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Temporomandibular joint osteoarthritis (TMJ-OA) is a degenerative disease of the joint. The early manifestations of TMJ-OA are abnormal remodeling of condylar subchondral bone. In bone tissue, bone marrow mesenchymal stem cells (BMSCs) and osteoblasts play important roles in the differentiation and maturation of most hematopoietic cells. MicroRNA-26b (miR-26b) is upregulated during the osteogenesis of BMSCs, and miR-26b overexpression leads to the activation of β-catenin and the enhancement of osteogenesis and cartilage formation. However, the pathologic mechanism remains unclear. In the present study, we used a rat model with OA-like changes in the TMJ induced by experimental unilateral anterior crossbite (UAC) and found that the level of miR-26b was markedly lower in BMSCs from the subchondral bones of UAC rats than in those from sham control rats. MiR-26b overexpression by agomiR-26b increased condylar subchondral bone osteogenesis in UAC rats. Notably, although agomiR-26b primarily affected miR-26b levels in the subchondral bone (but not in cartilage or the synovium), the overexpression of miR-26b in BMSCs in UAC rats largely rescued OA-like cartilage degradation, while the inhibition of miR-26b in BMSCs exacerbated cartilage degradation in UAC rats. We measured the expression levels of β-catenin and related osteogenic and osteoclastic factors after using miR-26b mimics and inhibitors in vivo. Moreover, BMSCs were treated with the β-catenin blocker Wnt-C59 and then transfected with miR-26b mimics or inhibitors. Then, we examined the expression of β-catenin as the direct target of miR-26b. The results of the present study indicate that miR-26b may modulate subchondral bone loss induced by abnormal occlusion and influence the osteogenic differentiation of subchondral BMSCs through β-catenin in the context of TMJ-OA progression., (Copyright © 2022. Published by Elsevier Inc.)

Temporomandibular joint osteoarthritis (TMJOA) is a chronic degenerative disease for which the underlying mechanism still remains unclear. Compared with apoptosis and autophagy, necroptosis causes greater harm to tissue homeostasis by releasing damage-associated molecular patterns (DAMPs). However, the role of necroptosis and downstream key DAMPs in TMJOA is unknown. Here, rodent models of TMJOA were established by the unilateral anterior crossbite (UAC). Transmission electron microscopy (TEM) and immunohistochemistry of receptor interacting protein kinase 3 (RIPK3)/phosphorylation of mixed lineage kinase domain-like protein (pMLKL) were conducted to evaluate the occurrence of necroptosis in vivo. The therapeutic effects of blocking necroptosis were achieved by intra-articularly injecting RIPK3 or MLKL inhibitors and using RIPK3 or MLKL knockout mice. In vitro necroptosis of condylar chondrocyte was induced by combination of tumor necrosis factor alpha (TNFα), second mitochondria-derived activator of caspases (SMAC) mimetics and carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (z-VAD-fmk). The possible DAMPs released by necroptotic chondrocytes were screened by quantitative proteomics and blocked by specific antibody. Translucent cytosol, swollen organelles, and ruptured cell membranes, features of necroptosis, were frequently manifested in chondrocytes at the early stage of condylar cartilage degeneration in TMJOA, which was accompanied by upregulation of RIPK3/pMLKL. Inhibiting or knocking out RIPK3/MLKL significantly prevented cartilage degeneration. DAMPs released by necroptotic condylar chondrocytes, such as syndecan 4 (SDC4) and heat shock protein 90 (HSP90), were verified. Furthermore, blocking the function of SDC4 significantly attenuated the expression of TNFα in cartilage and synovium, and accordingly increased cartilage thickness and reduced synovial inflammation. Thus, the necroptotic vicious cycle of TNFα-SDC4-TNFα contributes to cartilage degeneration and synovitis, and can serve as a potential therapeutic target for treating TMJOA. © 2022 American Society for Bone and Mineral Research (ASBMR)., (© 2022 American Society for Bone and Mineral Research (ASBMR).)

