Trabecular Anisotropy in the Primate Mandibular Condyle Is Associated with Dietary Toughness.

Past attempts to establish a relationship between mandibular morphology and different dietary categories (e.g., frugivore, folivore, insectivore) have had mixed results, possibly because descriptive dietary categories are too broad and obscure variation within primate diets. Another potential reason is that not all aspects of skeletal architecture, especially trabecular anisotropy, have factored into functional assessments of dietary inputs into jaw form. Recent emphasis on quantifying food mechanical properties (FMPs) has provided an alternative to reliance on dietary categories. We used data on FMPs to test for correlations among dietary toughness and Young's modulus and the trabecular structure of the mandibular condyle, which is loaded during feeding and should reflect differences in masticatory stresses associated with different dietary FMPs. Adult primate mandibles from 11 species were imaged using high-resolution X-ray computed tomography, and trabecular structure was analyzed with BoneJ and Quant3D to assess common three-dimensional trabecular parameters. Results of phylogenetic generalized least squares analysis suggested a positive correlation between the degree of anisotropy (DA) and toughness, and weaker correlations between FMPs and various other trabecular variables. Because the DA contributes to the mechanical properties of bone, these results suggest a functional relationship between dietary toughness and trabecular anisotropy in the mandibular condyle. Such a perspective underscores the need to consider all aspects of skeletal morphology in evaluating the links between diet and jaw biomechanics. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Development of a Rat Model of Mechanically Induced Tunable Pain and Associated Temporomandibular Joint Responses.

PURPOSE: Although mechanical overloading of the temporomandibular joint (TMJ) is implicated in TMJ osteoarthritis (OA) and orofacial pain, most experimental models of TMJ-OA induce only acute and resolving pain, which do not meaningfully simulate the pathomechanisms of TMJ-OA in patients with chronic pain. The aim of this study was to adapt an existing rat model of mechanically induced TMJ-OA, to induce persistent orofacial pain by altering only the jaw-opening force, and to measure the expression of common proxies of TMJ-OA, including degradation and inflammatory proteins, in the joint. MATERIALS AND METHODS: TMJ-OA was mechanically induced in a randomized, prospective study using 2 magnitudes of opening loads in separate groups (ie.,. 2-N, 3.5-N and sham control [no load]). Steady mouth opening was imposed daily (60 minutes/day for 7 days) in female Holtzman rats, followed by 7 days of rest, and orofacial sensitivity was measured throughout the loading and rest periods. Joint structure and extent of degeneration were assessed at day 14 and expression of matrix metalloproteinase-13 (MMP-13), hypoxia-inducible factor-1α (HIF-1α), and tumor necrosis factor-α (TNF-α) in articular cartilage was evaluated by immunohistochemistry and quantitative densitometry methods at day 7 between the 2 loading and control groups. Statistical differences of orofacial sensitivity and chondrocyte expression between loading groups were computed and significance was set at a P value less than .05. RESULTS: Head-withdrawal thresholds for the 2 loading groups were significantly decreased during loading (P < .0001), but that decrease remained through day 14 only for the 3.5-N group (P < .00001). At day 14, TMJs from the 2-N and 3.5-N groups exhibited truncation of the condylar cartilage, typical of TMJ-OA. In addition, a 3.5-N loading force significantly upregulated MMP-13 (P < .0074), with nearly a 2-fold increase in HIF-1α (P < .001) and TNF-α (P < .0001) at day 7, in 3.5-N loaded joints over those loaded by 2 N. CONCLUSION: Unlike a 2-N loading force, mechanical overloading of the TMJ using a 3.5-N loading force induced constant and nonresolving pain and the upregulation of inflammatory markers only in the 3.5-N group, suggesting that these markers could predict the maintenance of persistent orofacial pain. As such, the development of a tunable experimental TMJ-OA model that can separately induce acute or persistent orofacial pain using similar approaches provides a platform to better understand the pathomechanisms involved and possibly to evaluate potential treatment strategies for patients with painful TMJ-OA.

