compressive strain in the temporal bone, although these correlations fell slightly short of statistical significance (Table 4). DISCUSSION TMJ Anatomy: Pigs vs. Humans Pigs (family Suidae) are hoofed mammals of the order Artiodactyla. Other familiar members of the order are hippopotamus, camels, deer, and bovids such as sheep and cattle. TMJ anatomy is quite variable within the artiodactyls. Hippos and peccaries (family Tayassuidae, the sister group of the suids) feature prominent pre- and postglenoid processes that constrain the excursions of the condyle (Herring, 1972, 1975). Deer and bovids differ from pigs in the reverse direction, permitting unrestricted free movements of the concave condyle in almost all directions. It is this laxity that makes the sheep TMJ so convenient as a TMJ OF PIGS 159 Fig. 5. A: Enlargement of the area outlined in Figure 2A, showing the posterior aspect of condyle at the area of disc attachment. Note that the site of attachment coincides with a qualitative change in the condylar surface. The cartilage layer (between black arrows) is largely replaced by bony trabeculae perpendicular to the surface (white arrow). B: Coronal section of the condyle, lateral segment, BrdU histochemistry with methyl green counterstain. The lateral pole is to the left. Low-power view, showing extensive invaginations of the perichondrium into the cartilage layer. The box is enlarged in C. C: Higher-power view of the outlined area. The chondrocytes are negative for BrdU, but labeled fibroblast-like cells are numerous in the invaginating perichondrium. surgical model (van Loon et al., 2000). Because of the diversity among artiodactyls and because artiodactyls are only very distantly related to primates, the resemblance of the pig and human TMJ is clearly one of evolutionary convergence, presumably due to similar function. The resemblance is far from exact. Nonhuman primates, such as rhesus macaques, which share a recent common ancestry with humans, are closer to humans in TMJ structure (Tong and Tideman, 2001). Nevertheless, pigs will clearly remain popular alternative research models, and it is important to understand the limits of interspecies comparisons. As in the TMJ of humans, the pig condyle articulates against the strongly curved articular eminence rather than the mandibular fossa. As pointed out by other workers, the pig TMJ disc closely resembles that of humans (Meister et al., 1973; Ström et al., 1986) in most details, including its attachments. Also similar are the articular surfaces that slope from lateral, dorsal, and anterior toward medial, ventral, and posterior. Internal architecture is probably also similar. Like pigs, human mandibular condyles are strongly trabecular with the trabeculae perpendicular to the condylar axis (Giesen and van Eijden, 2000). Less attention has been paid to the human articular eminence, but most photographs show it as having a much thicker cortex than the condyle (e.g., Scapino, 1983). This suggests that the robust construction of the eminence in pigs is not due to its cantilevered position, because the 160 HERRING ET AL. Fig. 6. Left TMJ, oblique inferior view. Capsular ligaments have been drawn (pink) on a photograph of the skull. The medial (MCL) and lateral (LCL) capsular ligaments are thickenings in the capsule. The anterior capsular ligament (ACL) is a delicate, elastic extension from the anterior capsule that inserts on the superior edge of the mandible from the mandibular notch to the tip of the coronoid process. The hatched areas on the condyle and squamosal part of the temporal bone indicate the attachment areas of the posterior disc/capsule. human eminence is buttressed by the braincase. Instead, the thick cortex of both human and pig articular eminences suggests a commonality, either of loading or of growth pattern. Pig-human differences include overall dimensions. The pig joint is larger relative to body size. Whereas adult humans have condyles 15–20 mm in width (Hylander, 1992), 8-month-old miniature pigs weighing 46 ⫾ 4 kg (comparable to a small human) have condyles 21–25 mm wide (Sindelar, personal communication). An additional difference is that the pig TMJ is more protected than the human laterally and medially. Laterally, the TMJ capsule is hidden by the overhanging zygomatic flange (Figs. 2– 4). Medially, the bony configuration is similar, but whereas the medial capsule of humans is composed of weak areolar tissue (Loughner et al., 1997), that of pigs is strengthened by a stout ligament (Fig. 6). The most striking morphological difference between pig and human TMJs is, however, the