Objective: The aim of this study was to demonstrate the biological function of Semaphorin 4D (Sema4D)/Plexin-B1 in the bone formation features of osteoblasts in early-stage temporomandibular joint (TMJ) osteoarthritis., Design: Sema4D/Plexin-B1, expressed by osteoclasts/osteoblasts, plays a balancing role in bone formation and resorption. However, previous studies have mainly focused on bone resorption by osteoclasts in early-stage osteoarthritis. This study used our reported experimental unilateral anterior crossbite (UAC) mouse model to explore subchondral bone changes, which were assessed by micro-CT analysis. The changes in osteoblasts were investigated after the inhibition of Sema4D by BMA-12 injection with the detection of bone formation-related markers. A Transwell migration assay was performed to reveal the specific impact of Sema4D on osteoblasts in vitro., Results: The data demonstrated that subchondral bone loss in early-stage TMJ osteoarthritis was accompanied by the upregulated expression of Sema4D in cartilage and subchondral bone and Plexin-B1 in subchondral bone. Reducing Sema4D levels could inhibit subchondral bone loss and cartilage degeneration in early-stage TMJ osteoarthritis. In vitro, the results revealed that Sema4D could reduce the expression of osteocalcin and alkaline phosphatase and increase the migrating capability of Plexin-B1-positive osteoblasts., Conclusions: Our results revealed that elevated Sema4D expression in early-stage TMJ osteoarthritis might decrease the bone formation activity of osteoblasts in the subchondral bone by binding to Plexin-B1 expressed by osteoblasts. Inhibiting Sema4D/Plexin-B1 signaling in early-stage osteoarthritis represents a promising strategy for new therapeutic approaches to osteoarthritis., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A switch from autophagy to apoptosis is implicated in chondrocytes during the osteoarthritis (OA) progression with currently unknown mechanism(s). In this study we utilized a flow fluid shear stress (FFSS) model in cultured chondrocytes and a unilateral anterior crossbite (UAC) animal model. We found that both FFSS and UAC actively induced endoplasmic reticulum stress (ERS) in the temporomandibular joints (TMJ) chondrocytes, as demonstrated by dramatic increases in expression of HSPA5, p-EIF2AK3, p-ERN1 and ATF6. Interestingly, both FFSS and UAC activated not only pro-death p-EIF2AK3-mediated ERS-apoptosis programs but also pro-survival p-ERN1-mediated autophagic flux in chondrocytes. Data from FFSS demonstrated that MTORC1, a downstream of p-ERN1, suppressed autophagy but promoted p-EIF2AK3 mediated ERS-apoptosis. Data from UAC model demonstrated that at early stage both the p-ERN1 and p-EIF2AK3 were activated and MTORC1 was inhibited in TMJ chondrocytes. At late stage, MTORC1-p-EIF2AK3-mediated ERS apoptosis were predominant, while p-ERN1 and autophagic flux were inhibited. Inhibition of MTORC1 by TMJ local injection of rapamycin in rats or inducible ablation of MTORC1 expression selectively in chondrocytes in mice promoted chondrocyte autophagy and suppressed apoptosis, and reduced TMJ cartilage loss induced by UAC. In contrast, MTORC1 activation by TMJ local administration of MHY1485 or genetic deletion of Tsc1 , an upstream MTORC1 suppressor, resulted in opposite effects. Collectively, our results establish that aberrant mechanical loading causes cartilage degeneration by activating, at least in part, the MTORC1 signaling which modulates the autophagy and apoptosis programs in TMJ chondrocytes. Thus, inhibition of MTORC1 provides a novel therapeutic strategy for prevention and treatment of OA. Abbreviations : ACTB: actin beta; ATF6: activating transcription factor 6; BECN1: beclin 1; BFL: bafilomycin A 1 ; CASP12: caspase 12; CASP3: caspase 3; DAPI: 4',6-diamidino-2-phenylindole; DDIT3: DNA-damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERS: endoplasmic reticulum stress; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; FFSS: flow fluid shear stress; HSPA5/GRP78/BiP: heat shock protein 5; LAMP2: lysosome-associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; OA: osteoarthritis; PRKAA1/2/AMPK1/2: protein kinase, AMP-activated, alpha 1/2 catalytic subunit; RPS6: ribosomal protein S6; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: thapsigargin; TMJ: temporomandibular joints; TSC1/2: tuberous sclerosis complex 1/2; UAC: unilateral anterior crossbite; UPR: unfolded protein response; XBP1: x-box binding protein 1.

