Differential gene expression in the perichondrium and cartilage of the neonatal mouse temporomandibular joint.

Our goal was to discover genes differentially expressed in the perichondrium (PC) of the mandibular condylar cartilage (MCC) that might enhance regenerative medicine or orthopaedic therapies directed at the tissues of the temporomandibular joint. We used targeted gene arrays (osteogenesis, stem cell) to identify genes preferentially expressed in the PC and the cartilaginous (C) portions of the MCC in 2-day-old mice. Genes with higher expression in the PC sample related to growth factor ligand-receptor interactions [FGF-13 (6.4x), FGF-18 (4x), NCAM (2x); PGDF receptors, transforming growth factor (TGF)-beta and IGF-1], the Notch isoforms (especially Notch 3 and 4) and their ligands or structural proteins/proteoglycans [collagen XIV (21x), collagen XVIII (4x), decorin (2.5x)]. Genes with higher expression in the C sample consisted mostly of known cartilage-specific genes [aggrecan (11x), procollagens X (33x), XI (14x), IX (4.5x), Sox 9 (4.4x) and Indian hedgehog (6.7x)]. However, the functional or structural roles of several genes that were expressed at higher levels in the PC sample are unclear [myogenic factor (Myf) 9 (9x), tooth-related genes such as tuftelin (2.5x) and dentin sialophosphoprotein (1.6x), VEGF-B (2x) and its receptors (3-4x) and sclerostin (1.7x)]. FGF, Notch and TGF-beta signalling may be important regulators of MCC proliferation and differentiation; the relatively high expression of genes such as Myf6 and VEGF-B and its receptors suggests a degree of unsuspected plasticity in PC cells.

Roles of Chondrocytes in Endochondral Bone Formation and Fracture Repair.

The formation of the mandibular condylar cartilage (MCC) and its subchondral bone is an important but understudied topic in dental research. The current concept regarding endochondral bone formation postulates that most hypertrophic chondrocytes undergo programmed cell death prior to bone formation. Under this paradigm, the MCC and its underlying bone are thought to result from 2 closely linked but separate processes: chondrogenesis and osteogenesis. However, recent investigations using cell lineage tracing techniques have demonstrated that many, perhaps the majority, of bone cells are derived via direct transformation from chondrocytes. In this review, the authors will briefly discuss the history of this idea and describe recent studies that clearly demonstrate that the direct transformation of chondrocytes into bone cells is common in both long bone and mandibular condyle development and during bone fracture repair. The authors will also provide new evidence of a distinct difference in ossification orientation in the condylar ramus (1 ossification center) versus long bone ossification formation (2 ossification centers). Based on our recent findings and those of other laboratories, we propose a new model that contrasts the mode of bone formation in much of the mandibular ramus (chondrocyte-derived) with intramembranous bone formation of the mandibular body (non-chondrocyte-derived).

Specificity of meal pattern analysis as an animal model of determining temporomandibular joint inflammation/pain.

Analyzing feeding behavior, and in particular meal duration, can be used as a biological marker for temporomandibular joint (TMJ) inflammation/pain. The present study determined the specificity of meal duration as a measure of TMJ inflammation/pain in a rodent model. The model was also used to test the efficacy of dexamethasone (DEX) as a treatment for TMJ inflammation/pain that was induced by TMJ injection of complete Freund's adjuvant (CFA). In the first study, anesthetized male Sprague-Dawley rats housed in computerized feeding modules received bilateral intra-articular knee injections of CFA or saline. The next day, CFA-injected rats had significant knee swelling and impaired mobility. Food intake in the CFA-injected group was reduced over the next two days and this was due to reduced meal number with no change in meal size. Notably, meal duration was normal in both the CFA and saline knee-injected groups. In the second study, male rats were assigned to one of four groups: Group 1, no CFA and no DEX treatment; Group 2, no CFA and treatment with DEX (0.4 mg/kg i.m. once daily); Group 3, bilateral TMJ CFA injection and no DEX treatment; and Group 4, bilateral TMJ CFA injection and treatment with DEX. CFA significantly increased TMJ swelling and stress-induced chromodacryorrhea in Group 3, but treatment with DEX attenuated these effects in Group 4. Compared to the controls, meal duration was significantly lengthened 24 and 48 h post-CFA injection in Group 3, whereas DEX treatment attenuated TMJ swelling, chromodacryorrhea and normalized meal duration. The data demonstrate that meal pattern analysis, and in particular meal duration, can be used as a non-invasive specific measure of TMJ inflammation/pain and can be used as a marker of DEX treatment efficacy.