An Immunohistochemical Study of Matrix Components in Early-Stage Vascular Canals Within Mandibular Condylar Cartilage in Midterm Human Fetuses.

Matrix components of vascular canals (VCs) in human fetal mandibular condylar cartilage (15-16 weeks of gestation) were analyzed by immunohistochemistry. Prevascular canals (PVCs), consisting of spindle-shaped cells without capillary invasion, were observed within the cartilage. Intense immunoreactivity for collagen type I, weak immunoreactivity for aggrecan and tenascin-C, weak hyaluronan (HA) staining, and abundant argyrophilic fibers in PVCs indicated that they contain noncartilaginous fibrous connective tissues that was different from those in the perichondrium/periosteum. These structural and immunohistochemical features of PVCs are different from those of previously reported cartilage canals of the long bone. Capillaries entered the VCs from the periosteum and ascended through VCs. Following capillary invasion, loose connective tissue had formed in the lower part of VCs, and immunoreactivity for collagen types I and III, tenascin-C, and HA staining was evident in the matrix of loose connective tissue. No chondroclasts or osteogenic cells were seen at the front of capillary invasion, although small, mononuclear tartrate-resistant acid phosphatase (TRAP)-positive cells were present. Meanwhile, TRAP-positive, multinucleated chondroclasts and flattened, osteoblast-like cells were observed in the loose connective tissue at the lower part of VCs. These results may indicate slow progress of endochondral ossification in human fetal mandibular condyle. Further, unique matrix components in PVCs/VCs, which were different from those in cartilage canals in long bone, may reflect the difference of speed of endochondral ossification in cartilage canals and human fetal mandibular condyles.

Analysis of pain in the rabbit temporomandibular joint after unilateral splint placement.

AIMS: To determine whether behavioral, anatomical, and physiologic endpoints widely used to infer the presence of pain in rodent models of temporomandibular disorders (TMD) were applicable to the rabbit model of TMD associated with altered joint loading. METHODS: Unilateral molar dental splints were used to alter temporomandibular joint (TMJ) loading. Changes in nociceptive threshold were assessed with a mechanical probing of the TMJ region on nine splinted and three control rabbits. Fos-like immunoreacitivty in the trigeminal subnucleus caudalis was assessed with standard immunohistochemical techniques from three splinted and six control animals. Retrogradely labeled TMJ afferents were studied with patch-clamp electrophysiologic techniques from three splinted and three control animals. Remodeling of TMJ condyles was assessed by histologic investigations of three splinted and three control animals. A Student t test or a Mann-Whitney U test was used with significance set at P < .05 to compare splinted to control samples. RESULTS: While variable, there was an increase in mechanical sensitivity in splinted rabbits relative to controls. The increase in Fos+ cells in splinted rabbits was also significant relative to naïve controls (86 ± 8 vs 64 ± 15 cells/section, P < .05). The rheobase (364 ± 80 pA) and action potential threshold (-31.2 ± 2.0 mV) were higher in TMJ afferents from splinted rabbits compared to controls (99 ± 22 pA and -38.0 ± 2.0 mV, P < .05). There was significant remodeling in the condylar fibrocartilage layers as manifested by a change in glycosaminoglycan distribution and a loss of defined cell layers. CONCLUSION: Behavioral and anatomical results were consistent with an increase in nociceptive signaling in concert with condylar remodeling driven by altered TMJ loading. Changes in excitability and action potential waveform were consistent with possible compensatory changes of TMJ afferents for an overall increase in afferent drive associated with joint degeneration. These compensatory changes may reflect pain-adaption processes that many patients with TMJ disorders experience.

Norepinephrine Regulates Condylar Bone Loss via Comorbid Factors.