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 Ca 2+ loading and CaSR localization in endoplasmic reticulum (ER), casually accelerating cell differentiation that could be abrogated by emptying ER Ca 2+ 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., (© 2018 American Society for Bone and Mineral Research.)

Aims: To investigate the changes in condylar cartilage and subchondral bone of the temporomandibular joint (TMJ) in a mouse model of incisor malocclusion., Methods: By bonding a single (single group) or a pair (pair group) of metal tube(s) to the left incisor(s), a crossbite-like relationship was created between left-side incisors in mice. The morphological changes in the TMJ condyles were examined by hematoxylin and eosin and toluidine blue staining. Indices of osteoclastic activity, including tartrate-resistant acid phosphatase (TRAP) staining and macrophage colony stimulating factor (M-CSF) were investigated by histochemistry or real-time polymerase chain reaction (PCR). The osteoblastic activity was indexed by osteocalcin expression. Expressions of semaphorin 4D and its receptor, Plexin-B1, were detected by real-time PCR. Two-way analysis of variance was used to assess the differences between groups., Results: One week and 3 weeks after bonding the metal tube(s), cartilage degradation and subchondral bone loss were evident histologically. Both indices of osteoclastic activity (TRAP and M-CSF) were significantly increased in cartilage and subchondral bone after bonding the metal tube(s). Osteocalcin expression in cartilage was significantly increased at week 3, while its expression in subchondral bone was significantly increased at week 1 but decreased at week 3. The semaphorin 4D expression in cartilage and subchondral bone was significantly decreased at week 1 but significantly increased at week 3. For Plexin-B1 expression, a significant increase was detected in subchondral bone at week 3., Conclusion: Bonding a single or a pair of metal tube(s) to left incisor(s) is capable of inducing remodeling in the TMJ, which involved cartilage degradation and alteration of osteoclastic and osteoblastic activity.

Objective: 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., Design: 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., Results: Compared with the age-matched controls, TMJ subchondral bone loss was observed in the UAC-treated rats (p<0.05). 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 (p<0.05), and the capability of proliferation, migration and osteoblast differentiation were all decreased in the locally isolated BMSCs from the UAC group (p<0.05)., Conclusions: 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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

Background: Abnormal lipid metabolism is involved in the development of osteoarthritis (OA). ATP‐binding cassette protein G1 (ABCG1) is crucial in mediating the outflow of cholesterol, phosphatidylcholine and sphingomyelin and reducing intracellular lipid accumulation. Objective: This study aimed to evaluate whether ABCG1 participates in the abnormal adipogenesis of chondrocytes in osteoarthritic cartilage of temporomandibular joint. Methods: Eight‐week‐old female rats were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical (IHC) staining, and qRT–PCR were performed. Primary condylar chondrocytes of rats were transfected with ABCG1 shRNA or overexpression lentivirus and then stimulated with fluid flow shear stress (FFSS). Cells were collected for oil red O staining, immunofluorescence staining, and qRT–PCR analysis. Results: Abnormal adipogenesis, characterized by increased expression of Adiponectin, CCAAT/enhancer‐binding protein α (Cebpα), fatty acid binding protein 4 (Fabp4) and Perilipin1, was enhanced in the degenerative cartilage of TMJ OA in rats with UAC, accompanied by decreased expression of ABCG1. After FFSS stimulation, we observed lipid droplets in the cytoplasm of cultured cells with increased expression of Adiponectin, Cebpα, Fabp4 and Perilipin1 and decreased expression of ABCG1. Knockdown of Abcg1 induced abnormal adipogenesis and differentiation of condylar chondrocytes. Overexpression of ABCG1 alleviated the abnormal adipogenesis and differentiation of condylar chondrocytes induced by FFSS. Conclusions: Abnormal adipogenesis of chondrocytes and decreased ABCG1 expression were observed in degenerative cartilage of TMJ OA. ABCG1 overexpression effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration. [ABSTRACT FROM AUTHOR]