Chondrocytes Directly Transform into Bone Cells in Mandibular Condyle Growth.

For decades, it has been widely accepted that hypertrophic chondrocytes undergo apoptosis prior to endochondral bone formation. However, very recent studies in long bone suggest that chondrocytes can directly transform into bone cells. Our initial in vivo characterization of condylar hypertrophic chondrocytes revealed modest numbers of apoptotic cells but high levels of antiapoptotic Bcl-2 expression, some dividing cells, and clear alkaline phosphatase activity (early bone marker). Ex vivo culture of newborn condylar cartilage on a chick chorioallantoic membrane showed that after 5 d the cells on the periphery of the explants had begun to express Col1 (bone marker). The cartilage-specific cell lineage-tracing approach in triple mice containing Rosa 26(tdTomato) (tracing marker), 2.3 Col1(GFP) (bone cell marker), and aggrecan Cre(ERT2) (onetime tamoxifen induced) or Col10-Cre (activated from E14.5 throughout adult stage) demonstrated the direct transformation of chondrocytes into bone cells in vivo. This transformation was initiated at the inferior portion of the condylar cartilage, in contrast to the initial ossification site in long bone, which is in the center. Quantitative data from the Col10-Cre compound mice showed that hypertrophic chondrocytes contributed to ~80% of bone cells in subchondral bone, ~70% in a somewhat more inferior region, and ~40% in the most inferior part of the condylar neck (n = 4, P < 0.01 for differences among regions). This multipronged approach clearly demonstrates that a majority of chondrocytes in the fibrocartilaginous condylar cartilage, similar to hyaline cartilage in long bones, directly transform into bone cells during endochondral bone formation. Moreover, ossification is initiated from the inferior portion of mandibular condylar cartilage with expansion in one direction.

Alterations in rat condylar cartilage following discectomy.

The purpose of this study was to examine the effect of unilateral extirpation of the articular disc of the temporomandibular joint on selected metabolic and compositional characteristics of the condylar cartilage. Unilateral discectomy was performed in one group of growing (28-day-old) rats, while another group received only unilateral arthrotomy. The effect of the discectomy procedure was assessed by comparison of ratios of the operated to unoperated sides in the discectomy and arthrotomy groups. Total uronic acid content (micrograms/mg dry tissue wt) of the condylar cartilage and [35S]-sulfate incorporation (dpm/micrograms uronic acid) into the condylar cartilage were decreased in discectomy-group animals at nine, 16, and 28 days following surgery, while hydration of the cartilage was increased in discectomy animals at both 16 and 28 days post-surgery. Wet and dry tissue weights of the cartilage were dramatically increased in discectomy-group animals at all post-operative intervals. These metabolic and compositional changes were accompanied by pronounced structural alterations in the cartilage, including chondrocyte clustering and tears parallel to the articular surface. With the exception of [35S]-sulfate incorporation, the changes demonstrated in the condylar cartilage following discectomy were similar to early osteoarthritic alterations reported in limb articular cartilage following partial meniscectomy or cruciate ligament resection.

Relationships between mandibular joint size and craniofacial size in human groups.

