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Objectives: Temporomandibular joint (TMJ) hypofunction secondary to feeding a liquid dietin the growing period leads to morphological hypoplasia. However, few studies haveevaluated the results ofmechanical loading onthe hypoplastic TMJ. Thisstudy investigatedwhether TMJ hypofunction in rats causes osteoarthritis (OA)-like changes when exposed tomechanical loading.Design: Male 21-day-old Wistar rats weredivided intofour groups.The first group (C)servedas the control. In the second group (W), mechanical loading was applied to the TMJ bycontinuous steady mouth opening (3 h/day for 5 days) from 63 days of age. A jaw-openingdevice was used to hold the mandible open in the maximal mouth-opening position with acobalt–chromium (Co–Cr) wire (w: 0.9 mm). Groups C and W both received a normal harddiet. The third group (L) and fourth group (LW) were fed a liquid diet and group LW weresubjected to the same loading as group W. We evaluated the TMJ using micro-CT, toluidineblue staining and immunohistochemistry of matrix metalloproteinase (MMP)-13.Results: In group LW in the superior and posterior regions of the condyle, bone volumefraction, trabecular thickness and trabecular number were significantly decreased andtrabecular spacing was significantly increased. The ratio of MMP-13 immunopositive cellswas significantly higher than in the other groups. OA-like changes were also observed,including reduced thickness of the cartilage, irregularities in the chondrocytic layer, andcell-free areas.Conclusions: TMJ hypofunction in rats is likely to lead to OA-like changes when exposed tomechanical loading.# 2014 Elsevier Ltd. All rights reserved.* Corresponding author. Tel.: +81 3 5803 5530.E-mail addresses: yoneman.orts@tmd.ac.jp, yoneorts@tmd.ac.jp (I. Yonemitsu).

Objective Planar cell polarity (PCP) refers to the cell polarity across the tissue plane and controls various cell behaviors and structures. Although the expression of several PCP signaling components has been detected in tooth germs, knowledge of the gene expression patterns of these PCP components during tooth development remains incomplete. The aim of this study is to characterize the temporal and spatial changes in PCP gene expression during tooth development. Design Expression of Celsr1 and 2 , Fzd3 and 6 , Vangl1 and 2 , and Dvl1-3 genes was analyzed in mouse molar germs from the bud to the bell stage using in situ hybridization. Results At the bud stage, all target genes were expressed in all areas of the tooth bud. In the enamel organ at the cap stage, expression of Fzd3 was suppressed in the enamel knot, whereas Fzd6 was strongly expressed there. Expression of Vangl2 was strongly expressed in the inner dental epithelium from the cap stage onwards. In the inner dental epithelium, strong expression of Fzd3 , Dvl2 and Vangl2 was noted at the early bell stage, and of Celsr1 , Fzd3 , Fzd6 , Vangl2 and Dvl2 at the bell stage. Furthermore, differentiated odontoblasts strongly expressed Celsr1 , Vangl2 , and Dvl2 . Conclusion The gene expression patterns delineated in this study improve our understanding of the role(s) of PCP components during tooth development.

Objective Versican is a large, aggregating chondroitin sulphate proteoglycan. In dental tissue, versican expression occurs primarily in mesenchymal tissue but rarely in epithelial tissue. We investigated the expression, localisation and synthesis of versican in the enamel organ of the developing tooth germ. Design To elucidate versican localisation in vivo , in situ hybridisation and immunohistochemistry were conducted in foetal ICR mice at E11.5–E18.5. Epithelium and mesenchyme from the lower first molars at E16.0 were enzymatically separated and versican mRNA expression was investigated by semi-quantitative RT-PCR. Organ culture of the separated samples combined with metabolic labelling with [ 35 S], followed by gel filtration, was performed to analyse secreted proteoglycans. Results Versican mRNA was first expressed in the thickened dental epithelium at E12.0 and continued to be expressed in the enamel organ until the bell stage. Versican immunostaining was detected in the stellate reticulum areas from the bud stage to the apposition stage. The enamel organ at E16.0 expressed versican mRNA at a level comparable to that in dental mesenchyme. Furthermore, when compared to dental mesenchyme, about 1/2–3/4 of the [ 35 S]-labelled versican-like large proteoglycan was synthesised and released into tissue explants by the enamel organ. Conclusions The dental epithelium of developing tooth germ is able to synthesise significant amounts of versican.