retrodiscal area. Pigs have no postglenoid wall, leaving the posterior aspect of the TMJ unprotected by bone and nearly subcutaneous. For this reason the posterior approach can be used to access the lateral pterygoid muscle (e.g., Liu and Herring, 2000a). The missing bony wall has consequences for posterior discal attachments. In humans the temporal and condylar attachments are separated by a delicate venous plexus, and the whole area is sheltered by the postglenoid process. Luder has described this area as serving to provide space for the distensible discal attachments (Luder, 1996, p 49). The equivalent space in the exposed pig TMJ is filled by a rubbery retrodiscal pad. If this tissue is capable of accommodating condylar movements, it must use elasticity rather than vascular volume compensation. The general construction of the pig TMJ permits a range of movements rather similar to those of humans. The soft posterior wall of the joint may allow more retrusion than in humans. In anesthetized animals, roughly the same amount of retrusion and protrusion can be achieved (4 – 6 mm) (Sun et al., in press). As in humans, masticatory Fig. 7. BrdU immunohistochemistry in parasagittal section, showing the surface of the articular eminence (A), superior surface of the TMJ disc (B), and surface of the condyle (C), all near the middle area. Anterior is to the left. BrdU-positive cells (black) were found almost exclusively in the subarticular perichondrial layers of the eminence and condyle. No replicating cells were present in the fibrous articular tissue or the disc, and very few were found in the cartilage. The cartilage layer of the eminence is much thinner than that of the condyle and replicating (BrdU-positive) cells are less numerous. opening is accompanied by protrusion and masticatory closing by retrusion (Herring and Scapino, 1973). The attachment of the disc to the poles of the condyle implies that, again as in humans, protrusion and retrusion are TMJ OF PIGS TABLE 2. Total counts of BrdU-labeled cells in the condyle and articular eminence (mean and standard deviation) Coronal sections Parasagittal sections Combined Eminence Condyle t-test 35 ⫾ 24 n ⫽ 13 112 ⫾ 73 n ⫽ 16 P ⬍ 0.02 83 ⫾ 73 n ⫽ 10 182 ⫾ 109 n ⫽ 15 P ⫽ 0.001 128 ⫾ 88 n⫽8 294 ⫾ 146 n ⫽ 15 P ⫽ 0.02 Fig. 8. Relative distribution of BrdU-labeled cells in the TMJ. The error bars show the mean ranks with 95% confidence intervals. Using the raw data in Table 1, ranks were assigned for each articular surface in each plane of section for each subject. The site with the least labeled cells was assigned the rank of 1, whereas the site with the most labeled cells was assigned a rank of 3 (for parasagittal sections) or 4 (for coronal sections). Significant regional differences were found for the condyle in parasagittal sections and the articular eminence in coronal sections (Friedman ANOVA followed by post hoc Wilcoxon signed-rank tests). *, pairs significantly different at P ⬍ 0.05; **, pairs significantly different at P ⬍ 0.01. movements of the condyle-plus-disc relative to the articular eminence, whereas opening rotations probably occur between the disc and the condyle. It seems likely that the lateral (bony) and medial (ligamentous) reinforcements of the TMJ would limit transverse condylar movements. However, lateral excursions are not solely produced by transverse condylar movements, but also by rotations arising from asymmetrical condylar protrusion/retrusion (Sun et al., in press). The elongated jaws of pigs also help compensate for limited TMJ movement by exaggerating angular motion. During chewing the incisors undergo lateral excursions of 4 –5 mm (Herring, 1976). Patterns of Cell Replication and Growth A number of our histological observations are best explained in the context of growth. While the articular eminence does not contribute to cranial dimensions, the condylar cartilage serves both as an articular surface and as a major growth site for the mandible. The 11-month-old pigs in the present study were postpubertal but not full- 161 grown. Epiphyses of long bones were still open, the dentition was still mixed, and the jaws were presumably still lengthening. Cell replication was active, as indicated by the BrdU results. Because the interval between BrdU injection and animal sacrifice was only 10 hr, it is likely that only a fraction of the replicating cells had time to incorporate the label. Nevertheless, the distribution of labeled cells is probably an accurate depiction of relative rates of cell division in different parts of the TMJ. The fact that these rates varied regionally is