Substantial evidence attests to the capability of the joint to undergo morphological alteration in response to biomechanical forces transmitted to it during function. Measurements expressing the size of mandibular condyles and fossae were obtained from skulls representative of a broad spectrum of subsistence practices and tooth use. Craniofacial dimensions were measured for some groups. Considerable differences in joint size were noted between groups roughly consistent with known or presumed intensity of masticatory stress. Size was largest in the hunter-gatherers, intermediate in aboriginal horticulturalists and smallest in 20th century American caucasoids and 17th century British. In each group, male joint size was absolutely larger than females. With the exception of condylar breadth, male joint dimensions were not relatively larger than female when corrected for differences in craniofacial size. In contrast, same-sex comparisons of Eskimo and American caucasoid means adjusted for differences in craniofacial size showed joint size in Eskimos to be significantly larger, both absolutely and relatively. Eskimo females had relatively larger joints than American caucasoid males. Thus, intergroup differences in joint size persist even when differences in craniofacial size are taken into account. Although the influence of genetic factors cannot be excluded, differences in the nature or intensity of tooth use during growth may account, at least in part, for the observed differences in joint size.

Effect of condylotomy on matrix synthesis and mineralization in the rat mandibular condylar cartilage.

Little information is available about the influence of biomechanical factors on the rate of matrix synthesis and mineralization in the condylar cartilage. In an attempt to temporarily disrupt articular forces at the temporomandibular joint, bilateral condylotomies were performed on 29 day-old male Sprague-Dawley rats. Animals which underwent a similar surgical procedure except for actual condylotomy comprised a sham-operated control group; a non-operated third group served as further controls. Incorporation of [35S]-sulphate did not differ among the groups at 18 h and 2 days after condylotomy, but was significantly reduced in condylotomy animals at 7 and 14 days. Total alkaline phosphatase activity was reduced at 18 h and increased at 14 days by condylotomy, with no differences at the intermediate time intervals; similarly 45Ca-uptake was increased at the 14-day interval. Thus both matrix synthesis and mineralization of the condylar cartilage were altered in the 2 weeks after condylotomy. The changes are in general similar to those noted in other studies after a lessening or removal of articular forces from the condyle.

Effect of condylotomy on DNA synthesis in cells of the mandibular condylar cartilage in the rat.

The role of articular forces and the periosteal envelope in the regulation of condylar cartilage growth is uncertain. Transection of the condylar neck, or condylotomy, influences both by a temporary reduction of articular forces and a transient release of the ramus periosteal envelope. To investigate the effects of this procedure on proliferative activity of the prechondroblast layer of the condylar cartilage, bilateral condylotomies were performed on 29-day-old male Sprague-Dawley rats. A surgical control group underwent identical incision without condylotomy and a non-operated third group also served as controls. Eighteen hours after surgery, there was no difference in [3H]-thymidine incorporation among the different treatment groups. However, by two days after surgery, [3H]-thymidine incorporation was significantly higher in the condylotomy group than in either surgical or untreated controls. Incorporation values remained significantly higher in condylotomy animals at seven days after surgery, but declined to control levels by 14 days after surgery. These findings support other radioautographic and cephalometric evidence of an increased growth rate of the condylar cartilage after condylotomy, but suggest that the increase may be transient and perhaps related to the disruption and restoration of articular function.

Age-associated changes in decorin in rat mandibular condylar cartilage.

The small proteoglycan decorin strongly binds the fibrils of collagen types I and II; this interaction is thought to play a part in the maintenance of tissue integrity and biomechanical properties. In limb articular cartilage, there is evidence that decorin synthesis increases with age and that it is elevated in response to increased loading or in osteoarthritic cartilage. The aim here was to characterize the presence and relative amount of decorin in the condylar cartilage of the temporomandibular joint (TMJ) with maturation by Western blotting, and to assess its tissue localization by immunohistochemistry. Comparative data were obtained from tibial articular cartilage, which has been extensively studied. Cartilage from the mandibular condyle and tibial plateau was harvested from 24-day-old (growing) and 161-day-old (young adult) female Sprague-Dawley rats. In growing animals, decorin appeared slightly more abundant in the mandibular condylar cartilage than in articular cartilage, whereas in young adult animals the decorin content in the TMJ cartilage was noticeably less than in limb articular cartilage. Although there was an increase in decorin abundance with age at the TMJ, the increase in decorin with age in limb articular cartilage was considerably more pronounced. These data indicate that, although decorin is present in mandibular condylar cartilage, its abundance in adults is less than in limb articular cartilage; thus, maturation-associated changes may be dissimilar in magnitude from those documented for limb articular cartilage.

