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Summary Temporomandibular joint (TMJ), a site that is often impacted by osteoarthritis (OA), is biomechanically linked with dental occlusion. Tissue responses in TMJ condyle to biomechanical stimulation could be investigated by intervention of the dental occlusion in animals. Unilateral anterior crossbite, an experimental malocclusion, has been demonstrated to induce TMJ-OA lesions, showing primarily as enhanced cartilage calcification and subchondral cortical bone formation at the osteochondral interface, causing the osteochondral interface thickening and stiffening. The changed interface would worsen the local biomechanical environment. At the cartilage side, the matrix degenerates. In the case of insufficient restoration of the matrix, the cells in the deep zone flow into the ones undergoing autophagy, apoptosis, and terminal differentiation while the cells in the superficial zone are promoted to differentiate to supply the loss of the deep zone cells. At the meantime, the bone marrow stromal cells are stimulated to bone formation in the subchondral cortical region which is uncoupled with the sites of the osteoclast-mediated resorption process that is predominantly observed at the subchondral trabecular bone region. Overall, the thickening and stiffening osteochondral interface, due greatly to the enhanced endochondral ossification in deep zone cartilage, should be a central pathological process that links with cartilage decay and subchondral bone remodelling in OA joints. The residual chondrocytes locating in the cartilage superficial zone have the progenitor-like qualities that can proliferate, and also differentiate into the deep zone chondrocytes, thus should be critical in progression and rehabilitation of TMJ-OA.

As a new type of cell death, necroptosis is initiated by tumor necrosis factor receptor 1(TNFR1), and then activated receptor-interacting protein kinase 1(RIP1) and receptor-interacting protein kinase 3 (RIP3), following by the activation of mixed lineage kinase domain-like protein(MLKL) to deliver cell death signal. When necroptosis happens, damage associated molecular patterns (DAMPs) enter into extracellular area through the ruptured cytomembrane, followed by the disordered tissue hemeostasis. In recent years, many researches showed that necroptosis playimportant roles in a few bone related diseases, such as osteoporosis, osteonecrosis, osteosarcoma, etc. Thus, we try to briefly review the researches in this field.

Objectives To detect whether early growth response 1 (EGR1) in peripheral blood leucocytes (PBLs) indicates temporomandibular joint (TMJ) osteoarthritis (OA) lesions. Materials and methods Egr1 mRNA expression levels in PBLs were detected in eight malocclusion patients without temporomandibular disorder (TMD) signs and 16 malocclusion patients with clinical TMD signs with (eight) or without (eight) imaging signs of TMJ OA. Twelve 6-week-old rats were randomized to a control group and a unilateral anterior crossbite (UAC) group and were sampled at 4 weeks. The Egr1 mRNA expression levels in PBLs and protein expression levels in different orofacial tissues were measured. Results Patients with TMD signs with/without TMJ OA diagnosis showed lower Egr1 mRNA expression levels in PBLs than patients without TMD signs. The lower Egr1 mRNA expression was also found in the PBLs of UAC rats, which were induced to exhibit early histo-morphological signs of TMJ OA lesions. In subchondral bone of UAC rats, EGR1 protein expression was decreased, co-localization of EGR1 with osterix or dentin matrix protein-1 was identified, and the number of EGR1 and osterix double-positive cells was reduced (all p Conclusion Egr1 reduction in PBLs potentially indicates subchondral bone OA lesions at an early stage.

OBJECTIVE We aimed to develop a mouse model predominating in a proliferative response in the articular cartilage of the temporomandibular joints. MATERIALS AND METHODS Bilateral anterior elevation of occlusion was developed by installing metal tubes onto the incisors of mice with edge-to-edge relation to prevent tooth wear, leading to an increase in the vertical height of the dental occlusion with time. Morphological changes and expression changes in Cyclin D1, Aggrecan, and type II and type X collagen in the mandibular condylar cartilage were detected. In addition, cells were isolated from the mandibular condylar cartilage and exposed to cyclic tensile strain (CTS). RESULTS Compared with age-matched controls, the tooth length was longer at 3 weeks, 7 weeks, and 11 weeks in BAE mice (p

Traumatic joint injuries produce osteoarthritic cartilage manifesting accelerated chondrocyte terminal differentiation and matrix degradation via unknown cellular and molecular mechanisms. Here we report the ability of biomechanical stress to increase expression of the calcium-sensing receptor (CaSR), a pivotal driver of chondrocyte terminal differentiation, in cultured chondrogenic cells subjected to fluid flow shear stress (FFSS) and in chondrocytes of rodent temporomandibular joint (TMJ) cartilage subjected to unilateral anterior cross-bite (UAC). In cultured ATDC5 cells or TMJ chondrocytes, FFSS induced Ca2+ loading and CaSR localization in endoplasmic reticulum (ER), casually accelerating cell differentiation that could be abrogated by emptying ER Ca2+ stores or CaSR knockdown. Likewise, acute chondrocyte-specific Casr knockout (KO) prevented the UAC-induced acceleration of chondrocyte terminal differentiation and matrix degradation in TMJ cartilage in mice. More importantly, local injections of CaSR antagonist, NPS2143, replicated the effects of Casr KO in preventing the development of osteoarthritic phenotypes in TMJ cartilage of the UAC-treated rats. Our study revealed a novel pathological action of CaSR in development of osteoarthritic cartilage due to aberrant mechanical stimuli and supports a therapeutic potential of calcilytics in preventing osteoarthritis in temporomandibular joints by targeting the CaSR. © 2018 American Society for Bone and Mineral Research.

Background The tooth morphology course is an important basic dental course. However, it is difficult to fully reflect the three-dimensional (3D) morphological characteristics of tooth structure in two-dimensional pictures in traditional textbooks. The aim of this study was to assess the effect of 3D-printed plastic model teeth in the teaching of tooth morphology. Methods Twenty-two undergraduate students who matriculated at the School of Stomatology, the Fourth Medical University, in 2014 and 23 who matriculated in 2016 participated in the study. Each student who matriculated in 2016 was given a full set of fourteen standard 3D-printed plastic model teeth for use during the learning process, and an anonymous questionnaire was used to evaluate the usefulness of the 3D-printed plastic model teeth from the perspective of the students. Results There was no significant difference between the two groups in the scores of the theoretical examination or the total score. However, for the score of the sculpted gypsum teeth, the students who used the 3D-printed plastic model teeth in their studies scored significantly higher (P = 0.002). More than 90% of the students thought that the 3D-printed plastic model teeth were of great help or were very helpful for mastering the anatomy of teeth and for carving the gypsum teeth. Conclusion Standard 3D-printed plastic teeth can effectively assist students in learning tooth morphology by transforming two-dimensional pictures and descriptions in the textbook into a 3D conformation, effectively promoting students’ learning and mastery of tooth morphology and structure. Additionally, the results suggest that 3D-printed plastic model teeth are of great help to the students in mastering and improving their carving skills.