in itself evidence that directional growth was taking place, rather than simple tissue turnover. The layering of the eminential and condylar articular surfaces is typical of that seen in humans and other animals at comparable ages. The location of the BrdU label between the fibrous articular layer and the underlying cartilage corresponds to the proliferative zone identified for the condyle (Luder, 1996) and presumably has the same role in the eminence. A previous BrdU study on the rat TMJ did not find labeling on the temporal bone articular surface (Sekine et al., 1991), but this may be due to the absence of an articular eminence in rats. Although some of the newly replicated cells probably contribute to the fibrous articular layer, it seems likely that most are prechondroblasts. A gradual conversion of fibrous tissue toward a cartilage phenotype has been described for the (presumed) most loaded surfaces in humans: articular eminence, intermediate zone of the disc, and anterior aspect of the condyle (Moffett et al., 1964; Luder, 1996, 1998). With the exception of the disc, these were also the locations of cartilage in the present sample of pigs. The posterior slope of the condyle displayed some unusual features that probably reflect the posterior-superior direction of condylar growth. These include thickening of articular and cartilage layers, invaginations of the perichondrium, and a sudden transition to a noncartilaginous, trabecular area at the posterior discal attachment. The number of replicating cells was far higher here than in any other location in the TMJ (Table 1). Some of these features are seen in other species, which of course grow in the same direction. Thickening of the articular layer on the posterior slope of the condyle has been noted in both humans and monkeys (Luder, 1996); only in this region do humans retain a cellular intermediate layer (Luder, 1998). The sudden loss of cartilage at the posterior discal attachment is also seen in humans (Luder, 1996), although the transition to spindly trabeculae (Fig. 5A) appears to be unique to pigs. In previous growth studies we have noticed that these trabeculae elongate at a remarkable rate (0.3– 0.4 mm/day in 4-month-old pigs) (Ferrari and Herring, 1995). Finally, the prominent invaginations of the posterior perichondrium also have a human equivalent, although only in fetuses and very young children. Connective tissue invasions of the human fetal TMJ condylar head were described by Vinogradoff (1910) as “crampons.” In Vinogradoff’s opinion the crampons served to fasten the perichondrium firmly to the cartilage. Later writers have considered them more as vascular channels, possibly analogous to cartilage canals (summarized by Luder, 1996). In the pig joints, Vinogradoff’s mechanical explanation seems more plausible than the nutritional idea; the invaginations we observed were not particularly vascular, but they were strongly associated with the attachments of the disc into the condyle. Even more striking, however, was their association with cell replication. Without excep- 162 HERRING ET AL. TABLE 3. Mean peak masticatory strains for the animals used for BrdU labeling* Temporal bone strain (ε) Pig 214 215 216 217 221 222 223 224 239 241 245 246 249 250 258 259 n Mean ⫾ S.D. Condylar strain (ε) Tension Compression Orientation Tension Compression Orientation – 66 44 67 152 282 62 338 351 100 251 – 251 400 198 243 14 200 ⫾ 120 – ⫺52 ⫺26 ⫺25 ⫺121 ⫺223 ⫺28 ⫺228 ⫺47 ⫺36 ⫺61 – ⫺76 ⫺65 ⫺98 ⫺116 14 ⫺86 ⫾ 67 – 80° 82° 55° 115° 128° 127° 129° 66° 87° 151° – 47° 131° 36° 131° 14 98° ⫾ 37 – 85 44 143 364 254 422 110 113 73 – 43 99 103 – 163 13 155 ⫾ 119 – ⫺174 ⫺68 ⫺200 ⫺578 ⫺624 ⫺740 ⫺324 ⫺207 ⫺197 – ⫺81 ⫺94 ⫺69 – ⫺111 13 ⫺267 ⫾ 231 – 167° 130° 158° 74° 100° 85° 124° 77° 121° – 116° 101° 86° – 90° 13 110° ⫾ 30 *These data are a subset of a previously published sample [Liu & Herring, 2000a]. Left and right chewing cycles (10 –20/ animal) were averaged. Missing data are indicated by dashes. Minimum principal strains were all compressive (expressed as negative values) and maximum principal strains were all tensile (positive values). The orientation of tensile strain is expressed as a clockwise angle relative to the occlusal plane with the skull viewed from the left (Fig. 9). Shear strain can be calculated by summing tensile strain and the absolute value of compressive strain. Fig. 9. Average bone strain during chewing for the 16 pigs used in the present study (data in Table 3). The strain gauges were located on the squamosal part of the temporal bone just lateral to the articular eminence (see Fig. 