Degenerative changes of condylar subchondral bone occur frequently in temporomandibular disorders. Although psychologic stresses and occlusal abnormalities have been implicated in temporomandibular disorder, it is not known if these risks represent synergistic comorbid factors that are involved in condylar subchondral bone degradation that is regulated by the sympathetic nervous system. In the present study, chronic immobilization stress (CIS), chemical sympathectomy, and unilateral anterior crossbite (UAC) were sequentially applied in a murine model. Norepinephrine contents in the subjects' serum and condylar subchondral bone were detected by ELISA; bone and cartilage remodeling parameters and related gene expression in the subchondral bone were examined. Subchondral bone loss and increased subchondral bone norepinephrine level were observed in the CIS and UAC groups. These groups exhibited decreased bone mineral density, volume fraction, and bone formation rate; decreased expressions of osterix, collagen I, and osteocalcin; but increased trabecular separation, osteoclast number and surface, and RANKL expression. Combined CIS + UAC produced more severe subchondral bone loss, higher bone norepinephrine level, and decreased chondrocyte density and cartilage thickness when compared to CIS or UAC alone. Sympathectomy simultaneously prevented subchondral bone loss and decreased bone norepinephrine level in all experimental subgroups when compared to the vehicle-treated counterparts. Norepinephrine also decreased mRNA expression of osterix, collagen I, and osteocalcin by mesenchymal stem cells at 7 and 14 d of stimulation and increased the expression of RANKL and RANKL/OPG ratio by mesenchymal stem cells at 2 h. In conclusion, CIS and UAC synergistically promote condylar subchondral bone loss and cartilage degradation; such processes are partially regulated by norepinephrine within subchondral bone.

Study of differential properties of fibrochondrocytes and hyaline chondrocytes in growing rabbits.

We aimed to build a culture model of chondrocytes in vitro, and to study the differential properties between fibrochondrocytes and hyaline chondrocytes. Histological sections were stained with haematoxylin and eosin so that we could analyse the histological structure of the fibrocartilage and hyaline cartilage. Condylar fibrochondrocytes and femoral hyaline chondrocytes were cultured from four, 4-week-old, New Zealand white rabbits. The production of COL2A1, COL1OA1, SOX9 and aggrecan was detected by real time-q polymerase chain reaction (RT-qPCR) and immunoblotting and the differences between them were compared statistically. Histological structures obviously differed between fibrocartilage and hyaline cartilage. COL2A1 and SOX9 were highly expressed within cell passage 2 (P2) of both fibrochondrocytes and hyaline chondrocytes, and reduced significantly after cell passage 4 (P4). The mRNA expressions of COL2A1 (p=0.05), COL10A1 (p=0.04), SOX9 (p=0.03), and aggrecan (p=0.04) were significantly higher in hyaline chondrocytes than in fibrochondrocytes, whereas the expression of COL1A1 (p=0.02) was the opposite. Immunoblotting showed similar results. We have built a simple and effective culture model of chondrocytes in vitro, and the P2 of chondrocytes is recommended for further studies. Condylar fibrocartilage and femoral hyaline cartilage have unique biological properties, and the regulatory mechanisms of endochondral ossification for the condyle should be studied independently in the future.

Higher number of pentosidine cross-links induced by ribose does not alter tissue stiffness of cancellous bone.

The role of mature collagen cross-links, pentosidine (Pen) cross-links in particular, in the micromechanical properties of cancellous bone is unknown. The aim of this study was to examine nonenzymatic glycation effects on tissue stiffness of demineralized and non-demineralized cancellous bone. A total of 60 bone samples were derived from mandibular condyles of six pigs, and assigned to either control or experimental groups. Experimental handling included incubation in phosphate buffered saline alone or with 0.2M ribose at 37°C for 15 days and, in some of the samples, subsequent complete demineralization of the sample surface using 8% EDTA. Before and after experimental handling, bone microarchitecture and tissue mineral density were examined by means of microcomputed tomography. After experimental handling, the collagen content and the number of Pen, hydroxylysylpyridinoline (HP), and lysylpyridinoline (LP) cross-links were estimated using HPLC, and tissue stiffness was assessed by means of nanoindentation. Ribose treatment caused an up to 300-fold increase in the number of Pen cross-links compared to nonribose-incubated controls, but did not affect the number of HP and LP cross-links. This increase in the number of Pen cross-links had no influence on tissue stiffness of both demineralized and nondemineralized bone samples. These findings suggest that Pen cross-links do not play a significant role in bone tissue stiffness.