Calcitonin gene-related peptide and substance P immunoreactivity in rat trigeminal ganglia and brainstem following adjuvant-induced inflammation of the temporomandibular joint.

The immunoreactivity of two inflammatory mediators, calcitonin gene-related peptide (CGRP) and substance P, was measured in the trigeminal ganglia and brainstem to characterize an adjuvant-induced inflammation within the rat temporomandibular joint at various acute (6, 24 and 48 h) and intermediate (10 day) time intervals. Concentrations of adjuvant-related neuropeptides were compared to those in both contralateral vehicle-related tissues and non-injected controls. By 6 h, CGRP immunoreactivity in the trigeminal ganglia was significantly above that in contralateral vehicle-injected tissue. The CGRP had decreased at each of the following time-points, but remained significantly elevated at 10 days. Substance P in the ganglion on the injected side was significantly increased for all four time periods. In brainstem subnucleus caudalis, CGRP was significantly increased for all four time periods. Substance P immunoreactivity in the subnucleus caudalis was significantly increased for the initial three time periods, but by day 10 had been reduced to that of the control. These data show that the pattern of changes in neuropeptides following the induction of inflammation is different between substance P and CGRP. Moreover, the pattern of change varies between the brainstem and the trigeminal ganglion. This suggests that the two neuropeptides may have different roles in the inflammatory process, and that this process may be modulated by different mechanisms at the brainstem and ganglion.

Capsaicin application to the temporomandibular joint alters calcitonin gene-related peptide levels in the trigeminal ganglion of the rat.

The aim of this investigation was to determine the temporal effect of an intra-articular injection of capsaicin to the temporomandibular joint on the levels of calcitonin gene-related peptide-like immunoreactivity (CGRP-ir) in the trigeminal ganglion of the rat. The temporomandibular joints of 26 adult female rats were injected on one side with capsaicin and contralaterally with a control vehicle. Another 8 animals served as an untreated control group and received no injections. Animals were sacrificed at time intervals of 4 hours, 48 hours, 10 days, and 21 days following treatment. The trigeminal ganglia were extirpated, and CGRP-ir levels were quantified using a radioimmunoassay. Results demonstrated that when the capsaicin-treated side and the vehicle-treated side were compared, CGRP-ir levels decreased initially at 4 hours and increased at 48 hours. At 10 days, CGRP-ir levels had again dropped below control levels, followed by an increase at 21 days. CGRP-ir levels for the first two time periods investigated, which simulate an acute inflammatory state, mimic results observed in studies using limb joints, while the other time periods, which represent an intermediate and a chronic condition, respectively, suggest a more complex interaction with capsaicin-sensitive primary afferents.

Reactive changes in the temporomandibular joint after mandibular midline osteodistraction.

The purpose of this study was to evaluate the histologic changes within the condyle in response to mandibular widening using osteodistraction. Mandibular midline osteotomies were made in nine Macaca mulatta monkeys and tooth-borne distraction devices were bonded to the mandibular dentition. Distraction was continued until a 3-5 mm widening was achieved. The appliances were then stabilized for a period of 4 weeks. Non-decalcified sagittal sections of the lateral, middle and medial thirds of the condyles were analyzed. Although three of the seven animals showed no unusual morphology, four others exhibited morphologic differences within the fibrous layer, cartilage layer or bone/cartilage interface. Histologic changes were seen to occur in the fibrous layer, cartilaginous layer and cartilage/bone interface. The severity of these changes were correlated with the likely rotational forces directed at the condyle on the postero-lateral and antero-medial surfaces.

Effect of age on the adaptive response of the adult temporomandibular joint. A study of induced protrusion in Macaca mulatta.