3) and on the lateral surface of the condyle (Liu and Herring, 2000a). The arrows depict the magnitude and orientation of the peak principal strains. Arrowheads pointing away from the gauge site indicate tensile strain, whereas arrowheads pointing toward the gauge site indicate compressive strain. The hatched triangles indicate 1 S.D. for orientation. Tensile strain, directed approximately vertically, dominates the temporal bone. For the condyle, compressive strain, oriented anterosuperior to posteroinferior, is dominant. tion, the invaginations were heavily labeled with BrdU. Conceivably, the infolded posterior perichondrium reflects the need for rapid cell replication in this fastest-growing part of the skull. The dividing cells occupy a layer of a curved surface but must provide new cells to fill a volume. Folding this layer would be an effective way to increase its area and the number of dividing cells without changing the volume enclosed. The distribution of BrdU labeling on the eminence (Fig. 8) suggests that the articular eminence was becoming flatter. The eminence is convex in the parasagittal section (Fig. 2A), and the tendency for higher replication anteriorly and posteriorly would decrease that convexity. In the coronal plane the eminence is concave (Fig. 2B), and the lower replication at the poles would reduce the concavity. The situation is less clear for the condyle, because the parasagittal sections are dominated by posterior growth, and little difference among locations was seen in the coronal plane. Although BrdU labeling in parasagittal sections of the condyle clearly indicates growth in the posterior direction, no such trend was apparent for the eminence (Fig. 8). Because the eminence becomes repositioned in space by growth at cranial synchondroses and sutures, there is no need for oriented expansion of the eminence cartilage. In the coronal plane, both the eminence and the condyle move laterally with growth. In the case of the condyle, this movement probably involves Enlow’s V-principle (Enlow and Hans, 1996), in which an enlarging V-shape (the condyle) shows remodeling resorption on its periphery but apposition centrally. The V-principle leads to the expectation that more central areas on the condylar surface would have more replicating cells than more polar areas. Our data (Fig. 8, lower right graph) are in the correct direction but are very far from statistical significance. Strangely, the data for the eminence (Fig. 8, upper right graph) do correspond with the expectation of the V-principle, but the eminence should not be growing in this manner. Rather, the eminence is thought to move laterally by simple osseous drift, with resorption on the medial side and apposition on the lateral side. If osseous drift were responsible 163 TMJ OF PIGS TABLE 4. Parametric (Pearson) and nonparametric (Spearman) correlations between BrdU labeling and peak strain magnitudes during mastication Straina Temporal bone Total labeled cells Articular eminence Pearson r Spearman rho Condylar surface Pearson r Spearman rho Tension Compression ⫺0.86** ⫺0.79* ⫺0.64† ⫺0.79* ⫺0.29 ⫺0.28 ⫺0.52† ⫺0.48† Condyle Shear Tension Compression Shear ⫺0.87** ⫺0.83* ⫺0.17 ⫺0.32 ⫺0.22 ⫺0.18 ⫺0.21 ⫺0.14 ⫺0.42 ⫺0.40 ⫺0.26 ⫺0.18 ⫺0.51† ⫺0.44 ⫺0.43 ⫺0.34 a Compression and tension are the principal strains illustrated in Figure 9. Shear strain is the sum of tensile strain and the absolute value of compressive strain. *P ⬍ 0.05; **P ⬍ 0.01; †0.05 ⬍ P ⬍ 0.10. Fig. 10. Scatter plots for the best correlations between cell replication and labeling (Table 4). All correlations were negative. BrdU labeling in the articular eminence correlated best with shear strain in the temporal bone (asterisks, r ⫽ – 0.87, P ⫽ 0.005), and labeling in the condylar surface correlated best with compressive strain in the condyle (diamonds, r ⫽ – 0.51, P ⫽ 0.07). for the pattern of cell replication in the eminence, then we would expect higher labeling laterally than medially. This was not seen; in fact, the trend was in the reverse direction. In short, the only aspect of BrdU labeling that strongly supported a known pattern of growth was that seen in parasagittal sections, specifically the large number of replicating cells near the posterior attachment on the condyle. Labeling in the coronal plane did not support the standard models of growth. This failure may reflect our lack of understanding of the growth in this plane, or perhaps our study was too limited in time to reflect a global picture of that growth. On the other hand, some aspects of cell replication may be better explained by function than