Robert J. Hinton

Histologic examination of the temporomandibular joint after mandibular advancement with and without rigid fixation: an experimental investigation in adult Macaca mulatta.

This study evaluated the histologic response of the temporomandibular joint (TMJ) following mandibular advancement using rigid and nonrigid fixation in monkeys. Twelve adult female rhesus monkeys underwent sagittal ramus osteotomies with advancement. Six of them were placed into maxillomandibular fixation (MMF); six underwent bicortical bone-screw fixation without MMF. Changes in condylar position were quantified using lateral cephalograms with the aid of bone markers. The animals were killed at 6 weeks and the TMJs were prepared for histologic analysis. Three measures of condylar cartilage thickness were obtained for each animal and were correlated to changes in position of the condyle. Animals who underwent MMF showed a tendency for anterior movement of the condyles; animals who underwent rigid fixation showed a tendency for posterior condylar position. Thicker cartilage layers were found in the MMF animals. Animals who had posterior displacement of the condyles showed evidence of resorption of the posterior surface of the condyle and anterior surface of the postglenoid spine. There was a significant correlation between a change in the horizontal position of the condyle and the thickness of the posterior aspect of the condylar cartilage. The results of this study indicate that alterations in condylar position may induce remodeling changes within the TMJ.

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.

Regulation of cell proliferation in rat mandibular condylar cartilage in explant culture by insulin-like growth factor-1 and fibroblast growth factor-2

Insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2) regulate the proliferation and differentiation of growth-plate chondrocytes, but surprisingly little is known of the mechanisms underlying growth regulation in secondary cartilages such as the mandibular condylar. The aims here were to investigate whether IGF-1 and FGF-2 receptors are present in mandibular condylar cartilage in vivo from 28-day-old male Sprague-Dawley rats (by immunohistochemistry), how proliferation in that cartilage responds to increasing concentrations of exogenous IGF-1 or FGF-2 in explant culture (by [3H]thymidine incorporation), and whether the expression of these growth factors and their receptors in the cartilage changes during the transition to puberty (quantitative reverse transcriptase-polymerase chain reaction). Immunoreactivity for receptors (R) for IGF-1 and FGF-2 (IGF-1R, FGFR1, and FGFR3) was most pronounced in chondroblasts and hypertrophic chondrocytes, while FGFR2 immunoreactivity was strongest in the articular and prechondroblastic zones. The proliferative response elicited by exogenous IGF-1 was considerably greater than that induced by FGF-2, although the threshold concentration for a significant response was lower for FGF-2. In the transition from prepuberty (31 days) to the beginning of late puberty (42 days), a pronounced trend of increasing IGF-1 and decreasing FGF-2 gene expression was evident. Of the receptors, only FGFR2 and FGFR3 expression increased. These data provide evidence that proliferation in the mandibular condylar cartilage might be regulated in part by IGF-1 and FGF-2, and that expression of these genes changes considerably at puberty. The data also suggest that mechanisms governing proliferation in mandibular condylar cartilage might have as much in common with those regulating cranial sutures as those regulating growth-plate.

Response of the Mandibular Joint to Loss of Incisal Function in the Rat

In a study of the rat mandibular joint (MJ), Simon [Acta anat. 97: 351-360 (1977)] suggested that reduction in condylar cartilage thickness noted in animals subjected to removal or trimming of incisors resulted from the lessening of joint reaction forces produced during incision. In order to explore this question further, the microanatomy of the MJ in 47-day-old rats whose incisors had been trimmed every other day was compared to that in control animals and in a third group fed a soft diet as a control for reduced joint reaction forces. Both the incisor-clipped and soft-diet groups exhibited reduced size and density of bony trabeculae underlying the condylar cartilage and diminished staining for alcian blue. The thickness of the prechondroblastic layer of the condylar cartilage was significantly (p less than or equal to 0.01) reduced relative to controls in both experimental groups on the superior aspect of the cartilage, but was reduced in the more posterior parts of the cartilage only in the incisor-clipped group. While not denying that joint reaction forces may affect MJ response, the reduced proliferative response noted in the posterior region of the condylar cartilage in incisor-clipped animals is perhaps best explained by a decrease in the frequency and extent of protrusion of the lower jaw due to a lack of incisal preparation of food items.