Objective The present purpose was to investigate the involvement of Derlin-3 in the endoplasmic reticulum stress pathway-mediated apoptosis of chondrocytes in biomechanically stimulated mandibular condylar cartilage. Design First, fluid flow shear stress (FFSS) was applied to ATDC5 cells with or without overexpression of Derlin-3 by lentiviral transduction or silencing of Derlin-3 by siRNA transfection. Apoptosis was evaluated by TUNEL assay. Molecular markers related to the endoplasmic reticulum stress-apoptosis pathway, including GRP78, CHOP, ATF6, Caspase-12, and cleaved Caspase-3, were detected by real-time polymerase chain reaction and Western blotting. Second, the expression of proteins related to the endoplasmic reticulum stress-apoptosis pathway of the chondrocytes in mandibular condylar cartilage of mice treated with unilateral anterior crossbite (UAC) prostheses was evaluated by immunohistochemical staining and TUNEL assay. Results FFSS induced the endoplasmic reticulum stress-apoptosis pathway in ATDC5 cells. This apoptosis was suppressed by overexpressing Derlin-3 but was enhanced by silencing Derlin-3. UAC increased Derlin-3 expression in mandibular condylar cartilage at 1 and 3 weeks but decreased Derlin-3 expression at 7 and 11 weeks. The reduction of Derlin-3 expression by UAC was associated with the increase in the endoplasmic reticulum stress pathway-mediated apoptosis in degenerative mandibular condylar cartilage. UAC elicited changes in Derlin-3 expression and the endoplasmic reticulum stress pathway-mediated apoptosis was reversed after the removal of the prosthesis. Conclusion Reduced Derlin-3 expression is associated with the biomechanically induced endoplasmic reticulum stress pathway-mediated apoptosis of chondrocytes in the mandibular condylar cartilage and could be a therapeutic target for the treatment of biomechanically stimulated cartilage degradation.

The striking predilection of temporomandibular disorders (TMD) in women, especially during gonad-intact puberty or reproductive years, indicates that oestrogen plays an important role in the progression of TMD, but the underlying mechanism remains unclear. In this study, unilateral anterior crossbite (UAC) was used to create temporomandibular joint osteoarthritis (TMJ OA) models in rats, while 17β-estradiol (E2) injections were applied to mimic patients with high-physiological levels of oestrogen. Micro-CT scanning, histological staining and real-time PCR assays were preformed to observe the degenerative changes in the mandibular condylar cartilage and subchondral bone. The results showed that obvious degradation was found in the condylar cartilage and subchondral bone of rats with UAC procedure, including decreased cartilage thickness, loss of extracellular matrix, increased apoptotic chondrocytes and expression of pro-inflammatory and catabolic factors, decreased bone mineral density and increased osteoclast activity. E2 supplements aggravated the condylar cartilage degradation but reversed the abnormal bone resorption in the subchondral bone induced by UAC. Our results revealed that high-physiological oestrogen plays a destructive role in condylar cartilage but a protective role in subchondral bone at the early stage of TMJ OA. These dual and distinct effects should be given serious consideration in future OA treatments.

Background Dental diseases are among the most frequently reported health problems in drug abusers. However, few studies have been conducted on oral health of methamphetamine (meth) abusers in China. The aim of the present study was to investigate the caries and periodontal health profile of former meth abusers in Eastern China. Methods A cross-sectional study was conducted on 162 former meth abusers in the male Zhoushan Compulsory Detoxification Center. A standardized questionnaire, which collected information about age, drug-use duration / pattern, oral hygiene habit and systemic diseases, was administered. Then, a dental examination was performed to investigate the severity of dental caries and periodontal diseases. In evaluating dental caries, the prevalence of dental caries, the scores of decayed teeth (DT), missing teeth (MT), filled teeth (FT), and decayed, missing, filled teeth (DMFT) were recorded. In evaluating periodontal diseases, community periodontal index (CPI), and the prevalence of gingival bleeding, dental calculus, periodontal pocket and loose teeth, were recorded. Additionally, the non-parametric test was adopted to analyze the potential risk factors via SPSS. Results All the participants abused meth by inhalation. The mean scores of DT, MT, FT and DMFT in the former meth users were 2.72 ± 2.78, 3.07 ± 3.94, 0.33 ± 1.03 and 6.13 ± 5.20 respectively. The prevalence of gingival bleeding, dental calculus, periodontal pocket and loose teeth was 97.53%, 95.68%, 51.23% and 9.26% respectively. The DT, DMFT and CPI scores in those who had abused meth for longer than 4 years were significantly higher than those who abused for less than 4 years (P = 0.039, 0.045, P

Cells in articular cartilage are zonal arranged. Cells in superficial zone cartilage are generally small and proliferative. Appropriate negative pressure stimulation is beneficial to cell survival and tissue repair. Whether negative pressure has promotive impact on the proliferation activity of the superficial zone chondrocytes is of interest. In this study, we isolated superficial chondrocytes from the mandibular condylar cartilage of rats. After negative pressure treatment, the cells were collected for RNA-sequencing, quantitative real-time PCR and western blotting assays, aiming to detect the proliferative responses of chondrocytes to negative pressure and explore the potential molecular mechanisms. Data from RNA-sequencing analysis indicated that the superficial chondrocytes responded to the 4 h −10 kPa treatment by a significant increase in proliferation. In addition, the expression of high-mobility group box 2 (HMGB2) and the phosphorylation of AKT were obviously promoted. Knockdown of HMGB2 decreased AKT phosphorylation and diminished the negative pressure-induced proliferation of chondrocytes, as shown by decreased expression of Ki67 and cyclin-dependent kinase 6 (CDK6). In contrast, overexpression of HMGB2 enhanced AKT phosphorylation and further promoted proliferative activity. Moreover, LY294002, an AKT inhibitor, suppressed the proliferative activity of chondrocytes under negative pressure, while SC79, an activator of AKT phosphorylation, enhanced the proliferation of chondrocytes. Our data demonstrated that HMGB2 exhibits a promotion impact on chondrocyte proliferation under negative pressure via the phosphorylation of AKT. These results provide a new perspective for superficial zone chondrocytes proliferation under negative pressure, which should be benefit for cartilage regeneration.

Biomarkers of temporomandibular joint (TMJ) osteoarthritis (OA) remain unknown. The objective was to detect whether molecular biomarkers from peripheral blood leucocytes (PBLs) engage in TMJ OA lesions. Thirty‐four six‐week‐old Sprague Dawley rats were used. The top upregulated gene ontology categories and gene‐fold changes in PBLs were detected by a microarray analysis comparing rats that received 20‐week unilateral anterior crossbite (UAC) treatment with age‐matched controls (n = 4). Twenty weeks of UAC treatment had been reported to induce TMJ OA‐like lesions. The other twenty‐four rats were randomly placed in the UAC and control groups at 12‐ and 20‐week time points (n = 6). The mRNA expression levels of the selected biomarkers derived from the microarray analysis and their protein expression in the alveolar bone and TMJ were detected. The microarray analysis indicated that the three most highly involved genes in PBLs were Egr1, Ephx1 and Il10, which were confirmed by real‐time PCR detection. The increased protein expression levels of the three detected molecules were demonstrated in cartilage and subchondral bone (P

Objective Dental occlusion are frequently changed in clinic. Molecular responses in jaw muscles to aberrant dental occlusion are changes are attractive, yet remain are obscure. Design Unilateral anterior crossbite (UAC) prostheses were applied to Sprague-Dawley rats and then ceased after two weeks to detect the reactions of the masseter, a representative jaw elevator, and the lateral pterygoid muscle (LPM), a representative jaw depressor. Results Two weeks of UAC elicited mild injury of the two muscles. Myogenesis and protective reactions were detected as increases in αB-crystallin expression in the masseter after 3 days and in the LPM after 2 weeks, and increases in desmin expression in both muscles after 2 weeks. A switch in fibre types from IIb to IIx occurred in the LPM but not in the masseter. Inflammatory responses, shown by the infiltration of inflammatory cells and increases in TNF-α mRNA expression, and fibrosis responses, shown by increased mRNA expression of Type I and III collagens, appeared very mild in the two muscles. These responses were partially recovered by the cessation of UAC. During the whole process, no obvious changes were observed in mitochondrial function, as indicated by the levels of proliferator-activated receptor γ coactivator 1α, mitofusin-2 and voltage-dependent anion channel. Conclusions UAC causes injury and very limited inflammatory and fibrosis adaption in the masseter and LPM. Both muscles respond with myogenesis and protective activity. The LPM responds also with muscle fibre isoform alternations. These alterations were partially recovered by the cessation of dental stimulation at an early stage.