by growth. TMJ Loading in Relation to Structure and Cell Replication The compressive loading applied to joints causes both hydrostatic (shape-preserving) and shear (distortional) stress. According to Carter and Beaupré (2001), increasing levels of hydrostatic compression should be associated with thicker articular cartilages (but not necessarily tissue growth), whereas shear loads result in endochondral ossification. Further, Carter and Beaupré (2001) have argued that hydrostatic compression is always greater in the convex than in the concave member of a synovial joint and that this difference causes the convex member to have a thicker layer of cartilage. Thus, hydrostatic loading may be the reason for the thicker cartilage and more active cell replication of the convex condyle than of the concave eminence. However, this explanation is only useful for the coronal plane. In the parasagittal plane, both elements are convex. Functional loading of the TMJ is probably dominated by shear stress, rather than hydrostatic compression. The strain gauge results always indicated distortion, i.e., simultaneous compression and tension at right angles to each other. In dentistry it is believed that increasing the loading on the condyle will reduce or redirect growth away from the compressive axis (Proffit, 1986). Thus, where distortion is present, cell replication is expected to favor the tensile axis (summarized by Heegaard et al., 1999). Our original selection of areas for BrdU quantification was intended to compare regions of ligamentous attachment with more central portions of the articular surfaces. We reasoned that the attachments would add a tensile component to the otherwise compressed cartilage and expected that cell labeling would be higher at the attachments. The expectation of higher replication at attachments was clearly wrong. It is supported only by the parasagittal sections of the eminence (Fig. 8, top left), and these results were not statistically significant. The high BrdU counts at the posterior attachment to the condyle are better explained by growth (see above), and the anterior attachment to the condyle actually had fewer labeled cells than the middle region (Fig. 8, bottom left). All results from the coronal sections were opposite to the expected findings. Cell replication at the poles was less than at the bearing surfaces, significantly so for the eminence (Fig. 8, right). Furthermore, overall density of labeling was higher in the central third of the joint than in the more polar lateral or medial thirds. This can be seen by dividing the counts in Table 1 by the area measured. Labeling density in the middle region of the parasagittally sectioned central third was 24 cells/mm2 in the eminence and 46 cells/mm2 in the condyle, whereas the coronally 164 HERRING ET AL. sectioned lateral and medial thirds showed labeling densities of 1–3 cells/mm2 in the eminence and 4 cells/mm2 in the condyle. In retrospect, the notion that compressive loading would be reduced at the attachment zones was probably incorrect because the orientations of these two loads would not coincide. The tensile forces applied by the disc and ligaments would be roughly parallel to the articular surfaces (Fig. 2), whereas the compressive loading presumably acts perpendicular to the surfaces. Thus tension applied by the attachments would not decrease the compressive loading, but rather would increase the shear stress, inhibiting cell division. Another possible explanation for the lower replication rates of the polar areas is that these regions may actually be loaded more heavily in compression than more central areas. The condyle and eminence are usually at their closest approach at the poles (Figs. 3C and 4B); such congruency may increase local loading and depress cell replication. In the absence of detailed information about the distribution of stress within the articular surfaces, it is impossible to test these alternatives rigorously. Although the distribution of stress within the articular surfaces is unknown, we do have data relevant to loading in the parasagittal plane. The present study is unique in providing bone strain information on the same animals used for BrdU labeling. The fact that we were able to establish correlations is in itself remarkable. Both BrdU labeling and in vivo bone strain have many sources of experimental variation that mitigate against detecting relationships. Furthermore, the BrdU results were obtained from the right TMJ and the strain information from the left TMJ. Despite these difficulties, the results show clearly that bone strain is negatively