Background: Temporomandibular joint osteoarthritis (TMJOA) is characterized by articular cartilage degeneration and progressive synovitis. How to effectively inhibit TMJOA in the early stage has been a hot topic in the biomedical field. As a non‐invasive physiotherapy, pulsed electromagnetic field (PEMF) treatment has shown great potential in the treatment of osteoarthritis (OA) in extremity joints. Objective: This study aims to investigate the biological effect of PEMF intervention on TMJ cartilage degeneration and synovium inflammation at the early stage of TMJOA. Methods: PEMF (2.0 mT, 15 Hz, 2 h/day) treatment was given to rats in which TMJOA was induced by applying the unilateral anterior crossbite (UAC). Histological and immunohistochemical staining, TUNEL assay, real‐time PCR and western blotting assay were performed to detect the changes of the morphology and the expression of pro‐inflammatory and degradative factors in condylar cartilage and synovium. Results: Obvious condylar cartilage degeneration, characterized by decreased cartilage thickness, degraded cartilage extracellular matrix, increased expression of pro‐inflammatory and degradative factors (TNF‐α, IL‐1β, MMP‐13, ADAMTS‐5, IL‐6, MMP‐3, MMP‐9 and COL‐X) and increased chondrocytes death, was observed in UAC group, accompanied by synovium hyperplasia and up‐regulation of pro‐inflammatory and degradative factors in synovium. PEMF intervention reversed the decreased cartilage thickness at 3 weeks and degraded cartilage extracellular matrix at 6 weeks. Moreover, the up‐regulation of pro‐inflammatory, degradative and hypertrophyic factors and chondrocytes death in condylar cartilage induced by UAC were inhibited to some extent. In addition, the synovium hyperplasia and the up‐regulation of pro‐inflammatory and degradative factors in synovium were inhibited at 3 weeks and 6 weeks. Conclusions: Appropriate PEMF stimulation can reverse the loss of cartilage extracellular matrix, the chondrocytes death, the increased expression of pro‐inflammatory and degradative factors in cartilage, the decreased cartilage thickness and synovium inflammation induced by UAC at the early stage of TMJOA to some extent. PEMF stimulation may be a promising method in clinical TMJOA treatment. [ABSTRACT FROM AUTHOR]

Objective: Incisors tubed prosthesis with bilateral anterior elevation (BAE) relation had been reported to stimulate the proliferative response in the mandibular condylar cartilage of mice, thus the prosthetic occlusion elevation had been proposed to treat cartilage degeneration. Currently, we aimed to detect the long‐term effect of BAE on temporomandibular joints (TMJs). Materials and Methods: Twelve 6‐week‐old female mice were assigned to age‐matched control and BAE groups (n = 6). Micro‐CT images and the macro‐ and micro‐morphology of the mandibular condyles were analyzed at 29 weeks. Results: Compared with the age‐matched controls, in BAE group, there were loss of subchondral cortical bone and heavy loss of the subchondral trabecular bone at the superior sites of the TMJ condyles, but hyperostosis at the inferior sites as revealed by micro‐CT images and histological slices. In BAE group, cartilage thickness and matrix area were increased with upregulated expression of type II, type X collagen, and Ki67, but the expression of cleaved caspase‐3 was downregulated (all, p < 0.05). Conclusion: In addition to cartilage thickening, long‐term BAE induces loss of the subchondral cortical bone and heavy loss of the underneath subchondral trabecular bone, but hyperostosis further underneath. Using BAE as a treatment remains double‐edged. [ABSTRACT FROM AUTHOR]

