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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The insulin-sensitive glucose transporter (GLUT4) is involved in early bone growth in control and diabetic mice, but is regulated through the insulin-like growth factor I receptor.

Children with uncontrolled type I (insulin-dependent) diabetes mellitus are characterized by a slow growth rate, which improves upon adequate therapy. While skeletal growth is an energy-consuming process involving high glucose utilization, the role of glucose transporters (GLUT) and their regulation in the bone formation process are not yet fully understood. Thus, we studied both in vivo and in vitro early endochondral bone formation in control and streptozotocin-induced young diabetic mice. Using in situ hybridization and immunohistochemistry techniques, we demonstrated the novel existence of the insulin-sensitive glucose transporter (GLUT4), as well as GLUT1, in juvenile-derived murine mandibular condyles and in the humeral growth plate-two models for endochondral bone formation. Insulin-like growth factor (IGF) I receptors (IGF-I-R), but not insulin receptors (IR), were shown to have cellular distribution similar to GLUT4, being more abundant in mature chondrocytes. Further, in the skeletal growth centers of streptozotocin-induced diabetic mice, GLUT4, IGF-I, and IGF-I and insulin receptor levels, but not GLUT1 were markedly reduced. The decrease in GLUT4 and in IGF-I and insulin receptors was associated with severe histological changes in the mandibular condyles and humeral growth plate. Insulin therapy restored IR levels to normalcy, whereas IGF-I-R and GLUT4 levels were only partially recovered. Thus, GLUT4 and IGF-I-R have a potential role in early bone growth in mice. Further, during early bone growth GLUT4 may be regulated through the IGF-I receptor rather than via the insulin receptor. We propose that skeletal growth retardation in type I diabetes may be associated with reduced expression of the GLUT4 and IGF-I receptor in the bone growth center.

Differential effects of hypothyroidism on the cartilage and the osteogenic process in the mandibular condyle: recovery by growth hormone and thyroxine.

Hypothyroidism was induced in young female Sprague-Dawley rats by the addition of methimazole (50 mg/kg BW.day) to drinking water for a period of 7 weeks (7-14 weeks of age). Replacement therapies of 0.7 U/kg BW human GH (hGH), 15 micrograms/kg BW L-T4 (T4), and a combination of hGH and T4 at the same doses were introduced during the last 2 weeks of the experiment. The responses of the cartilage and subchondral spongiosa of mandibular condyles were assessed by morphological and morphometric parameters. In addition, immunohistochemical localization of the GH receptor and insulin-like growth factor-I was determined by the streptavidin-biotin-peroxidase technique. In the hypothyroid rats, the trabecular bone volume of the subchondral spongiosa increased by 49%, indicating osteopetrosis. Along the ossification front, bone trabeculae occupied 18% and vascular elements 82% in mandibular condyles of control rats, whereas in hypothyroid rats, the percentage occupied by bone trabeculae increased to 89%. The cellularity of the cartilage in hypothyroid condyles was markedly reduced and was fully restored by T4, but not by GH replacement. Immunohistochemistry revealed the presence of GH receptors throughout the condyle regardless of the thyroid state of the animal. On the other hand, insulin-like growth factor-I immunohistochemical localization revealed the peptide to be present at all maturational stages of the cells in condyles from control and T4-treated rats, but to be lacking in young chondrocytes of hypothyroid and hGH-treated rats. This result demonstrates that the hypothyroid cartilage is compromised in its response to hGH.

Differential expression of fibroblast growth factor receptor-1, -2, and -3 and syndecan-1, -2, and -4 in neonatal rat mandibular condyle and calvaria during osteogenic differentiation in vitro.