correlated with cell replication. All correlations were negative, with high significance for the articular eminence and borderline significance for the condyle (Table 4, Fig. 10). Thus, individual pigs with high masticatory loading had low rates of cell division in the TMJ. This may be a causal relationship; high loading, especially compressive loading, has been associated with decreased chondrocyte proliferation in many in vivo and in vitro studies (Hamrick, 1999; Farnum et al., 2000). Because the strain data also include information about orientation in the parasagittal plane, we can also investigate the relationship between the directionality of loading and of cell replication in parasagittal sections, at least for the condyle. As discussed above, the articular eminence did not show directionality of cell replication. Furthermore, the magnitude of compressive strain (thought to be the main mechanical influence on growth) in the squamosal part of the temporal bone was relatively low, averaging only – 86 ⑀ (Table 3). By contrast, the condyle was strongly directional in its growth, with the posterior location being far more active than the anterior and middle areas, and its strain pattern was strongly compressive, averaging –267 ⑀. This compression was directed anterosuperiorly (Fig. 9). Thus, in the condyle, cell replication is oriented in the direction of tensile strain, posterosuperiorly. This direction is orthogonal to the compressive axis. This finding is again consistent with the notion that loading (compression) decreases cell division. Even on an individual basis, there are indications that the direction of condylar compression may be associated with the direction of growth. There were only two pigs in which the posterior area of the condyle did not have the most BrdUlabeled cells (pigs 215 and 217); both showed slightly heavier labeling in the middle than in the posterior site (Table 1). These were also the only two animals that had an unusually anterior-posterior (⬎1 S.D. from the mean) orientation of condylar tensile strain (Table 3). Thus the compressive axis of these two animals was almost vertical, resulting in a more symmetrically distributed compressive load over the top of the condyle and a more even spread of replicating cells. In summary, these data indicate that individual differences in loading may be a strong influence on growth in the TMJ. In particular, high strains are associated with low cell replication rates, and animals with unusual loading orientations have unusual growth patterns as well. In general, the findings were consistent with the hypothesis that local replicative activity would be lowest in the direction of the compressive load. However, areas of ligamentous attachment, rather than having high replication rates as expected, had relatively low numbers of labeled cells; the mechanical correlates of this difference are still unknown. Direct mechanical effects on cell replication provide a mechanism whereby altered function, such as eating a soft diet, can cause changes in craniofacial dimensions (Ciochon et al., 1997). ACKNOWLEDGMENTS We are grateful to Patricia Emry for histological preparation, to Katherine Rafferty, Scott Pedersen, and Christopher Marshall for their help with the experiments, and to the Diagnostic Imaging Science Center at the University of Washington for its assistance with CT and MRI. LITERATURE CITED Albu I, Brâtucu L, Roth HK. 1979. Vergleichend-Morphologische Untersuchungen über die Struktur des Discus Articularis des Kiefergelenkes. Morphol Embryol (Buch.) 25:197–204. Amor FB, Carpentier P, Foucart JM, Meunier A. 1998. Anatomic and mechanical properties of the lateral disc attachment of the temporomandibular joint. J Oral Maxillofac Surg 56:1164 –1167. Anton E. 1999. Detection of apoptosis by a modified trichrome technique. J Histotechnol 22:301–304. Barer R. 1953. Lecture notes on the use of the microscope. Springfield, IL: C.C. Thomas. 76 p. Berg R, Meister R. 1974. Lichtmikroskopische Untersuchungen über den Discus articularis des Kiefergelenks einiger Haussäugetiere under besonderer Berücksichtigung seiner Resektion. Deutsche Zahn-, Mund- und Kieferheilkunde 62:12–17. Bermejo A, González O, González JM. 1993. The pig as an animal model for experimentation on the temporomandibular articular complex. Oral Surg, Oral Med, Oral Pathol 75:18 –23. Carter DR, Beaupré GS. 2001. Skeletal function and form. Cambridge, UK: Cambridge University Press. 