The distribution of hyaluronan and versican in the dental pulp of the young rat was mapped histochemically. The pattern of staining showed considerable variation between different teeth and different specimens. The most common pattern was a strong reaction for hyaluronan and a weak reaction for versican in the subodontoblastic region, with the reverse deeper in the pulp. This was not an entirely consistent pattern and there was considerable regional variation in the staining intensity for both molecules. The localization of these molecules at similar sites could thus indicate related roles in the connective tissue matrix rather than any chemical bonding between them.

The first indication of cartilage formation was detected on day 14.5 of pregnancy in close connection to the ossifying mandible. A separated blastema was never observed. Thus the findings support the concept that the condylar cartilage develops from already differentiated cells, not from primary undifferentiated mesenchymal cells. Moreover, endochondral bone formation started on day 16 of pregnancy. Although the condylar cartilage basically undergoes the same processes of endochondral bone formation as the long bone, many hypertrophic chondrocytes may survive and are released into the primary spongiosa.

Apoptotic reactions were demonstrated with a commercial detection kit in the reduced ameloblasts around the tip of the cusp just before and after exposure to the mouth but, as tooth eruption advanced, they were no longer seen in the remainder of the reduced ameloblasts. Although some of the reduced ameloblasts appear to be expelled as a consequence of apoptosis, the remainder appear to constitute the junctional epithelium.

Mitotic cells in the rat mandibular condyle (proliferative mandibular cells) were compared with mitotic cells in the rat tibial growth plate (proliferative tibial cells). In the tibial proliferative cells, the rough endoplasmic reticulum mostly became vacuolated during the latter stage of mitosis, whereas the rough endoplasmic reticulum of the proliferative mandibular cells rarely underwent disorganization. Further, a wide area remained outside of the mitotic spindle in the proliferative tibial cells at the metaphase, while only a narrow area remained in the proliferative mandibular cells. This finding might account for the difference of disorganization of the rough endoplasmic reticulum.

Large light cells (more than 20 μm dia), including some with mitotic figures, appeared to be surviving hypertrophic chondrocytes. Thus at least a few hypertrophic chondrocytes in the rat mandibular condyle may survive, be released into the primary spongiosa, and divide.

One hypothesis for the mechanism of tooth eruption is that the periodontal ligament fibroblasts generate the eruptive force. To assess the force generated, these fibroblasts were obtained by explant culture of ligament from mandibular premolars of a dog and were cultured in collagen gel matrices. The forces generated by them under isometric conditions were continuously measured for 120 h with a strain gauge. At the same time the number of cells in the gel was counted and the force measured was calculated as the force generated by 104 cells. Shortly after the start of culture, the force per 104 cells increased rapidly; it reached 5.2 × 10−4N at 8 h, and then remained at the same level for about 48 h. Our findings suggest that fibroblasts of the periodontal ligament may generate sufficient force for tooth eruption.

Objective Previous studies indicate that hypertrophic chondrocytes can transdifferentiate or dedifferentiate and redifferentiate into bone cells during the endochondral bone formation. Mandibular condyle in aged c-src -deficient mice has incremental line-like striations consisting of cartilaginous and non-cartilaginous layers, and the former contains intact hypertrophic chondrocytes in uneroded lacunae. The purpose of this study is to determine the phenotype changes of uneroded hypertrophic chondrocytes. Design Immunohistochemical and ultrastructural examinations of the pericellular matrix of hypertrophic chondrocytes in the upper, middle, and lower regions of the mandibular condyle were conducted in aged c-src -deficient mice, using several antibodies of cartilage/bone marker proteins. Results Co-localisation of aggrecan, type I collagen, and dentin matrix protein-1 (DMP-1) or matrix extracellular phosphoprotein (MEPE) was detected in the pericellular matrix of the middle region. Ultrastructurally, granular substances in the pericellular matrix of the middle region were the remains of upper region chondrocytes, which were mixed with thick collagen fibrils. In the lower region, the width of the pericellular matrix and the amount of collagen fibrils were increased. Versican, type I collagen, DMP-1, and MEPE were detected in the osteocyte lacunae. Additionally, DMP-1 and MEPE were detected in the pericellular matrix of uneroded hypertrophic chondrocytes located in the lower, peripheral region of the mandibular condyle in younger c-src -deficient mice, but not in the aged wild-type mice. Conclusions These results indicate that long-term survived, uneroded hypertrophic chondrocytes, at least in a part, acquire osteocytic characteristics.