Fibroblast growth factors (FGFs) play important roles in the control of skeletal cell growth and differentiation. To identify the mechanisms of regulation of FGF actions during chondrogenesis and osteogenesis, we investigated, by immunohistochemistry, the spatiotemporal expression of the high-affinity FGF receptors (FGFR-1, -2, and -3) and coreceptors (syndecans-1, -2, and -4) in newborn rat condyle and calvaria during chondrogenesis and osteogenesis in vitro. During chondrogenesis at 4 days of culture, condyle chondrocytes showed weak FGFR-1, FGFR-2, and syndecan-1 immunoreactivity; stronger syndecan-2 expression; and marked FGFR-3 and syndecan-4 immunolabeling. At a later stage (i.e., 9 days of culture), FGFR-1, -2, and -3 were coexpressed with syndecan-4 in chondrocytes. Condyle progenitor cells located in the condyle perichondrium initially expressed strong syndecan-2 and -4 and weak syndecan-1 labeling, whereas no FGFR was detectable. When these cells differentiated into osteoblasts, they expressed syndecan-2 and -4 coincidently with FGFR-1, -2, and -3 at 9 days of culture. In newborn rat calvaria, syndecan-1, -2, and -4 were coexpressed mainly with FGFR-1 and -2 in osteoblasts. In the two models, treatment with FGF-2 (100 ng/mL) at 4-9 days of culture increased cell growth and decreased glycosaminoglycan or collagen synthesis, respectively, suggesting interactions of FGF-2 with distinct FGFRs and syndecans during chondrogenesis and osteogenesis. The coincident or distinct spatiotemporal expression pattern of FGFRs and syndecans in chondrocytes, progenitor cells, and osteoblasts represents a dynamic mechanism by which FGF effects on skeletal cells may be controlled in a coordinate manner during cartilage and bone formation in vitro.

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.

The early postnatal development of the murine mandibular condyle is regulated by endogenous insulin-like growth factor-I.

Skeletal growth during the early postnatal period is thought to be GH-independent, and is probably regulated by intrinsic growth factors. We studied the involvement of locally produced insulin like growth factor-I (IGF-I) in the growth of the neonatal mandibular condyle. Immunofluorescence studies revealed intense staining with antibodies to IGF-I in the mandibular condyle of 2-day-old ICR mice. We have also shown that these mandibular condyles contain specific high-affinity binding sites (Kd = 0.157 nmol/l) for IGF-I (427 fmol/mg). Autoradiographical studies of iodinated IGF-I revealed that the distribution of the receptors for IGF-I was parallel to that of IGF-I production, mainly in the younger zones of the condyle, namely the chondroprogenitor and the chondroblast cell layers. Immunoinhibition of IGF-I resulted in an almost complete inhibition (-91%) of thymidine incorporation into DNA, as well as in marked degenerative changes in the morphological appearance of the condyle. Our studies support the hypothesis that early postnatal growth is dependent on the paracrine activity of endogenous GH-independent IGF-I.

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.

Presence of chondroid bone on rat mandibular condylar cartilage. An immunohistochemical study.

Immunohistochemical techniques were used to examine the locations of type I and type II collagens in the the most anterior and the posterosuperior regions of the mandibular condylar cartilages of young and adult rats. Large ovoid and polygonal cells, which were morphologically different from any of the neighboring cells, e.g., mature or hypertrophied chondrocytes, osteoblasts, or fibroblasts, were observed at the most anterior margin of the young and adult condylar cartilages. In the extracellular matrix (ECM) of this area, an eosinophilic staining pattern similar to that in bone matrix was observed, while the peripheral ECM showed basophilic staining and very weak reactivity to Alcian blue. Immunohistochemical examination showed that the ECM was stained heavily and diffusely for type I collagen, while a staining for type II collagen was faint and limited to the peripheral ECM. Two different staining patterns for type II collagen could be recognized in the ECM: one pattern revealed a very faint and diffuse reaction while the other showed a wak rim-like reaction. These staining patterns were markedly different from those in the cartilaginous cell layer in the posterosuperior area of the condylar secondary cartilage, which showed faint staining for type I collagen and a much more intense staining for type II collagen. These observations reveal the presence of chondroid bone, a tissue intermediate between bone and cartilage tissues, in the mandibular condylar cartilage, and suggest the possibility of osteogenic transdifferentiation of mature chondrocytes.

Age-related changes in the localization of glycosaminoglycans in condylar cartilage of the mandible in rats.