β2-adrenergic signal transduction in mesenchymal stem cells (MSCs) induces subchondral bone loss in osteoarthritis (OA) of temporomandibular joints (TMJs). However, whether conditional deletion of β2-adrenergic receptor (Adrb2) in nestin+ MSCs can alleviate TMJ-OA development remains unknown. In this study, nestin-Cre mice were crossed with Adrb2 flox mice to generate mice lacking Adrb2 expression specifically in the nestin+ MSCs (Adrb2-/-), and TMJ-OA development in such mice was investigated. Adrb2 flox mice (Adrb2+/+) and Adrb2-/- mice were subjected to unilateral anterior crossbite (UAC), while mice in the control group were subjected to sham operation. Adrb2+/+ and Adrb2-/- mice in the control group showed no distinguishable phenotypic changes in body weight and length, mandibular condylar size, and other histomorphological parameters of the condylar subchondral bone. A significant increase in subchondral bone loss and cartilage degradation was observed in Adrb2+/+ UAC mice; the former was characterized by decreased bone mineral density, bone volume fraction, and trabecular plate thickness, and increased trabecular separation, osteoclast number and osteoclast surface, and pro-osteoclastic factor expression; the latter was characterized by decreased cartilage thickness, chondrocyte density, proteoglycan area, and collagen II and aggrecan expression, but increased matrix metalloproteinase and alkaline phosphatase expression and percentage area of calcified cartilage. Adrb2 deletion in nestin+ MSCs largely attenuated UAC-induced increase in condylar subchondral bone loss, cartilage degradation, and aberrant calcification at the osteochondral interface. Thus, Adrb2-expressing MSCs in the condylar subchondral bone play an important role in TMJ-OA progression and may serve as novel therapeutic targets for TMJ-OA.

Oestrogen and hypoxia inducible factor-2α (HIF2α) are key regulators in the pathogenesis of osteoarthritis (OA). However, the cellular interaction between oestrogen and HIF2α in articular cartilage during OA process remains unknown. Our previous study has revealed that high-physiological level of oestrogen aggravates the degradation of condylar cartilage in the early stage of temporomandibular joint osteoarthritis (TMJ OA). Here, we hypothesize that HIF2α involves the effect of oestrogen on mandibular condylar cartilage in the progression of TMJ OA. Our experiment in vivo found that the degeneration of condylar cartilage caused by unilateral anterior crossbite (UAC) model, characterized by obvious degenerative morphology, loss of cartilage extracellular matrix, up-regulation of TNF-α, HIF2α and its' down-stream OA-related cytokines (MMP-13, VEGF and Col X), could be alleviated by lack of oestrogen while aggravated by high level of oestrogen in rats. Meanwhile, our in vitro study found that 17β-estradiol stimulation resulted in the loss of extracellular matrix, increased expression of TNF-α, IL-1, HIF2α and its' down-stream OA-related cytokines (MMP-13, VEGF and Col X) in primary condylar chondrocytes via oestrogen receptor beta (ERβ), which could be reversed by ER antagonist, selective estrogen receptor modulators (SERMs) and HIF2α translation inhibitor. Our results reveal that high level of oestrogen can aggravate the degenerative changes of mandibular condylar cartilage, while lack of oestrogen can alleviate it via oestrogen-ERβ-HIF2α pathway during TMJ OA progression.

Activated calcium-/calmodulin-dependent protein kinaseII (CaMKII) is important to promote chondrocytes from proliferative to pre-hypertrophic state, which probably plays a role in osteoarthritis (OA), a widespread degeneration disease with enhanced aberrant chondrocyte differentiation. Our aim was to detect the role of CaMKII, and its relationship with the feedback loop of Indian hedgehog (Ihh) and Parathyroid-related peptide (PTHrP) in the temporomandibular joints (TMJs) OA. KN93, the competitive inhibitor of CaMKII, was added to the culture medium in vitro and was locally injected to rats TMJs (n = 54, female) every other day for 4 weeks from the beginning of the 5th and 9th week after installing of unilateral anterior crossbite (UAC), termed as 4 wk+4 wk and 8 wk+4 wk, accordingly. The RNA expression of CaMKII α (1.49 ± 0.09), CaMKII β (3.36 ± 0.20), Ihh (1.88 ± 0.06) and PTHrP (1.87 ± 0.12) was all enhanced, especially at 24 dyn/cm2 in vitro (all P < .05), accompanied with downregulated expression of cartilage matrix, but upregulated markers of chondrocytes differentiation (all P < 0.05). Similarity was observed in the 4 wk+4 wk group in vivo. In the 8 wk+4 wk group, UAC upregulated the RNA expression of CaMKII α (1.81 ± 0.24), CaMKII β (1.36 ± 0.07) and Ihh (1.70 ± 0.21), however, down-regulated PTHrP (0.53 ± 0.04) (all P < .05), in consonance with the protein expression. All these changes were attenuated by KN93 (all P < .05). In conclusion, CaMKII took a role, via Ihh and PTHrP pathways, in promoting biomechanically induced TMJ chondrocytes differentiation, the initiation issue of UAC stimulated osteoarthritic changes in rodent TMJs. Inhibiting CaMKII is helpful to rescue the biomechanically stimulated cartilage degradation and prospective to be a target treatment of OA.

Osteoarthritis (OA) of the temporomandibular joint (TMJ) is associated with dental biomechanics. A major change during OA progression is the ossification of the osteochondral interface. This study investigated the formation, radiological detectability, and mechanical property of the osteochondral interface at an early stage, the pathogenesis significance of which in OA progression is of clinical interest and remains elusive for the TMJ. Unilateral anterior crossbite (UAC) was performed on 6-wk-old rats as we previously reported. TMJs were harvested at 4, 12, and 20 wk. The progression of TMJ OA was evaluated using a modified Osteoarthritis Research Society International (OARSI) score system. Osteochondral interface was investigated by quantifying the thickness via von Kossa staining of histological slices and in vivo calcium deposition by calcein injection. Tissue ossification was imaged by micro-computed tomography (CT). Mechanical properties were measured at nanoscale using dynamic indentation. Time-dependent TMJ cartilage lesions were elicited by UAC treatment. Geometric change of the condyle head and increased value of the OARSI score were evident in UAC TMJs. At the osteochondral interface, there was not only enhanced deep-zone cartilage calcification but also calcium deposition at the osseous boundary. The thickness, density, and stiffness of the osteochondral interface were all significantly increased. The enhanced ossification of the osteochondral interface is a joint outcome of the aberrant deeper cartilage calcification at the superior region and promoted formation of subchondral cortical bone at the inferior region. The micro-CT detectable ossification from an early stage thus is of diagnostic significance. Although the environment of the cartilage and subchondral bone could be changed due to the stiffness of the interface, whether or not the stiffened interface would accelerate OA progress remains to be confirmed. With that evidence, the osteochondral interface could be a new diagnostic and therapeutic target of the mechanically initiated OA in the TMJ.