Levels and distribution of organochlorine pollutants in primary dental tissues and bone of lamb.

This study examined the bioconcentration of selected organochlorine pollutants, tetra- and hexa-chlorobiphenyls with planar (PCB-80, PCB-169) and non-planar (PCB-54, PCB-155) structure, and persistent organochlorine pesticides with planar [hexachlorobenzene (HCB)] and non-planar [1,1-bis (4-chlorophenyl)-2,2-dichloroethene (4,4'-DDE)] structure in primary dental tissues (pulp, dentine, and enamel) and mandibular bone of lactationally exposed lambs, and compared it with the organochlorines distribution pattern in permanent dental tissues and bone. Also, the role of pollutants physicochemical properties and tissue specific characteristics in the bioconcentration was assessed. Residual levels of individual pollutants were analyzed by high-resolution gas chromatography with electron-capture detection. Our results showed that transfer of organochlorines to primary hard dental tissues was higher than to permanent hard dental tissues. Metabolically more stable, planar, and toxic organochlorines (e.g. PCB-169 and HCB) predominated in primary hard dental tissues, where they may represent a potential risk for developmental dental defects.

Multi-tissue analysis of oxygen isotopes in wild rhesus macaques (Macaca mulatta).

Oxygen isotopes in animal tissues are directly related to body water composition and thus the environment. Accurate measurement of animal tissue δ(18)O provides information about local climate, an animal's geographical origin and subsequent movements, with wide applications in palaeobiology and forensic science. The genesis and evolution of tissue-based oxygen isotopes within species and within individuals are complex. We present the first data, for non-human primates, rhesus macaques (Macaca mulatta), on the relationship between oxygen isotope sources in bio-apatite (PO(4) and PCO(3)) and hair taken from six sample sites in Asia, ranging from western India to northern Vietnam. The range of values is similar within each tissue type, with good correlation between tissues (r = 0.791 to 0.908), allowing cross-tissue extrapolations. This is important when the availability of suitable tissues is limited. Biological interpretation of the small data set is difficult: macaque diets are eclectic, and the samples are from various locations. However, factors such as overall climate, precipitation quantity and source, and altitude are clearly influencing the results for each discrete geographical grouping. Future work could be aimed at assessing δ(18)O tissue associations for other species as the relationships appear to be species-specific.

Histological investigation of osseous changes of mandibular condyles with backscattered electron images.

OBJECTIVES: This study aims to confirm the radiographic morphology of mandibular condyles with microscopic observation and to investigate the mechanism of osseous changes of adult human mandibular condyles. METHODS: Following the radiographic examinations of ten temporomandibular joints (TMJs) from five cadavers using conventional tomography, helical CT and limited cone beam CT for dentistry (limited CBCT), ten mandibular condyles were removed. Micro CT images were made of the undecalcified dissected joints in the sagittal plane, perpendicular to the long axis of the condyles, to produce images similar in orientation to the radiographic images. Tissue morphology was observed through backscattered electron images and elemental analysis using electron probe microanalysis (EPMA). The microscopic findings were compared with the radiographic findings. RESULTS: Limited CBCT images most accurately depicted erosive change of the bone cortex of the mandibular condyle. EPMA indicated bone remodelling in all mandibular condyles analysed, which showed cartilage calcification different from typical enchondral ossification. No finding of dissolution or disruption of the superficial soft tissue was observed in all specimens, including those presenting radiographic findings of osteoarthrosis. Active bone remodelling with hypertrophic changes of superficial soft tissue was observed in mandibular condyles, which implied radiographic findings of osteoarthrosis. CONCLUSIONS: The high detectability of limited CBCT images on bony morphology of mandibular condyles was confirmed. It is speculated that bone remodelling of adult human mandibular condyles is brought about by cartilage calcification. It was suggested that the osseous changes classified as osteoarthrosis with radiographic images are normal bone remodelling.