Temporomandibular joints (TMJs) have a biomechanical relationship with dental occlusion. Aberrant occlusion initiates degenerative remodeling responses in TMJ condyles. Aging is a promoting factor of osteoarthritis (OA) development. The aim of this study was to assess the effect of aging on degenerative remodeling in TMJ condyles in response to occlusal biomechanical stimulation caused by the installation of aberrant prostheses and observe rehabilitation after their removal. The experiments involved 84 female C57BL/6J mice (42 at 6 weeks old and 42 at 28 weeks old). A bilateral anterior crossbite (BAC) model was developed, and the TMJs were sampled at 3, 7, and 11 weeks. BAC was removed at 7 weeks in a subset of mice, which accepted BAC treatment at 6 week of age, and maintained for another 4 weeks after BAC removal. TMJ changes were assessed with micro-CT, histomorphology, immunohistochemistry (IHC), and immunofluorescence staining assays. The results showed that BAC induced typical OA-like TMJ lesions that were more severe in the elder groups as evaluated by the acellular zones, clustered chondrocytes, fissures between cartilage and subchondral bone, reductions in matrix amount and the cartilage thickness as revealed by histomorphological measurements, and subchondral bone loss as detected on micro-CT images. IHC indicated significant increases in cleaved caspase-3-expressing cells and decreases in ki67-positive cells in the BAC groups. There were obvious age-dependent changes in the numbers of superficial zone cells and CD90-expressing cells. Supportively, cleaved caspase-3-expressing cells obviously increased, while ki67-expressing cells significantly decreased with aging. In the elder BAC groups, the superficial zone cells such as CD90-expressing cells were greatly reduced. At 11 weeks, the superficial zone cells were almost non-existent, and there were clear serrated injuries on the cartilage surface. BAC removal attenuated the degenerative changes in the condylar cartilage and subchondral bone. Notably, the rescue effect was more pronounced in the younger animals. Our findings demonstrate the impacts of aging on both TMJ degenerative changes in response to BAC and regenerative changes following BAC removal. The reduced number of chondro-progenitor cells in aged TMJ cartilage provides an explanation for this age-related decline in TMJ rehabilitative behaviors. [ABSTRACT FROM AUTHOR]

Objective: To explore the suppressing impact of low phosphorus intake on osteoarthritic temporomandibular joint and the possible mechanisms of nuclear acid injury in the insulted chondrocytes.Design: Chondrocytes were loaded with fluid flow shear stress (FFSS) with or without low phosphorus medium. Seventy-two mice (sampled at 3-, 7- and 11-wk, n = 6) and forty-eight rats (sampled at 12-wks for different testing purpose, n = 6) were applied with unilateral anterior crossbite (UAC) with or without low phosphorus diet. In the FFSS model, the Ca and P content, molecules related to nucleic acid degradation and the mineral-producing responses in chondrocytes were detected. The effect of culture dish stiffness on chondrocytes osteogenic differentiation was measured. In the UAC model, the content of Ca and P in serum were tested. The condylar cartilage ossification and stiffness were detected using micro-CT, scanning electron microscope and atomic force microscope.Results: FFSS induced nucleic acid degradation, Pi accumulation and mineral-producing responses in the cultured chondrocytes, all were alleviated by low P medium. Stiffer dish bottoms promoted the osteogenic differentiation of the cultured chondrocytes. UAC stimulated cartilage degeneration and chondrocytes nucleic acid damage, increased PARP 1 and serum P content, and enhanced ossification and stiffening of the cartilage, all were suppressed by low phosphorus diet (all, P < 0.05).Conclusion: Nucleic acid damage takes a role in phosphorus production in osteoarthritic cartilage, contributing to the enhanced mineralization and stiffness of the cartilage that in turn promotes cartilage degradation, which can be alleviated by low phosphorus intake. [ABSTRACT FROM AUTHOR]

Objective: The temporomandibular joint (TMJ) displays a high remodelling capability in response to occlusion changes. The purpose of the current study was to investigate the responses of TMJ condyles of growing mice to the installation of a unilateral anterior crossbite (UAC) prosthesis and the replacement of the UAC prothesis with a bilateral anterior elevation (BAE) prosthesis. Materials and Methods: C57BL/6J mice were randomly assigned to the blank control and experimental groups. In mice in the experimental groups, UAC was created, while in others, BAE was created after the creation of UAC or removal of UAC. Changes in TMJ condylar cartilage and subchondral bone were assessed. Results: The degradation of condylar cartilage induced by UAC was reversed by BAE, as evaluated by cartilage histochemical changes, collagen II‐positive area, collagen X‐positive chondrocytes and expression levels of Adamts‐5, Mmp13, Tnf‐α and Il‐1β. Subchondral bone was assessed based on the subchondral bone volume, the number of TRAP‐positive cells and the Opg/Rankl ratio. Conclusion: The growing mouse TMJ condyle displays a high remodelling capability, which can be degenerative or rehabilitative in response to the creation of UAC and the replacement of UAC with BAE. Early correction of occlusion is beneficial for the recovery of degenerative condyles. [ABSTRACT FROM AUTHOR]