Mandibular condylar cartilage is sensitive to masticatory force, while mice lacking the c-src gene (c-src-deficient mice) have osteopetrosis and tooth eruption failure. The purpose of this study was to investigate the morphology of the mandibular condyle in these mice, which were maintained with a soft-food diet for 240 days after birth. The condylar head in the c-src-deficient mice showed slight deformity in shape before weaning, but showed remarkable undergrowth after weaning. No significant morphological or histological differences were detected between the mandibular condyle in wild-type mice fed soft food and those fed hard food, indicating that osteopetrosis, as well as abnormal masticatory force, influences the morphology of the mouse mandibular condyle, and that malocclusion rather than dietary consistency may have greater influence. After 70 days, incremental line-like striations consisting of cartilaginous and non-cartilaginous layers were detected in the mandibular condyle of the c-src-deficient mice, but not in the tibial growth plate. Immunostaining of aggrecan, collagen types II and X, and osteopontin was detected in the cartilaginous layers, but not in the non-cartilaginous layers showing collagen type I immunostaining. Chondrocyte lacunae were not eroded in the cartilaginous layers, and complete circumferential mineralisation around the lacunae and impaired osteoclast (chondroclast) function can account for this phenomenon. However, repeated cessation of chondrocyte differentiation may be required to completely explain the formation of the striations. These results indicate that the mandibular condyle in the c-src-deficient mice has unique structural features, adding to its deformity.

The biosynthesis of proteoglycans by these cultured pulp cells was investigated by metabolic labelling, using [ 35 S ]sulphate, [ 3 H ]glucosamine and [ 3 H ]leucine as precursors. Versican-like large proteoglycan, decorin- and biglycan-like small proteoglycans and a small amount of sulphated protein were released into the culture medium. Heparan sulphate species were also identified in cell-layer extracts. Versican-like proteoglycan had an average molecular mass of approximately 800 kDa. The molecular mass of chondroihnase ABC-digested core protein exhibited heterogeneity, ranging from 250 to 400 kDa, and the glycosaminoglycan chains had an average molecular mass of approximately 42 kDa. These results indicate the presence of 10–13 glycosaminoglycan chains per core protein, consistent with the characteristics of versican. This glycosaminoglycan chain contained approximately 63% 4-sulphated disaccharides.

Versican is a large interstitial proteoglycan that is believed to be able to bind hyaluronan to form large aggregate structures, but no study has isolated native versican aggregates from any tissue. In this study, ternary aggregate structures consisting of versican, hyaluronan, and link protein were isolated from rat dental pulp by associative extractions followed by caesium sulphate rate zonal sedimentation centrifugation. Fractions from the centrifugation were analysed by dot blot and Western blot using monoclonal antibodies and hyaluronan-binding protein. About 60% of the hexuronic acid was extracted by associative extractions. Positive reactions for versican, hyaluronan and link protein were clearly detected in the bottom fractions from the centrifugation, but were barely detectable in the top fractions. These results suggest that the majority of the versican, hyaluronan, and link protein forms ternary aggregate structures in the rat dental pulp.

Pulp tissue was obtained from maxillary incisors of young adult male Wistar rats, minced and digested with 0.5% trypsin and 0.02% EGTA at 37 degrees C for 30 min. Dissociated cells were cultured with or without 10 nM dexamethasone using Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 50 micrograms/ml ascorbic acid. Confluent cells were subcultured at 7 days and the medium further supplemented with beta-glycerophosphate (beta-GP). Dexamethasone in primary culture and/or secondary culture enhanced the formation of mineralized tissue while5 mM beta-GP was necessary for mineralization to occur. Biochemical analysis of the radiolabelled medium revealed that these cells produced type I, type I trimer and type III collagens. Analysis of [32PO4]-labelled medium, using DEAE-Sephacel ion-exchange chromatography and sodium dodecylsulphate-polyacrylamide gel electrophoresis, showed that these cells produced phosphophoryn-like protein. These results indicate that some of the rat dental pulp cells in culture express an odontoblast-like phenotype.