Relationship between tissue stiffness and degree of mineralization of developing trabecular bone.

It is unknown how the degree of mineralization of bone in individual trabecular elements is related to the corresponding mechanical properties at the bone tissue level. Understanding this relationship is important for the comprehension of the mechanical behavior of bone at both the apparent and tissue level. The purpose of the present study was, therefore, to determine the tissue stiffness and degree of mineralization of the trabecular bone tissue and to establish a relationship between these two variables. A second goal was to assess the change in this relation during development. Mandibular condylar specimens of four fetal and four newborn pigs were used. The tissue stiffness was measured using nanoindentation. A pair of indents was made in the cores of 15 trabecular elements per specimen. Subsequently, the degree of mineralization of these locations was determined from microcomputed tomography. The mean tissue stiffness was 11.2 GPa (+/-0.5 GPa) in the fetal group and 12.0 GPa (+/-0.8 GPa) in the newborn group, which was not significantly different. The degree of mineralization of the fetal trabecular cores was 744 mg/cm3 (+/-28 mg/cm3). The one in the newborn bone measured 719 mg/cm(3) (+/-34 mg/cm3). Again, the difference was statistically insignificant. A significant relationship between tissue stiffness and degree of mineralization was obtained for fetal (R = 0.42, p < 0.001) and newborn (R = 0.72, p < 0.001) groups. It was concluded that woven bone tissue in fetal and newborn trabecular cores resembles adult trabecular bone in terms of tissue properties and is strongly correlated with degree of mineralization.

Forward deviation of the mandibular condyle enhances endochondral ossification of condylar cartilage indicated by increased expression of type X collagen.

OBJECTIVE: Using type X collagen as a marker, this research was designed to examine the alteration of condylar growth in response to mandibular condylar forward positioning. METHODS: One hundred female Sprague-Dawley rats with 5 weeks of age were randomly divided into five experimental and five control groups. In the experimental groups, bite jumping appliances created forward positioning of the condyle. The experimental rats, together with the age-matched controls, were sacrificed on days 3, 7, 14, 21 and 30, respectively. Tissue sections were cut in the sagittal plane through the mandibular condyle and were processed for in situ hybridization and immunostaining of type X collagen and then for quantitative imaging analyses. RESULTS: (1) Both type X collagen mRNA in situ hybridization signals and type X collagen immunostaining were localized within the hypertrophic zone of the condylar cartilage. (2) With condylar forward positioning, the level of type X collagen mRNA signals (8,541 +/- 74 microm(2) at peak) was 300% higher than that in the controls (2,117 +/- 78 microm(2) at peak); type X collagen immunostaining in condylar advancing groups (54,864 +/- 134 microm(2) at peak) was 254% more than that in the controls (15,470 +/- 121 microm(2) at peak). (3) The amount of type X collagen mRNA signals and immunostaining in experimental and control groups reached the highest levels at day 14 and day 21, respectively, indicating that an increase in endochondral ossification occurred 21 days after condylar forward deviation. CONCLUSION: Condylar forward repositioning provokes an enhanced maturation of condylar chondrocytes resulting in increased synthesis of type X collagen, a extracellular protein that attributes to endochondral ossification.

Type II collagen and aggrecan mRNA expressions in rabbit condyle following disc displacement.