There is little information available regarding the morphological and biomolecular characteristics of mandibular condylar cartilage. The purpose of this study was to determine the age-related changes in the morphology and immunolocalization of glycosaminoglycans (GAGs) in mandibular condyles. The mandibular condylar cartilages from 4-, 8-, 16-, 32-, and 64-week-old Wistar male rats were examined to verify the localization of chondroitin-4-sulfate (Ch-4S), chondroitin-6-sulfate (Ch-6S) and keratan sulfate (KS) using an indirect immunofluorescent technique with three monoclonal antibodies for glycosaminoglycans, 2-B-6, 3-B-3 and 5-D-4, respectively. Morphologically, the condylar cartilage was a growth cartilage during growing periods, began to differentiate into articular cartilage from the central area of 16-week-old condyles, and became mature articular cartilage at 32 weeks of age. A regional difference was found in the morphological features and distribution of GAGs between the anterior, central, postero-superior and posterior areas of the condyles at each age. The immunohistochemical localizations of these three glycosaminoglycans showed age-related, morphology-dependent changes, from growth cartilage to articular cartilage-like cartilage. Immunoreactions for all of the antibodies decreased progressively with age in the interterritorial matrix, while the pericellular and territorial matrix in the condylar cartilage of the mandible maintained relatively higher immunoreactivity. In conclusion, age-related and regional differences in the localization of glycosaminoglycans Ch-4S, Ch-6S, and KS were found in the mandibular condyles in rats, and these changes are believed to be related to functional and developmental requirements.

Localization of types I, II and III collagen and glycosaminoglycans in the mandibular condyle of growing monkeys: an immunohistochemical study.

In order to analyse the regional and age-related variations of primate condyles, immunohistochemical techniques were used to examine the localization of types I, II and III collagen and a variety of glycosaminoglycans in distinct anteroposterior regions of the mandibular condyle of two growing female rhesus monkeys (Macaca mulatta). In the juvenile monkey staining for types I and III collagen was weak in the fibrous tissue layer, intense in the pre-cartilaginous tissue layer and faint in the cartilaginous tissue layer; staining was significantly more intense in the posterosuperior and posterior regions than in the anterior region. Similarly, staining for cartilage-characteristic extracellular matrices, including type II collagen and keratan sulfate, was intense in the cartilaginous tissue layer of the posterior condyle. In contrast, in the late-adolescent monkey staining for the extracellular matrices was more intense in the anterior half of the condyle (i.e. from the anterior to the posterosuperior region) than in the posterior region, and most intense in the posterosuperior region. The results demonstrate that marked regional differences exist in the phenotypic expression of the extracellular matrices in the mandibular condyles of growing monkeys and that these differences vary between different developmental stages. The variations probably reflect the predominance of competing growth and articulatory functions in the mandibular condyles.

An immunohistochemical study of regional differences in the distribution of type I and type II collagens in rat mandibular condylar cartilage.

The mammalian temporomandibular joint is a highly specialized diarthrodial joint under multidirectional compressive and tensile forces. In such a complicated biomechanical environment, the phenotypic expression of extracellular matrix may vary in different regions of the mandibular condylar cartilage. To test this hypothesis, immunohistochemical techniques were used to examine the localization of type I and type II collagens in various anterioposterior regions of the condylar cartilage of 4-week-old rats. In the posterosuperior region, which is mainly subjected to compressive forces, a strong reaction for type II collagen was observed in the cartilaginous layer (maturative and hypertrophic cell layers), and a rather weak reaction was observed for type I collagen in the precartilaginous and cartilaginous layers, compared with the reactions in other peripheral regions. Proceeding anteriorly, staining for type I collagen increased, while that for type II collagen decreased. In posteroinferior cartilage, which is subjected mainly to tensile forces because of its direct attachment to the retrodiscal pad, staining for type I collagen was strong, and that for type II collagen was faint in the cartilaginous layer. These results demonstrate that marked regional differences exist in the phenotypic expression of two major collagen components in mandibular condylar cartilage, which may reflect the local functional environment and cellular response.