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 < 0.05), with increased condylar cartilage thickness, matrix amount, and cell number (p < 0.05). Compared with the deep zone cells, CTS stimulated the superficial zone cells to express a higher level of proliferating cell nuclear antigen, Cyclin D1, Aggrecan, and type II collagen but a lower level of type X collagen and alkaline phosphatase. Conclusion: Bilateral anterior elevation stimulated the proliferative response in the mandibular condylar cartilage, offering a new therapeutic strategy for cartilage degeneration. [ABSTRACT FROM AUTHOR]

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 < 0.05), and increased levels of EPHX1 were reported in discs (P < 0.05); however, increased levels were not present in the alveolar bone. Immunohistochemistry revealed the increased distribution of EGR1‐positive, EXPH1‐positive and IL10‐positive cells predominantly in the osteochondral interface, with EXPH1 also present in TMJ discs. In conclusion, the increased mRNA expression of Egr1, Ephx1 and Il10 in PBLs may serve as potential biomarkers for developed osteoarthritic lesions relating to osteochondral interface hardness changes induced by dental biomechanical stimulation. [ABSTRACT FROM AUTHOR]

Summary: Background: The temporomandibular joint (TMJ) disc plays a role in joint movement and in load absorbance and distribution. An experimental unilateral anterior crossbite (UAC) prosthesis induces mandibular condylar cartilage degeneration in rats. However, the changes in the articular disc are still unknown. Objective: To describe changes in the TMJ discs of UAC rats. Methods: The discs of fifty‐four Sprague‐Dawley rats, equally distributed into a UAC group and an age‐matched sham‐operated control group at 4, 12 and 20 weeks (n = 9), were evaluated by gross and histomorphological observation and by detection at the mRNA or protein expression levels of the markers related to the matrix elements. Results: No macro‐ or micro‐morphological differences were observed between groups. However, there were catabolic degradative changes at the molecular level in the UAC group, showing a significant reduction in the mRNA and/or protein expression levels of many molecules. The reduction became worse with time (P < 0.05). The reduced molecules included: (a) those related to the extracellular matrix, such as type I collagen, decorin and fibromodulin; (b) those related to chondrogenesis, such as type II collagen and aggrecan; and (c) those related to osteogenesis, such as alkaline phosphatase and runt‐related transcription factor 2. The mRNA expression of vascular endothelial growth factor did not change. In contrast, fibronectin, which can promote wound healing, and its N‐terminal fragment, which can induce cartilage degradation, were accumulated (P < 0.05). Conclusion: TMJ discs were stimulated to catabolic changes by the aberrant dental occlusion and seemed to go to inanimate with time. [ABSTRACT FROM AUTHOR]

Objective To study the effects of experimentally created unilateral anterior crossbite prosthesis on the expression of parathyroid hormone-related peptide (PTHrP) and parathyroid hormone receptor-1 (PTHIR) in mandibular condylar cartilage of SD rat. Methods In experimental groups, the unilateral anterior crossbite metal prosthesis was cemented to the left incisors of the maxilla and mandible of 6-week-old SD rats, respectively. Animals were sacrificed at 2, 4, 8 weeks. Hematoxylin-essin(HE) staining were carried out for studying the morphological changes of the condylar cartilage. lmmunohistoehemical staining and real-time polymerase chain reaction (PCR) analysis were performed to detect the levels of expression of PTHrP and PTHIR in the condylar cartilages. Results The obvious degenerative changes were found in the condylar cartilages in experimental group at 8 weeks. Comparing to the control group, the expression of PTHrP mRNA(P<0.01) and protein(P<0.01) in the experimental group were increased, whereas PTHIR mRNA(P<0.01) and protein (P<0.01) levels were decreased. Conclusion The expression of PTHrP was increased in the condylar cartilage of rat with unilateral anterior crossbite metal prssthesis but its effects might be limited because of decreased expression of PTHIR in the condylar cartilage. The low level expression of PTHIR should be a part of the constitution of the molecular pathomechanism of temporomandibular joint osteoarthritis (TMJOA)-like lesion. [ABSTRACT FROM AUTHOR]