Mechanical stress is essential for bone development. Mechanical stimuli are transduced to biochemical signals that regulate proliferation, differentiation, and cytoskeletal reorganization in osteoblasts. In this study, we used proteomics to evaluate differences in the protein expression profiles of untreated Saos-2 osteoblast cells and Saos-2 cells subjected to mechanical stress loading. Using 2-D electrophoresis, MALDI–TOF mass spectroscopy, and bioinformatics, we identified a total of 26 proteins differentially expressed in stress loaded cells compared with control cells. Stress loaded Saos-2 cells exhibited significant upregulation of 17 proteins and significant downregulation of 9 proteins compared with control cells. Proteins that were most significantly upregulated in mechanically loaded cells included those regulating osteogenesis, energy metabolism, and the stress response, such as eukaryotic initiation factor 2 (12-fold), mitochondrial ATP synthase (8-fold), and peptidylprolyl isomerase A (cyclophilin A)-like 3 (6.5-fold). Among the proteins that were significantly downregulated were those involved in specific signaling pathways and cell proliferation, such as protein phosphatase regulatory (inhibitor) subunit 12B (13.8-fold), l-lactate dehydrogenase B (9.4-fold), Chain B proteasome activator Reg (Alpha) PA28 (7.7-fold), and ubiquitin carboxyl-terminal esterase L1 (6.9-fold). Our results provide a platform to understand the molecular mechanisms underlying mechanotransduction.

Autophagy is a cell protective mechanism for maintaining cellular homeostasis. The present study aimed to investigate whether autophagy is enhanced in the biomechanically induced degenerative cartilage of the temporomandibular joint (TMJ) and the potential role of mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) and mammalian Target of rapamycin (mTOR) in this observation. To induce degenerative changes in the TMJs, rats were subjected to biomechanical dental stimulation by moving 4 molars away from their original position as we previously reported. The ultrastructure of autophagosome was observed by transmission electron microscopy. The number of lysosomes was analyzed by flow cytometry. The expression levels of Beclin1 and LC3 and the involvement of MAP4K3 activity were detected by immunohistochemistry, real-time PCR and western blot. The activity of the mTOR pathway indicated by p-mTOR and p-p70S6 K was assayed by western blot. TMJ degeneration, characterized by irregular cell arrangement and cell-free area, was induced in the experimental groups. Under transmission electron microscopy, we observed the presence of autophagosomes, small patches of condensed chromatin, abundant rough endoplasmic reticulum and Golgi apparatus. The number of lysosomes and the expression levels of Beclin1 and LC3 increased, while the activity of mTOR and the expression level of MAP4K3 decreased in the experimental groups. Cartilage in TMJ which was induced to be degenerative biomechanically exhibited autophagy accompanied by reduced mTOR and MAP4K3 activity.

The pathological changes of subchondral bone during osteoarthritis (OA) development in the temporomandibular joint (TMJ) are poorly understood. In the present study, we investigated the longitudinal alterations of subchondral bone using a rat TMJ-OA model developed in our laboratory. Changes in bone mass were examined by micro-CT, and changes in osteoblast and osteoclast activities were analyzed by real-time PCR, immunohistochemistry, and TRAP staining. Subchondral bone loss was detected from 8 weeks after dental occlusion alteration and reached the maximum at 12 weeks, followed by a repair phase until 32 weeks. Although bone mass increased at late stages, poor mechanical structure and lower bone mineral density (BMD) were found in these rats. The numbers of TRAP-positive cells were increased at 12 weeks, while the numbers of osteocalcin-expressing cells were increased at both 12 and 32 weeks. Levels of mRNA expression of TRAP and cathepsin K were increased at 12 weeks, while levels of ALP and osteocalcin were increased at both 12 and 32 weeks. These findings demonstrated that there is an active bone remodeling in subchondral bone in TMJs in response to alteration in occlusion, although new bone was formed with lower BMD and poor mechanical properties.

Introduction Information about the effect of tooth movement on the myelinated nerve in the periodontal ligament is limited. In this study, we aimed to investigate what responses of the periodontal myelinated nerve can be evoked during experimental tooth movement. Methods In experimental-I group, the maxillary left and mandibular right third molars were moved distally. In experimental-II group, the maxillary left third molar but not the right one was moved, and the bilateral mandibular third molars were extracted. The ultrastructures of the myelinated nerve in the periodontal ligament of the bilateral maxillary third molars were observed under a transmission electron microscope. The expression of myelin basic protein was evaluated by immunohistochemistry. Results Degenerative ultrastructural changes of the myelinated nerve in the periodontal ligament were noticed mainly in the myelin sheath; these were observed earlier and were recoverable in the experimental-I group. In contrast, the ultrastructural changes of the myelinated nerve occurred mainly in the axons, were observed later, and were unrecoverable in the experimental-II group. A concomitant decrease of myelin basic protein expression was observed in both groups. Conclusions Both experimental tooth movement and occlusal changes accompanying it caused changes of the myelinated nerve in the periodontal ligament.

The effect of occlusion on the temporomandibular joint (TMJ) is debated. By inserting rubber-bands that were replaced by self-curing resin one week later, the left maxillary and the right mandibular first-molars were moved and kept mesially in Sprague-Dawley (SD) rats in both experimental I (EXP-I) and II (EXP-II) groups, aiming to establish a non-matching cusp-to-fossa occlusal relation. Four weeks later, the left maxillary and the right mandibular third-molars were moved and kept distally in the EXP-II group. Degenerative changes, typically as a cell-free area, were observed in TMJs of the EXP groups. Binary logistical analysis indicated that the odds ratio of EXP group, EXP-II vs. EXP-I, on the incidence of a cell-free area, was 2.8 (p=.036). Time point, gender, and side did not have such effects (p>0.05). The results indicate that the persistence of more scattered non-matching cusp-to-fossa occlusion is more harmful to the condylar cartilage in terms of the incidence of degenerative changes.

Summary This study was designed to investigate the expression differences of insulin-like growth factor-1 (IGF1), IGF type 1 receptor (IGFR1) and IGF-binding protein-3 (IGFBP3) in mandibular condylar cartilage between male and female rats with experimentally created malocclusion. A total of 40 male and 40 female rats were used, and malocclusion was created by moving the first molars mesially and the third molars distally in the experimental group. Animals were killed at the end of the second and fourth weeks. Haematoxylin and eosin (HE) staining was performed to monitor the changes in cartilage morphology and thickness. Immunohistochemistry and real-time PCR were used to detect the expression of IGF1, IGFR1 and IGFBP3. Osteoarthritis (OA)-like changes were observed in the experimental groups, with 2-week females showing larger OA-like regions than 2-week males (P