The aim of this investigation was to study the remodelling of cartilage in the mandibular condyle following disc displacement (DD) of the temporomandibular joint (TMJ). Forty adult Japanese white rabbits were used in this study. The right joints of 28 of the 40 rabbits had their discs surgically displaced. Four of the 28 were killed at 4 days or 1, 2, 4, 6, 8 and 12 weeks after surgery. The messenger RNA (mRNA) expression levels of aggrecan and type II collagen in cartilages were measured using in situ hybridization techniques. Results showed that aggrecan mRNA expression reduced in the first week after DD. The expression began to recover after 4 weeks and reached a normal level after 6 weeks. Type II collagen mRNA expression reduced from 4 weeks and the expression recovered after 8 weeks. This suggests that the chondrocyte reacting to the displacement of the TMJ disc, alters its matrix gene expression patterns and it is may be the cause of the shape changes of TMJ after DD.

Incisor disocclusion in rats affects mandibular condylar cartilage at the cellular level.

UNLABELLED: The effect of altered occlusion on the mandibular condylar cartilage remains unclear. OBJECTIVE: This study investigated the effect of unilateral incisor disocclusion on cartilage thickness, on mitotic activity and on chondrocytes maturation and differentiation in the mandibular condylar cartilage of rats. DESIGN: The upper and lower left incisors were trimmed 2mm every second day in five rats. In other five rats, the incisor occlusion was not altered. Condylar tissues from both sides of each mandible were processed and stained for Herovici's stain and immunohistochemistry for bromodeoxyuridine (BrdU), transforming growth factor-beta1 (TGF-beta1), alkaline phosphatase (ALP) and osteocalcin (OCN). Measurements of cartilage thickness and the numbers of immunopositive cells for each antibody were analysed by one-way analysis of variance (ANOVA). RESULTS: No significant differences were observed in cartilage thickness after 7 days of unilateral incisor disocclusion. However, the numbers of immunopositive cells for BrdU as a marker of DNA synthesising cells, TGF-beta1 as a marker of chondrocytes differentiation, and ALP and OCN as markers of chondrocytes maturation, were significant higher in the cartilage cells on both sides when incisor occlusion was unilaterally altered. Interestingly, alkaline phosphatase was highly expressed on the condylar side of incisor disocclusion, whereas osteocalcin was highly expressed on the side opposite to the incisor disocclusion. CONCLUSIONS: It is demonstrated that after 7 days, unilateral incisor disocclusion affects the mandibular condylar cartilage at the cellular level by increasing the mitotic activity and by accelerating chondrocytes maturation. Chondrocytes maturation appears more accelerated on the side opposite to incisor disocclusion.

Immunohistochemical study of the upper surface layer in rat mandibular condylar cartilage.

Both hyaluronic acid and fibronectin localizations were examined in the upper surface layer of rat mandibular condylar cartilages by immunohistochemical techniques. Their delicate structure was successfully preserved by preparation procedures of joint condyles with disks. Paraformaldehyde-fixed cartilaginous tissues were cut in a cryostat, and cryosections were analyzed using streptavidin-peroxidase and indirect immunofluorescence methods. Another immunogold method with conventional preparation procedures and a quick-freezing method was performed for their ultrastructural analyses. Both hyaluronic acid-binding protein and anti-fibronectin antibody were used to localize hyaluronic acid and fibronectin in the mandibular condylar cartilage, respectively. Some cryosections were pre-treated with hyaluronidase and chondroitinase before such labeling. The upper surface layer was composed of double laminar structures. One bordered with the cartilage matriceal surface, which was positive for fibronectin. The hyaluronic acid was localized over the fibronectin layer. Therefore, the hyaluronic acid in vivo was bound with fibronectin in the cartilaginous matrix, performing lubrication for the mandibular joint movement.

Immunohistochemical analysis of Bcl-2 and Bax oncoproteins in rabbit craniomandibular joint.