318 p. Christensen LV. 1975. Elastic tissue in the temporomandibular disc of miniature swine. J Oral Rehab 2:373–377. Ciochon RL, Nisbett RA, Corruccini RS. 1997. Dietary consistency and craniofacial development related to masticatory function in minipigs. J Craniofac Gen Dev Biol 17:96 –102. Dannhauer K-H. 1992. Die Wachstumsreaktion des mandibulären Gelenkknorpels auf biomechanische Reize und ihre Bedeutung für die Funktionskiefer-orthopädie—Ergebnisse tierexperimenteller und biophysikalischer Untersuchungen. Fortschr Kieferorthop 53: 53– 60. Decker JD, Marshall JJ, Herring SW. 1996. Differential cell replication within the periosteum of the pig mandibular ramus. Acta Anat 157:144 –150. Enlow DH, Hans MG. 1996. Essentials of facial growth. Philadelphia: Saunders. 303 p. TMJ OF PIGS Farnum CE, Nixon A, Lee AO, Kwan DT, Belanger L, Wilsman NJ. 2000. Quantitative three-dimensional analysis of chondrocytic kinetic responses to short-term stapling of the rat proximal tibial growth plate. Cells Tissue Org 167:247–258. Ferrari CS, Herring SW. 1995. Use of a bite-opening appliance in the miniature pig: modification of craniofacial growth. Acta Anat 154: 205–215. Frost HM. 1990. Skeletal structural adaptations to mechanical usage (SATMU): 3. The hyaline cartilage modeling problem. Anat Rec 226:423– 432. Giesen EBW, van Eijden TMGJ. 2000. The three-dimensional cancellous bone architecture of the human mandibular condyle. J Dent Res 79:957–963. Gratzner HG. 1982. Monoclonal antibody to 5-bromodeoxyuridine and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218:474 – 475. Greene CS, Mohl ND, McNeill C, Clark GT, Truelove EL. 1999. Temporomandibular disorders and science: a response to critics. Am J Orthod Dentofac Orthop 116:430 – 431. Hamrick MW. 1999. A chondral modeling theory revisited. J Theor Biol 201:201–208. Heegaard JH, Beaupré GS, Carter DR. 1999. Mechanically modulated cartilage growth may regulate joint surface morphogenesis. J Orthopaed Res 17:509 –517. Herring SW. 1972. The role of canine morphology in the evolutionary divergence of pigs and peccaries. J Mammal 53:500 –512. Herring SW, Scapino RP. 1973. Physiology of feeding in miniature pigs. J Morphol 141:427– 460. Herring SW. 1975. Adaptations for gape in the hippopotamus and its relatives. Forma et Functio 8:85–100. Herring SW. 1976. The dynamics of mastication in pigs. Arch Oral Biol 21:473– 480. Herring SW. 1995. Animal models of temporomandibular disorders: how to choose. In: Sessle BJ, Bryant PS, Dionne RA, editors. Temporomandibular disorders and related pain conditions. Seattle: IASP Press. p 323–328. Herring SW, Liu ZJ. 2001. Loading of the TMJ: anatomical and in vivo evidence from the bones. Cells Tissue Org 169:193–200. Hylander WL. 1992. Functional anatomy. In: Sarnat BG, Laskin DM, editors. The temporomandibular joint: a biological basis for clinical practice. Philadelphia: W.B. Saunders Co. p 60 –92. Kuboki T, Shinoda M, Orsini MG, Yamashita A. 1997. Viscoelastic properties of the pig temporomandibular joint articular soft tissues of the condyle and disc. J Dent Res 76:1760 –1769. Liu ZJ, Herring SW. 2000a. Masticatory strains on osseous and ligamentous components of the jaw joint in miniature pigs. J Orofacial Pain 14:265–278. Liu ZJ, Herring SW. 2000b. Bone surface strains and internal bony pressures at the jaw joint of the miniature pig during masticatory muscle contraction. Arch Oral Biol 45:95–112. Loughner BA, Gremillion HA, Mahan PE, Watson RE. 1997. The medial capsule of the human temporomandibular joint. J Oral Maxillofac Surg 55:363–369. Luder HU. 1996. Postnatal development, aging, and degeneration of the temporomandibular joint in humans, monkeys, and rats. Ann Arbor, MI: Center for Human Growth and Development, University of Michigan. 260 p. Luder HU. 1998. Age changes in the articular tissue of human mandibular condyles from adolescence to old age: a semiquantitative light microscopic study. Anat Rec 251:439 – 447. Marks L, Teng S, Årtun J, Herring S. 1997. Reaction strains on the condylar neck during mastication and maximum muscle stimulation in different condylar positions: an experimental study in the miniature pig. J Dent Res 76:1412–1420. McNeill C. 1997. Management of temporomandibular disorders: concepts and controversies. J Prosthet Dent 77:510 –522. Meister R, Berg R, Berg P. 1973. Beiträge zur topographischen und angewandten Anatomie des Kiefergelenkes (Articulatio temporomandibularis) einiger Haussäugetiere unter besonderer Berück- 165 sichtigung der Resektionsmöglichkeiten des Discus articularis. 