To investigate angiogenesis at the osteochondral junction and changes in expression of pro- and anti-angiogenic factors in rat mandibular condyles with osteoarthritis-like changes.In order to evoke osteoarthritis-like lesions in mandibular condyles, disordered occlusion was created experimentally in rats. Osteochondral vascularity was assessed histologically at 20 and 24 weeks. Protein and mRNA levels of pro-angiogenic factors including vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and matrix metalloproteases 9 (MMP9), and anti-angiogenic factor chondromodulin-I (CHM-I) were investigated by means of immunohistochemical staining and real-time PCR.Osteochondral angiogenesis was demonstrated as increased numbers of vascular channels terminating in the calcified cartilage and non-calcified cartilage in 20- and 24-week experimental groups compared with controls (all P0.05). In the experimental groups, VEGF, CTGF and MMP9 were highly expressed in the tissues adjacent to the osteochondral junction. However, CHM-I was more expressed in the superior but not deep hypertrophic chondrocytes. Compared to their age-matched controls, the protein levels of VEGF and CTGF were higher in 20-week experimental group, and the protein and mRNA levels of CTGF, MMP-9, and CHM-I increased in the 24-week experimental group (all P0.05).In the present rat model, osteochondral angiogenesis was observed in mandibular condyles with osteoarthritis-like changes, accompanied with local upregulation of VEGF, CTGF and MMP9. Although the increase in CHM-I may moderate pro-angiogenic factors effects in the superior cartilage, the deficiency of deep hypertrophic chondrocytes to express CHM-I may permit vascular invasion into condylar cartilage.

Summary Objective To investigate whether mechanical stress induces mineral deposits that contribute to matrix degradation at the onset of osteoarthritis (OA) in temporomandibular joint (TMJ) cartilage. Design Female Spraguee–Dawley rats were subjected to an unilateral anterior crossbite (UAC) procedure. Histology, electron microscopy, and energy dispersive spectrometer (EDS) were used to examine cartilage matrix structures and composition of mineral deposit in the affected TMJ cartilage. Protein and/or RNA expression of phenotypic markers and mineralization modulators and matrix degradation was analyzed by immunohistochemistry and/or real-time PCR. Synthetic basic calcium phosphate (BCP) and calcium pyrophosphate dehydrate (CPPD) crystals were used to stimulate ATDC5 cells for their impact on cell differentiation and gene expression. Results Fragmented and disorganized collagen fibers, expanded fibrous spaces, and enhancement of matrix vesicle production and mineral deposition were observed in matrices surrounding hypertrophic chondrocytes in cartilage as early as 2-weeks post-UAC and exacerbated with time. The mineral deposits in TMJ cartilage at 12- and 20-weeks post-UAC had Ca/P ratios of 1.42 and 1.44, which are similar to the ratios for BCP. The expression of mineralization inhibitors, NPP1, ANK, CD73, and Matrix gla protein (MGP) was decreased from 2 to 8 weeks post-UAC, so were the chondrogenic markers, Col-2, Col-X and aggrecan. In contrast, the expression of tissue-nonspecific alkaline phosphatase (TNAP) and MMP13 was increased 4-weeks post-UAC. Treating ADTC5 cells with BCP crystals increased MMPs and ADAMTS5 expression, but reduced matrix production in a time-dependent manner. Conclusion UAC induces deposition of BCP-like minerals in osteoarthritic cartilage, which can stimulate matrix degradation by promoting the expression of cartilage-degrading enzymes to facilitate OA progression.

To quantify the age- and sex-related changes in the rat condylar cartilage and subchondral bone.SD rats were obtained at the ages of 2, 3, 4, 5, 6 and 7 months. For each sex, the temporomandibular joints tissue blocks from four rats were subjected to histological assessment of cartilage thickness and subchondral bone architecture; for the remaining three rats, the mandibular condyles were delivered for gross measurement and evaluation of the mineralization and architecture properties of the subchondral bone by means of micro-CT.Rapid decrease of cartilage thickness but increase of subchondral bone density occurred respectively from 2 to 3 and 3 to 4 months old in female and 2 to 4 and 3 to 5 months old in male (P0.05), whereas rapid changes of subchondral bone architecture occurred from 3 to 4 months old in both sexes (P0.05). The significant enlargement of condyle size occurred at 4 or 5 months old in female but at 5 or 6 months in male (P0.05).This study demonstrated that the rapid developmental changes of rat condylar cartilage and subchondral bone primarily occurred before 4 months of age, resulting in thinner cartilage but larger and thicker subchondral bone, and they were followed by rapid growth in condylar size. Sex differences were identified that the endochondral ossification of fibrocartilage and formation of subchondral bone were faster in female than in male rats, leading to the earlier enlargement of condyle in female than in male.

Introduction The purpose of this study was to investigate the canal morphology of 504 maxillary permanent teeth of subjects of Han nationality in Chinese Guanzhong area. Methods Maxillary permanent teeth were randomly collected in Guanzhong area. After regular preparation, the teeth were immersed into ink without preparing access cavities and then put into hyperbaric oxygen chamber (0.6 Mpa) for 2 hours to let the ink penetrate into root canal from apical foramen, apical deltas and foramen of lateral canals under stable positive pressure. After demineralization and clearing, the following observations were made: (1) number of root canals, (2) root canal configuration by using Vertucci's classification, (3) presence of lateral canals, and (4) frequency of apical deltas. Results All the teeth were well-stained, and the fine details were well-revealed. Apical deltas (12.2%–83.3%) and lateral canals (13.7%–68.8%) could be frequently found in all types of maxillary teeth. Most of central incisors (95.8%), lateral incisors (91.4%), and canines (75.4%) displayed type I canal configuration, whereas most of first premolars (87.3%) and second premolars (72.3%) possessed 2 canals with type II, IV, or VI canal configuration. The majority of distobuccal roots and palatal roots of first molars (88.9%, 97.8%), second molars (92.0%, 94.0%), and third molars (87.5%, 91.6%) possessed type I canal configuration. The prevalence of mesiobuccal roots with type I configuration was 66.7% in maxillary first molars, 82% in second molars, and 62.5% in third molars. Conclusions The modified technique of canal staining can effectively reveal detailed root canal system. The canal configuration of maxillary teeth in subjects of Han nationality in Chinese Guanzhong area is consistent with previous reports in other races.

Increased subchondral trabecular bone turnover due to imbalanced bone-resorbing and bone-forming activities is a hallmark of osteoarthritis (OA). Wnt5a/Ror2 signaling, which can derive from bone marrow stromal cells (BMSCs), takes a role in modulating osteoblast and osteoclast formation. We showed previously that experimentally unilateral anterior crossbites (UACs) elicited OA-like lesions in mice temporomandibular joints (TMJs), displaying as subchondral trabecular bone loss. Herein, we tested the role of BMSC-derived Wnt5a/Ror2 signaling in regulating osteoclast precursor migration and differentiation in this process. The data confirmed the decreased bone mass, increased tartrate-resistant acid phosphatase (TRAP)–positive cell number, and enhanced osteoclast activity in TMJ subchondral trabecular bone of UAC-treated rats. Interestingly, the osteoblast activity in the tissue of TMJ subchondral trabecular bone of these UAC-treated rats was also enhanced, displaying as upregulated expressions of osteoblast markers and increased proliferation, migration, and differentiation capabilities of the locally isolated BMSCs. These BMSCs showed an increased CXCL12 protein expression level and upregulated messenger RNA expressions of Rankl, Wnt5a, and Ror2. Ex vivo data showed that their capacities of inducing migration and differentiation of osteoclast precursors were enhanced, and these enhanced capabilities were restrained after blocking their Ror2 signaling using small interfering RNA (siRNA) assays. Reducing Ror2 expression in the BMSC cell line by siRNA or blocking the downstream signalings with specific inhibitors also demonstrated a suppression of the capacity of the BMSC cell line to promote Wnt5a-dependent migration (including SP600125 and cyclosporine A) and differentiation (cyclosporine A only) of osteoclast precursors. These findings support the idea that Wnt5a/Ror2 signaling in TMJ subchondral BMSCs enhanced by UAC promoted BMSCs to increase Cxcl12 and Rankl expression, in which JNK and/or Ca2+/NFAT pathways were involved and therefore were engaged in enhancing the migration and differentiation of osteoclast precursors, leading to increased osteoclast activity and an overall TMJ subchondral trabecular bone loss in the UAC-treated rats.