Osterix Controls Cementoblast Differentiation through Downregulation of Wnt-signaling via Enhancing DKK1 Expression

Osterix (Osx), a transcriptional factor essential for osteogenesis, is also critical for in vivo cellular cementum formation. However, the molecular mechanism by which Osx regulates cementoblasts is largely unknown. In this study, we initially demonstrated that overexpression of Osx in a cementoblast cell line upregulated the expression of markers vital to cementogenesis such as osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP) at both mRNA and protein levels, and enhanced alkaline phosphatase (ALP) activity. Unexpectedly, we demonstrated a sharp increase in the expression of DKK1 (a potent canonical Wnt antagonist), and a great reduction in protein levels of β-catenin and its nuclear translocation by overexpression of Osx. Further, transient transfection of Osx reduced protein levels of TCF1 (a target transcription factor of β-catenin), which were partially reversed by an addition of DKK1. We also demonstrated that activation of canonical Wnt signaling by LiCl or Wnt3a significantly enhanced levels of TCF1 and suppressed the expression of OPN, OCN, and BSP, as well as ALP activity and formation of extracellular mineralized nodules. Importantly, we confirmed that there were a sharp reduction in DKK1 and a concurrent increase in β-catenin in Osx cKO mice (crossing between the Osx loxP and 2.3 Col 1-Cre lines), in agreement with the in vitro data. Thus, we conclude that the key role of Osx in control of cementoblast proliferation and differentiation is to maintain a low level of Wnt-β-catenin via direct up-regulation of DKK1.

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 (μg/mg dry tissue wt) of the condylar cartilage and [35S]-sulfate incorporation (dpm/μg 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.

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 26tdTomato (tracing marker), 2.3 Col1GFP (bone cell marker), and aggrecan CreERT2 (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.

Genes that regulate morphogenesis and growth of the temporomandibular joint: A review

Compared with the joints of the limbs, our understanding of the genes that regulate development and growth in the temporomandibular joint (TMJ) is fairly limited. Because the morphogenesis of the secondary cartilage and other intra‐articular structures in the TMJ occurs later and in a different manner than in the limbs, the genetic control of TMJ development might reasonably be assumed to differ from that in the limbs. However, studies of the specific genes regulating TMJ morphogenesis and growth have only begun to appear in the literature within the last decade. This review attempts to survey and interpret the existing knowledge on this topic and to suggest fruitful avenues of investigation for the future. Studies to date using knockout and over‐expression of candidate genes suggest that a developmental hierarchy of joint structures exists, with condyle development primary. A hierarchy of gene expression also exists: Runx2 and Sox9 expression is critical for condylar cartilage formation. Several of the other genes discussed in this report may regulate TMJ morphogenesis by affecting Sox9 and Runx2 expression and control the ihh‐PTHrP axis by means of these genes. Developmental Dynamics 243:864–874, 2014.

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.

Effect of Altered Masticatory Function on [3H]-Thymidine and [35S]-Sulfate Incorporation in the Condylar Cartilage of the Rat

The response of the condylar cartilage to alterations in compressive joint reaction forces in vivo has been little studied. In an attempt to reduce or eliminate the occlusal forces resulting from mastication or incision, male weanling rats were fed a soft diet requiring little chewing and/or had their incisors clipped every other day. Incorporation (dpm/micrograms DNA) of [3H]-thymidine and [35S]-sulfate was significantly decreased relative to controls in the incisor-clipped group, but not in the soft-diet group. Animals having both treatments also exhibited significantly lower incorporation values than controls, suggesting the importance of incision for loading at the mandibular joint. These data corroborate in vitro studies which suggest that compressive forces can affect mitotic activity and synthesis of proteoglycans in the condylar cartilage. However, additional factors, both hormonal and biomechanical in nature, may be important in the in vivo environment.