The purpose of this study was to elucidate the expression of proto-oncogene Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) in fibrocartilage of the disc and hyaline cartilage of the condyle in the rabbit craniomandibular joint (CMJ). Ten New Zealand white rabbit heads were used. Sections were processed by the immunohistochemical techniques using mouse anti-Bcl-2 and anti-Bax antibodies. Intensity levels of immunostaining in condylar cartilage were quantified by a computer-image system. Immunoreactivity for Bcl-2 was mainly observed in the cytoplasm of the reserve cell and chondrocytic cell layers. A mild heterogeneous Bax expression was detected in the cytoplasm of chondrocytes of the upper hypertrophic layer and a few cells of the chondrocytic layer. The cytoplasm of chondrocytes in the disc exhibited a high intensity for Bcl-2, while Bax activity was only sporadically observed. We have shown that Bcl-2 and Bax proteins are present in CMJ cartilage and their expression patterns suggest that these oncoproteins are involved in chondrocyte survival or death via apoptotic pathways.

Connective tissue growth factor mRNA expression pattern in cartilages is associated with their type I collagen expression.

Connective tissue growth factor (CTGF) has been identified as a secretory protein encoded by an immediate early gene and is a member of the CCN family. In vitro CTGF directly regulates the proliferation and differentiation of chondrocytes; however, a previous study showed that it was localized only in the hypertrophic chondrocytes in the costal cartilages of E 18 mouse embryos. We described the expression of CTGF mRNA and protein in chondrocytes of different types of cartilages, including femoral growth plate cartilage, costal cartilage, femoral articular cartilage, mandibular condylar cartilage, and cartilage formed during the healing of mandibular ramus fractures revealed by in situ hybridization and immunohistochemistry. To characterize the CTGF-expressing cells, we also analyzed the distribution of the type I, type II, and type X collagen mRNA expression. Among these different types of cartilages we found distinct patterns of CTGF mRNA and protein expression. Growth plate cartilage and the costal cartilage showed localization of CTGF mRNA and protein in the hypertrophic chondrocytes that expressed type X collagen mRNA with less expression in proliferating chondrocytes that expressed type II collagen mRNA, whereas it was also expressed in the proliferating chondrocytes that expressed type I collagen mRNA in the condylar cartilage, the articular cartilage, and the cartilage appearing during fracture healing. In contrast, the growth plate cartilages or the costal cartilages were negative for type I collagen and showed sparse expression of CTGF mRNA in the proliferating chondrocytes. We found for the first time that CTGF mRNA could be differentially expressed in five different types of cartilage associated with those expressing type I collagen. Moreover, the spatial distribution of CTGF mRNA in the cartilages with type I collagen mRNA suggested its roles in the early differentiation, as well as in the proliferation and the terminal differentiation, of those cartilages.

In situ hybridization and immunohistochemistry of bone sialoprotein and secreted phosphoprotein 1 (osteopontin) in the developing mouse mandibular condylar cartilage compared with limb bud cartilage.

Mandibular condylar cartilage is often classified as a secondary cartilage, differing from the primary cartilaginous skeleton in its rapid progress from progenitor cells to hypertrophic chondrocytes. In this study we used in situ hybridization and immunohistochemistry to investigate whether the formation of primary (tibial) and secondary (condylar) cartilage also differs with respect to the expression of two major non-collagenous glycoproteins of bone matrix, bone sialoprotein (BSP) and secreted phosphoprotein 1 (Spp1, osteopontin). The mRNAs for both molecules were never expressed until hypertrophic chondrocytes appeared. In the tibial cartilage, hypertrophic chondrocytes first appeared at E14 and the expression of BSP and Spp1 mRNAs was detected in the lower hypertrophic cell zone, but the expression of BSP mRNA was very weak. In the condylar cartilage, hypertrophic chondrocytes appeared at E15 as soon as cartilage tissue appeared. The mRNAs for both molecules were expressed in the newly formed condylar cartilage, although the proteins were not detected by immunostaining; BSP mRNA in the condylar cartilage was more extensively expressed than that in the tibial cartilage at the corresponding stage (first appearance of hypertrophic cell zone). Endochondral bone formation started at E15 in the tibial cartilage and at E16 in the condylar cartilage. At this stage (first appearance of endochondral bone formation), BSP mRNA was also more extensively expressed in the condylar cartilage than in the tibial cartilage. The hypertrophic cell zone in the condylar cartilage rapidly extended during E15-16. These results indicate that the formation process of the mandibular condylar cartilage differs from that of limb bud cartilage with respect to the extensive expression of BSP mRNA and the rapid extension of the hypertrophic cell zone at early stages of cartilage formation. Furthermore, these results support the hypothesis that, in vivo, BSP promotes the initiation of mineralization.