To elucidate inhibition of protein kinase C α (PKC α) activity by staurosporine on apoptosis of oral cancer cell line tongue squamous cell carcinoma (TSCCa) cells and to clarify the role of survivin and caspase-3 in mediating apoptosis. TSCCa cell viability was measured by MTT assay after 100 nmol/L staurosporine treatment. Apoptotic cells were identified by using phase contrast microscopy, acridine orange/ethidium bromide staining, and flow cytometry. Level of PKC α and its subcellular location were investigated using Western blot analysis. Expression of survivin and caspase-3 were evaluated using immunocytochemistry. Staurosporine significantly inhibited the cell viability of TSCCa cells in a dose- and time-dependent manner. Marked cell accumulation in G2/M phase was observed after 100 nmol/L staurosporine exposure for 6 h and 12 h. In addition, the percentage of apoptosis increased in a time-dependent manner, from 2.9% in control cultures to approximately 27.4% at 100 nmol/L staurosporine treatment for 24 h. Staurosporine displayed difference in inhibitory efficacy between cytosolic and membrance-derived PKC α. The content of PKCα in membrane versus cytosol decreased quickly, from 0.45 in ethanol-treated control cultures to 0.18 after staurosporine exposure for 24 h (P

Subchondral bone loss in mandibular condyles was reported to be induced by experimentally created unilateral anterior crossbite (UAC) which altered the occlusal load distribution and hereafter the temporomandibular joint (TMJ) remodelling process. However, the initial cellular responses are poorly understood. In the present study, changes in osteoblast and osteoclast activities in TMJ subchondral bone were investigated using the rats treated with UAC.Forty rats were randomly divided into UAC and control groups, and sampled at 2 weeks after the operation. Subchondral bone loss was evaluated by micro-CT. Osteoclast and osteoblast activities were analyzed by real-time PCR. The osteoblast differentiation of the locally isolated BMSCs from TMJ subchondral bone was assessed by Alizarin red staining. The migration of BMSCs was detected by transwell assays.Compared with the age-matched controls, TMJ subchondral bone loss was observed in the UAC-treated rats (p0.05). The osteoblast activity evaluated by real-time PCR and osteoblast number revealed by immunohistochemical staining were reduced in the TMJ subchondral bone of UAC rats (p0.05), and the capability of proliferation, migration and osteoblast differentiation were all decreased in the locally isolated BMSCs from the UAC group (p0.05).The present data demonstrated an involvement of reduced BMSCs activity in the initiation of the mandibular subchondral bone loss at the early stage of installation of the aberrant prostheses.

Estrogens have been suggested to play an important role in the development of temporomandibular disorders (TMD). However, a growing body of epidemiological, clinical and experimental researches focusing on the relationship between TMD and exogenous estrogen or serum estrogen has produced conflicting results. Recently, locally synthesized estrogens have been found and proved to contribute greatly to the function of cartilage. We hypothesize that estrogens synthesized locally in condylar cartilage have a profound effect on the development of TMD. Future investigation of local estrogen in condylar cartilage may give, at least partially, valuable evidences for the etiology and treatment strategy of TMD. In our opinion, regulating the amount and effect of locally synthesized estrogen seems to hold interesting future prospects for the treatment of TMD.

To study the effect of estrogen deficiency and altered temporomandibular joint loading on the histomorphology of condylar cartilage and on the expression of types II and X collagen and matrix metalloproteinase-3 (MMP-3).Thirty-six female rats were divided into four groups: ovariectomized rats on a normal diet, nonovariectomized control rats on a normal diet, ovariectomized rats on a soft diet, and nonovariectomized control rats on a soft diet. Ovariectomy was performed at the age of 60 days. Repeated-measures ANOVA was used to analyze the data.The condylar cartilage in the ovariectomized normal diet group showed a significantly higher number of cells than in the nonovariectomized control rats (P.001). The proportional amount of MMP-3 expression was significantly higher in the ovariectomized rats than in the nonovariectomized control rats in both diet groups (P.001). The area covered by types II and X collagen was significantly higher in the experimental groups than in the control groups (P.01).Condylar cartilage is sensitive to both estrogen level and dietary loading.

Background Estrogens play an important role in modulating the morphology and function of temporomandibular joints (TMJs), which is suggested to act via estrogen receptors (ERs). The present study was to investigate the expression of aggrecan, collagen type II (Col II), Col X, aromatase, ERα and ERβ in degenerative changes of mandibular condylar cartilage. Methods Forty male and 40 female 8-week-old rats were enrolled in this study. In experimental groups, the disordered occlusion was created by moving the first molars mesially and the third ones distally. Immunohistochemistry and real-time PCR were performed at the end of the second or fourth week. Results Degenerative changes, characterized by interrupted continuity of hypertrophic layer, pyknotic and eosinophilic lesion with few nuclei, areas filled with eosinophilic nuclei, were observed in more joints from female experimental groups than male ones. However, thickening changes in hypertrophic layer were only found in male experimental groups. The gene expression of Col II, Col X and aggrecan increased in 4-wk male experimental subgroup (both P < 0.01), but decreased in 2-wk and 4-wk female subgroups (P < 0.05). The gene expression of ERα decreased in 2-wk male and female experimental subgroups (both P < 0.01), however, that of ERβ increased except the 2-wk female experimental subgroup (all P < 0.01). The expression of aromatase decreased in both male and female experimental subgroups (all P Conclusions Mandibular condylar cartilage responses differently to the disordered occlusion in male and female rats. The levels of locally synthesized estrogen, ERα and ERβ may have limited attribution, if any, to the sex-specific cartilage response.

To investigate the effect of excess genistein on the extracellular matrix in mandibular condylar cartilage of female rats in vivo.Female SD rats were administered through oral gavage with genistein (50 mg/kg) or placebo daily for 6 weeks. The morphological changes of temporomandibular joints were studied with HE staining. The expression of cartilage matrix compounds (aggrecan and collagen type II), estrogen-related molecules (aromatase, estradiol, ERα and ERβ) and proliferating cell nuclear antigen (PCNA) in mandibular condylar cartilage was detected using immunohistochemistry, ELISA and real-time PCR.The genistein treatment significantly reduced the thickness of the posterior and middle regions of mandibular condylar cartilage, and decreased the expression of collagen type II, aggrecan and PCNA. Compared with the control group, the estradiol content and expression levels of the key estradiol-synthesizing enzyme aromatase in the genistein-treatment group were significantly decreased. The genistein treatment significantly increased the expression of ERβ, but decreased the expression of ERα.Excess genistein suppresses extracellular matrix synthesis and chondrocytes proliferation, resulting in thinner mandibular condylar cartilage. These effects may be detrimental to the ability of mandibular condylar cartilage to adapt to mechanical loads.