4. Schwein (Sus scrofa domesticus). Z Exp Chirurg 6:437– 448. Moffett BCJ, Johnson LC, McCabe JB, Askew HC. 1964. Articular remodeling in the adult human temporomandibular joint. Am J Anat 115:119 –142. Moroco JR, Hinton R, Buschang P, Milam SB, Iacopino AM. 1997. Type II collagen and TGF-␤s in developing and aging porcine mandibular condylar cartilage: immunohistochemical studies. Cell Tissue Res 289:119 –124. Müller K, Roth S, Fischer D-C, Walther S, Dannhauer K-H. 1996. The soft tissue cover of the mandibular condyle: age-related changes in high buoyant density proteoglycans, free tissue water and remodelling activity. J Orofac Orthop 57:310 –321. Ngan P, Kleeman B, Jordan F, Rosol T, Yousefian J, Shanfeld J, Davidovitch Z. 1992. Effect of intermittent pressure on periodontal ligament cell-mediated bone resorption in vitro. In: Davidovitch Z, editor. The biological mechanisms of tooth movement and craniofacial adaptation. Columbus, OH: Ohio State University. p 331–339. Nickel JC, McLachlan KR. 1994. In vitro measurement of the stressdistribution properties of the pig temporomandibular joint disc. Arch Oral Biol 39:439 – 448. Nickel JC, Beatty MW, Iwasaki LR, Leiker M, Simetich B, Kimmel M. 2000. Mechanical behavior of the porcine TMJ disc under repeated tensile loading. J Dent Res 79:592. Osborn JW. 1995. Internal derangement and the accessory ligaments around the temporomandibular joint. J Oral Rehab 22: 731–740. Proffit WR. 1986. Contemporary orthodontics. St. Louis: CV Mosby Co. 579 p. Rafferty KL, Herring SW, Artese F. 2000. Three-dimensional loading and growth of the zygomatic arch. J Exp Biol 203:2093–3004. Roth S, Müller K, Fischer D-C, Dannhauer K-H. 1997. Specific properties of the extracellular chondroitin sulphate proteoglycans in the mandibular condylar growth centre in pigs. Arch Oral Biol 42:63– 76. Roth TE, Goldberg JS, Behrents RG. 1984. Synovial fluid pressure determination in the temporomandibular joint. Oral Surg Oral Med Oral Pathol 57:583–588. Sato I, Shindo K, Ezure H, Shimada K. 1996. Morphology of the lateral ligament in the human temporomandibular joint. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 81:151–156. Scapino RP. 1983. Histopathology associated with malposition of the human termporomandibular joint disc. Oral Surg Oral Med Oral Pathol 55:382–397. Schmolke C. 1994. The relationship between temporomandibular joint capsule, articular disc and jaw muscles. J Anat 184:335– 345. Sekine J, Hironaka R, Inokuchi T. 1991. Application of bromodeoxyuridine immunohistochemistry to the rat temporomandibular joint. Okaj Folia Anat Jpn 68:219 –224. Sindelar BJ, Evanko SP, Alonzo T, Herring SW, Wight T. 2000. Effects of intraoral splint wear on proteoglycans in the temporomandibular joint disc. Arch Biochem Biophys 379:64 –70. Sindelar BJ, Edwards S, Herring SW. 2002. Morphologic changes in the TMJ following splint wear. Anat Rec 266:167–176. Ström D, Holm S, Clemensson E, Haraldson T, Carlsson GE. 1986. Gross anatomy of the mandibular joint and masticatory muscles in the domestic pig (Sus scrofa). Arch Oral Biol 31:763–768. Sun Z, Liu ZJ, Herring SW. 2002. Movement of TMJ tissues during mastication and passive manipulation: a study in miniature pigs. Arch Oral Biol, in press. Teng S, Herring SW. 1995. A stereological study of trabecular architecture in the mandibular condyle of the pig. Arch Oral Biol 40: 299 –310. Teng S, Choi IW, Herring SW, Rensberger JM. 1997. Stereological analysis of bone architecture in the pig zygomatic arch. Anat Rec 248:205–213. Timmel R, Grundschober F. 1982. The interposition of Lyodura in operations for ankylosis of the temporomandibular joint. J Maxillofac Surg 10:193–199. 166 HERRING ET AL. Tong AC, Tideman H. 2001. The microanatomy of the rhesus monkey temporomandibular joint. J Oral Maxillofac Surg 59:46 – 52. Ulrici V, Händel L, Vogel A, Reissig D. 1988. Microscopical-anatomical changes of the temporomandibular joint of miniature pig due to the unilateral occlusal disturbances. Anat Anz 167:329 – 333. van Loon J-P, de Bont LGM, Spijkervet FKL, Verkerke GJ, Liem RSB. 2000. A short-term study in sheep with the Groningen temporomandibular joint prosthesis. Int J Oral Maxillofac Surg 29:315–324. Vinogradoff A. 1910. Development de l’articulation temporomaxillaire chez l’homme dans la periode intrauterine. Internat Monatsschr Anat Physiol 27:490 –523. Yen EHK, Pollit DJ, Whyte WA, Suga DM. 1990. Continuous stressing of mouse interparietal suture fibroblasts in vitro. J Dent Res 69:26 –30. Yousefian JZ, Ngan PW, Miller B, Shanfeld J, Davidovitch Z. 1992. Effect of different types of stress on human periodontal ligament cells in vitro. In: Davidovitch Z, editor. The biological mechanisms of tooth movement and craniofacial adaptation. Columbus, OH: Ohio State University. p 319 –329.