Parathyroid hormone-related protein regulates proliferation of condylar hypertrophic chondrocytes.

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, e.g., it shows delayed appearance in development relative to the limb bud cartilage, originates from the periosteum rather than from undifferentiated mesenchymal cells, and shows rapid differentiation into hypertrophic chondrocytes as opposed to the epiphyseal growth plate cartilage, which has resting and proliferative zones. Recently, attention has been focused on the role of parathyroid hormone-related protein (PTHrP) in modulating the proliferation and differentiation of chondrocytes. To investigate further the characteristic modes of growth and differentiation of this cartilage, we used mice with a disrupted PTHrP allele. Immunolocalization of type X collagen, the extracellular matrix specifically expressed by hypertrophic chondrocytes, was greatly reduced in the condylar cartilage of homozygous PTHrP-knockout mice compared with wild-type mice. In contrast, immunolocalization of type X collagen of the tibial cartilage did not differ. In wild-type mice, proliferative chondrocytes were mainly located in both the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but were limited to the proliferative zone of the tibial cartilage. The number of proliferative chondrocytes was greatly reduced in both cartilages of homozygous PTHrP-knockout mice. Moreover, apoptotic chondrocytes were scarcely observed in the condylar hypertrophic cell layer, whereas a number of apoptotic chondrocytes were found in the tibial hypertrophic zone. Expression of the type I PTH/PTHrP receptor was localized in the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but was absent from the tibial hypertrophic chondrocytes. It is therefore concluded that, unlike tibial hypertrophic chondrocytes, condylar hypertrophic chondrocytes have proliferative activity in the late embryonic stage, and PTHrP plays a pivotal role in regulating the proliferative capacity and differentiation of these cells.

Immunohistochemical localization of fibroblast growth factor receptors in the rat mandibular condylar cartilage and tibial cartilage.

The fibroblast growth factor receptors (FGFRs), members of the tyrosine-kinase receptor family, are known to play a crucial role in the growth and development of cartilaginous tissues. The mandibular condylar cartilage has been suggested to have a characteristic growth pattern compared with the tibial growth plate cartilage, e.g., cell alignment, mode of proliferation and differentiation, and response to humoral and mechanical factors. To examine the mRNA expression and localization of fibroblast growth factor receptor (FGFR)-1, -2, and -3 in the condylar and tibial growth plate cartilages, reversed transcribed polymerase chain reaction (RT-PCR) assay and immunohistochemistry were carried out using growing rats. The enzymatically isolated rat condylar and tibial chondrocytes expressed mRNA of aggrecan and type II collagen, which are together known as the major cartilaginous extracellular matrices. Both types of cells expressed mRNA of FGFR-1, -2, and -3 by RT-PCR. In the neonatal rat, immunolocalization of FGFR-1, -2, and -3 was found in the middle of the condylar cartilage, mainly in the hypertrophic zone of the tibial cartilage. At 3 weeks old, the three FGFRs were broadly observed in both cartilages. At 8 weeks old, localization of FGFR-3 was absent in the hypertrophic cell layer of the condyle, whereas it was still broadly observed in the tibial growth plate cartilage. In the same stage, FGFR-1 and FGFR-2 showed similar localization in both cartilages to that at 3 weeks of age. All these observations suggest that FGFRs play an important role in the differential growth pattern of the condylar cartilage.