To investigate the effect of the experimentally created malocclusion (ECDO) on the expression of IL-1β, IL-6 and TNF-α in mandibular condylar cartilage of Sprague Dawley (SD) rats.In the experimental group, the malocclusion was created by moving the first and third molars 0.8 mm away. HE and immunohistochemical staining were performed on the mandibular condyle at the end of the 8th or 12th week. Gene expression was analyzed by real-time PCR. The data was processed using SPSS12.0 software package.Degenerative changes were found in the experimental group, the level of IL-1β was not significantly different among the groups (P0.05), while the expression of IL-6 in 8-week ECDO subgroup was significantly higher than that in 8-week control subgroup (P=0.009), and the expression of TNF-α in 8-week ECDO subgroup was significantly higher than that in 8-week control subgroup (P=0.001) and 12-week ECDO subgroup (P=0.004).At the early period, IL-6 and TNF-α, but not IL-1β, play an important role in the degenerative remodeling procedure in rat condylar cartilage induced by ECDO.

To examine the effect of decreased estrogen level and altered diet hardness on condylar cartilage morphology of the rat temporomandibular joint (TMJ) and on the expression of condylar cartilage estrogen receptor alpha (ERa) and matrix metalloproteinase-8 (MMP-8).A total of 36 female rats was divided into four groups: ovariectomized rats fed a normal diet, non-ovariectomized controls fed a normal diet, ovariectomized rats fed a soft diet, and non-ovariectomized controls fed a soft diet. Ovariectomy was performed at the age of 60 days. Seven days after the operation, the rats were sacrificed. Repeated measures ANOVA and Duncan's multiple comparison tests were used for statistical analysis.The ovariectomized rats had thicker cartilage layers than the controls, both in the normal diet and soft diet groups. The thinnest cartilage layers were found in the control rats fed with the soft diet. The thickness of the chondroblastic layer was significantly higher (P.001) in the normal-diet rats than in the soft-diet rats in both ovariectomized and non-ovariectomized groups. The thickness of the proliferative layer was significantly higher (P.001) in the ovariectomized soft-diet rats than in the soft-diet control rats. The proportional amount of ERa was statistically significantly higher (P.001) in the condylar cartilage of the ovariectomized rats than in the non-ovariectomized control rats both in the normal- and soft-diet groups. The proportional amount of ERa was statistically significantly higher (P.001) in the ovariectomized normal-diet rats than in the ovariectomized soft-diet rats. The proportional number of MMP-8-positive cells was statistically significantly higher (P.001) in the condylar cartilage of ovariectomized rats fed the soft diet than in non-ovariectomized control rats fed the soft diet. Control rats fed with the normal diet had a higher proportional amount of MMP-8 positive cells than control rats fed with the soft diet (P.05).The rat TMJ condylar cartilage is sensitive to changes in estrogen levels and altered diet hardness.

Immunotherapy is a promising strategy for the treatment of human epidermal growth factor receptor 2 (HER2)-positive tumors. Previously, we constructed an immuno-carboxy terminal fragment of Bid (immuno-tBid) and reported its specific and effective destruction of HER2-positive tumor cells. In this study, in order to further reduce the immunogenicity of the previous immuno-proapoptotic protein, we constructed a novel immuno-tBid by replacing domain II of Pseudomonas exotoxin A with a short furin cleavage sequence from the diphtheria toxin. In order to explore the possible application of this novel immuno-tBid in the treatment of osteosarcoma, we first examined the expression of the HER2 protein in a subclone of a human osteosarcoma cell line with relatively high metastatic potential (SOSP-9607-E10), as well as in clinical specimens of osteosarcoma. Quantitative real-time PCR and Western blot analysis revealed that the expression of HER2 was up-regulated in the SOSP-9607-E10 cells, while immunohistochemical analysis revealed that HER2 was overexpressed in 37% of the tissue specimens examined. Both HER2-positive SOSP-9607-E10 and SKBR-3 cells, as well as HER2-negative HeLa cells were transiently transfected with the novel immuno-tBid in order to study its specific pro-apoptotic effect. We demonstrate here that this novel immuno-tBid induces the specific destruction of HER2-overexpressing SOSP-9607-E10 cells through the release of cytochrome C. These results suggest that the novel immuno-tBid with a minimized exogenous fragment could represent a competitive approach for the treatment of HER2-positive osteosarcoma.

Osteoarthritis (OA) is a degenerative joint disease generally characterized by progressive cartilage degradation and subchondral bone changes. Subchondral bone changes have been proposed to initiate or accompany with cartilage degradation in OA. The purpose of this study was to characterize cartilage damage, subchondral bone remodeling, and the possible mechanism involved in these morphological changes in our reported rat model with OA-like lesions in the mandibular condyle. In experimental groups, the dental occlusion was orthodontically disturbed. By histological analysis, transmission electron microscopy (TEM), micro-CT scanning and serum tests, changes in condylar cartilage and subchondral bone were analyzed at 8 and 12 weeks after treatment. The mRNA and protein levels of bone pro-resorptive and pro-formative factors by chondrocytes were investigated. Increased degraded cartilage areas and obvious cartilage calcification were observed in 8- and 12-week treated (EXP) groups compared to the age-matched controls. Subchondral bone loss, characterized as decreased bone mineral density (BMD), bone volume fraction (BV/TV) and trabecular thickness (Tb.Th), but increased trabecular separation (Tb.Sp), was observed in the 12-week but not the 8-week EXP group, respectively, versus their age-matched controls. The subchondral bone loss in the 12-week EXP group was accompanied with decreased new bone formation rate, but increased serum carboxy terminal telopeptides (CTXs), and increased osteoclast numbers and proportion of surface area in the subchondral bone regions. Increased mRNA and protein levels of M-CSF, VEGF, RUNX and RANKL/OPG ratio, but decreased OPG, were found in condylar cartilage in the 12-week EXP group versus its age-matched controls, and those of RANKL/OPG ratios were significantly higher in the 12-week EXP group than the 8-week EXP. In addition, increased mRNA levels of VEGF, RUNX and RANKL/OPG ratio, but decreased OPG, were also found in condylar cartilage in the 8-week EXP group versus its age-matched controls (All P

To investigate the effects of experimentally created disordered occlusion on the mandibular condylar cartilage in terms of histological morphology and expression of MMP-3, MMP-9, TIMP-1 and aggrecan.Eighty 8-week-old Sprague-Dawley rats were randomly divided into two experimental (Exp) and two control (Con) groups, with equal sex and number distribution as subgroups. In the Exp group, the disordered occlusion was created by orthodontically moving the first and third molars 0.8mm away. Hematoxylin-eosin and immunohistochemical staining were performed on the mandibular condyles at the end of the 8th or 12th week. Gene expression was analysed by real-time PCR.Osteoarthritis-like lesions, typically seen as a cell-free area, were detected in the Exp group, predominantly in females. In the cell-free area, the immunopositive expression of MMP-3, MMP-9, TIMP-1 and aggrecan were absent. Hyper-proliferation changes, typically seen as conjunctive invaginations of chondrocytes, were also observed where immunopositive expression of the tested materials was strong. There were sex and time point related differences in gene expression. In the 8-week subgroup, the expression of MMP-3 decreased, while aggrecan increased in males; however, both MMP-9 and TIMP increased in the female group (P0.05). In the 12-week subgroup, the expression of MMP-3 increased, while TIMP, MMP-9 (male only) and aggrecan (female only) decreased (P0.05).The present results indicate that the experimentally created disordered occlusion led to osteoarthritis-like lesions accompanied by changes in the expression of MMP-3, MMP-9, TIMP-1 and aggrecan in mandibular condyle cartilage with gender differences.