Using occlusal and interproximal tooth wear as indicators of age, it is found that those animals with evident hypertrophy and hyperplasia of the condylar cartilage are among the youngest in the sample, supporting the view that adaptive potential of the temporomandibular joint may diminish with age.

Osterix couples chondrogenesis and osteogenesis in post-natal condylar growth.

Osterix (Osx) is a transcription factor essential for osteoblast differentiation and bone mineralization. Although there are indications that Osx also plays a regulatory role in cartilage, this has not been well-studied. The goal of this study was to define the function of Osx in the post-natal growth of the secondary cartilage at the mandibular condyle. Conditional Osx knockout (cKO) mice that were missing Osx only in cartilage were generated by crossing Osx-loxP mice to Aggrecan-Cre mice. Cre activity was induced by tamoxifen injection twice a week from day 12 to 1 mo of age, and specimens were collected at 1 and 5 mo of age. At 1 mo of age, the condylar hypertrophic chondrocyte zone in the cKO-mice was > three-fold thicker than that in the age-matched control, with little sign of endochondral bone formation. Immunohistochemistry and analysis of histological data revealed a defect in the coupling of chondrogenesis and osteogenesis in the cKO mice. In five-month-old mice examined to address whether late-stage removal of the Cre-deletion event would alleviate the phenotype, the hypertrophic chondrocyte zone in the cKO condyles was considerably larger than in wild-type mice. There were large discrete areas of calcified cartilage in the hypertrophic zone, few signs of endochondral bone formation, and large regions of disorganized intramembranous bone. Analysis of these data further strengthens the notion that Osterix is essential for the coupling of terminal cartilage differentiation and endochondral ossification in mandibular condylar cartilage.

Distribution of small integrin-binding ligand, N-linked glycoproteins (SIBLING) in the condylar cartilage of rat mandible.

The Small Integrin-Binding LIgand, N-linked Glycoprotein (SIBLING) family is one category of non-collagenous proteins closely related to osteogenesis. In this study, the authors systematically evaluated the presence and distribution of four SIBLING family members, dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) and osteopontin (OPN), in rat mandibular condylar cartilage using protein chemistry and immunohistochemistry. For protein chemistry, SIBLING proteins in the dissected condylar cartilage were extracted with 4M guanidium-HCl, separated by ion-exchange chromatography, and analyzed by Western immunoblotting. Immunohistochemistry was employed to assess the distribution of these four SIBLING proteins in the condylar cartilage of 2-, 5- and 8-week-old rats. Results from both approaches showed that all four members are expressed in the condylar cartilage. DSPP, unlike that observed in dentin and bone, exists as a full-length form (uncleaved) in the condylar cartilage. The NH(2)-terminal fragment of DMP1 is mainly detected in the matrix of the cartilage while the COOH-terminal fragment is primarily localized in the nuclei of cells in the chondroblastic and hypertrophic layers. The data obtained in this investigation provide clues about the potential roles of these SIBLING proteins in chondrogenesis.

Dielectrically induced sensitivity enhancements in electro-optic field sensors.

The sensitivity of an electro-optic (EO) field sensor depends inversely on the dielectric constant of the nonlinear crystal. In EO sensors based on lithium niobate the effective value of this dielectric constant is affected by dielectric relaxation effects and is identified with its smaller, high-frequency component. Because of this effect, the EO modulation is significantly enhanced, thus improving the field strength sensitivity.

Cellular response to force application at craniofacial sutures.

OBJECTIVES: To provide a comprehensive review of the literature describing research done on the responses of suture cells to force application in vitro and in vivo. DESIGN AND RESULTS: This review outlines the types of forces that can be applied, methods of applying the forces, the sutures used in experiments, and the changes in morphology, molecular biology (gene and protein expression), and cell biology (proliferation, differentiation, apoptosis) in response to these forces. CONCLUSION: The molecular response of sutures to force needs to be further investigated as these molecules can be used to enhance the way in which craniofacial sutures respond to mechanical force during orthopedic-orthodontic treatment.