To investigate the effect of experimentally created occlusal disorders (ECOD) on the expression of estrogen in rat condylar cartilage.The model of ECOD was created by moving right upper and left lower first molars anteriorly. The animals in ECOD were sacrificed at 2, 4, 6, 8 weeks later. In removed occlusal disorders group, the moved first molars were extracted at 6 weeks later, and the animals were sacrificed 2 weeks later. The expression of estrogen was detected by SABC technique of immunocytochemistry, and then was analyzed by the density of estrogen-positive chondrocytes.1) Estrogen was abundant in mature layer and hypertrophic layer of rat mandibular condylar cartilage. 2) In control group, the expression of estrogen decreased gradually from 6-week-old to 16-week-old. 3) In both childhood and puberty rats, the expression of estrogen in experiment group was significantly higher at 2 weeks after treatment, while no difference was found at 4, 6, 8 weeks after treatment. However, the expression in removed occlusal disorder group was higher than that in control group and 8 weeks of ECOD group.In rat condylar cartilage, the expression of estrogen de-creases with age. Induced by ECOD, the expression of estrogen increases in early stage of remodelling activity.

Estrogens are suggested to play an important role in the development of osteoarthritis (OA). Though many clinical and experimental researches have been carried out to investigate the relationship between OA and circulating estrogen or exogenous estrogen, the discrepancy among the results of these studies was also too large to get reconcilable conclusion. In the past several years, locally synthesized estrogens have been found and proved to contribute significantly to the turnover and function of bone tissues independent of circulating estrogen concentrations. We hypothesize that estrogens synthesized locally in bone or synovial tissues have a profound effect on the development OA. Future investigation of local estrogen in bone tissues may give, at least partially, valuable evidences for the etiology and treatment strategy of OA. In our opinion, regulating the amount and effect of locally synthesized estrogen may hold intriguing prospects for the treatment of OA.

To examine the expression of molar root patterning gene 1 (Mrp1) and predict the Mrp1 structure by bioinformatics analysis.A pair of Mrp1-specific PCR primers were designed, and RT-PCR method was used to study the mRNA's expression pattern in rat molar root and other organs. Gene positioning and other protein sequence prediction were carried out by chromosome analysis and other bioinformatics analysis.Mrp1 was expressed not only in the molar but also in the developing pancreas, liver, lung and kidney tissues. Mrp1 was located in the 18q12.3 chromosome of the rats and the Mrp1 amino acids sequence had about 37% homology with a known protein Uroplakin IIIb (p35) which was an urothelial differentiation membrane molecular marker. A trans-membrane structure, 5 PKC phosphorylation sites and 4 CKII phosphorylation sites in Mrp1 were found.Mrp1 has a broad expression in different developing organs, and it may have a important function in the rat tooth root development.

To investigate the effect of experimentally created disordered occlusion (ECDO) on cell death and proliferation in rat mandibular condylar cartilage.Sprague-Dawley rats were randomly assigned to experimental and control groups. In the experimental groups, ECDO was created by the dental orthodontic method. By means of histological evaluation, immunohistochemistry and TUNEL staining, we studied the histomorphological changes, the death and proliferation of chondrocytes.Time- and sex-related progressive histologic degradation was observed in the condylar cartilage of ECDO rats, accompanied with diminished chondrocyte proliferation in the female 12-week ECDO subgroup (P0.05). An increase in the number of apoptotic chondrocytes was seen in both the female 8- and 12-week ECDO subgroups and in the male ECDO 12-week subgroup (all P0.05), but not in the male ECDO 8-week subgroup (P0.05).ECDO induces degradation in the rat condylar cartilage accompanied by an increase in chondrocyte death.

Objective: Oestrogen expression may indicate a difference in resistance potential to mechanical strain. The purpose of this study was to investigate the expression of oestrogen and oestrogen receptors in mandibular condylar cartilages in male and female Sprague– Dawley rats at different ages. Materials and methods: One-hundred SD rats at the age of 2, 4, 8 weeks and 4, 12 months in both sexes, 10 in each age–sex group, were enrolled in this study. The expression of oestradiol, ERa and ERb was detected in mandibular condylar cartilages by the method of immunohistochemistry, and enzyme-linked immunosorbent assay or western blot. Results: Oestradiol and ERs immunoreactivity were obvious in mandibular condylar cartilages of SD rats. Oestradiol and ERa were observed in hypertrophic and mature layers, while ERb only in hypertrophic layer. There was no sex difference of same age (except 8-week age group) in the expression of oestradiol. The expression of both ERs, however, was usually higher in male than in age-matched female rats (P < 0.05), except that the 8-week-old female rats showed a higher ERa expression and the 4- and 8-week-old female rats showed a higher ERb expression than the age-matched male ones in western blot results (P < 0.05). Conclusions: The results that oestradiol, ERa and ERb are co-expressed in rat mandibular condylar cartilage, indicate that mandibular condylar cartilage is a target for oestrogen. The age and sex related differences in ERs expression may indicate a difference in potential to resist mechanical loading between genders at different ages.

This article was to study the effect of induced occlusal disorders and removed occlusal disorders on the expression of bone morphogenetic protein-2 (BMP-2) of condylar cartilage.Young and adult female Sprague-Dawley rats were divided respectively into induced occlusal disorders group, removed occlusal disorders group and control group, 3 rats every group. For induced occlusal disorders rats, the elastic rubbers were inserted between the first and second molar in the left upper side and right lower side to form the disordered occlusion. They were killed under anaesthesia 8 weeks after the treatment. For removed occlusal disorders rats, the first molars that caused disordered occlusion were extracted 6 weeks after forming disordered occlusion. 2 weeks later, they were killed under anaesthesia. For normal rats, they were killed at the same time with experimental rats. Hibateral temporomandibular joints of each rat were removed and stained with HE and monoclone antibody of BMP-2. The thickness of condylar cartilage was measured. The expression of BMP-2 in condylar cartilage was detected by half-quantity immunohistochemical analysis.For adult induced occlusal disorders group, the thickness of cartilage in intermediate part of condyle decreased. However, it increased in the posterior part. After removing occlusal disorders, the thickness of posterior condylar cartilage returned to normal level. But it was still thinner than control group in the intermediate part. The expression of BMP-2 in anterior, intermediate, posterior part of condylar cartilage of young induced occlusal disorders group was higher than that of young removed occlusal disorders group expression of BMP-2 showed induced occlusal disorders group was higher than removed occlusal disorders group, which was higher than control group. and control group. No difference of the expression of BMP-2 was found in young removed occlusal disorders group and control group. For the expression of BMP-2 in intermediate part of condylar cartilage, both adult induced and removed occlusal disorders groups were higher than adult control group. For the posterior part of adult condyle cartilage, the expression of BMP-2 showed induced occlusal disorders group was higher than removed occlusal disorders group, which was higher than control group.Induced occlusal disorders can lead higher expression of BMP-2 in condylar cartilage of young and adult rats. Adaptability of condylar cartilage of adult rats is weaker than young rats, especially the intermediate part.