The temporomandibular joint (TMJ), which is biomechanically related to dental occlusion, is often insulted by osteoarthritis (OA). This study was conducted to clarify the relationship between Indian hedgehog (Ihh) and parathyroid hormone receptor 1 (PTH1R) signaling in modulating the enhanced chondrocyte terminal differentiation in dental stimulated TMJ osteoarthritic cartilage. A gain- and loss-of-function strategy was used in an in vitro model in which fluid flow shear stress (FFSS) was applied, and in an in vivo model in which the unilateral anterior cross-bite (UAC) stimulation was adopted. Ihh and PTH1R signaling was modulated through treating the isolated chondrocytes with inhibitor/activator and via deleting Smoothened (Smo) and/or Pth1r genes in mice with the promoter gene of type 2 collagen (Col2-CreER) in the tamoxifen-inducible pattern. We found that both FFSS and UAC stimulation promoted the deep zone chondrocytes to undergo terminal differentiation, while cells in the superficial zone were robust. We demonstrated that the terminal differentiation process in deep zone chondrocytes promoted by FFSS and UAC was mediated by the enhanced Ihh signaling and declined PTH1R expression. The FFSS-promoted terminal differentiation was suppressed by administration of the Ihh inhibitor or PTH1R activator. The UAC-promoted chondrocytes terminal differentiation and OA-like lesions were rescued in Smo knockout, but were enhanced in Pth1r knockout mice. Importantly, the relieving effect of Smo knockout mice was attenuated when Pth1r knockout was also applied. Our data suggest a chondrocyte protective effect of suppressing Ihh signaling in TMJ OA cartilage which is dependent on PTH1R signaling. [ABSTRACT FROM AUTHOR]

Highlights • Insulin like growth factor-1 is increased in the degenerative cartilage. • Insulin like growth factor-1 receptor is increased in degenerative cartilage. • Insulin like growth factor binding protein-3 is dual in degenerative cartilage. • Insulin like growth factor binding protein-5 is increased in degenerative cartilage. • Occlusion stimulates insulin like growth factor-1 expression in jaw joint cartilage. Abstract Objective To investigate the changes in insulin-like growth factor-1 (IGF-1) expression levels in the degenerative mandibular condylar cartilage. Design Thirty-six rats were divided into the unilateral anterior crossbite and control groups. The expression levels of IGF-1; IGF-1 receptor (IGF-1R); IGF-binding protein-3 and -5 (IGFBP-3 and -5); proliferating cell nuclear antigen (PCNA); aggrecan; type-I, -II, -VI, and -X collagen; tissue inhibitor of metalloproteinases-1 and -3 (TIMP-1 and -3); metalloproteinases of matrix metalloproteinases-3 and-13 (MMP-3 and -13); a disintegrin and metalloproteinase thrombospondin-4 and -5 (ADAMTS-4 and -5); alkaline phosphatase (ALP); β-glucuronidase; and N-acetyl-β-glucosaminidase in the mandibular condylar cartilage were assessed. Results The protein expression levels of IGF-1and IGF-1R were increased from week 4 in the unilateral anterior crossbite group. The mRNA expression level of IGFBP-3 and -5 was upregulated from week 4 and week 2, respectively; that of IGFBP-3 was downregulated at week 8; and that of PCNA, type-II collagen, type-X collagen, aggrecan, TIMP-1, and TIMP-3 was downregulated, whereas that of MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, β-glucuronidase, and N-acetyl-β-glucosaminidase were upregulated from week 2. The positive area size of type-I collagen was increased and that of type VI collagen was decreased from week 2. The positive area size of type X collagen was increased at week 2 but decreased at week 8. The percentage of ALP-positive cells was increased from week 4. Conclusions Unilateral anterior crossbite stimulated the multifarious expression of IGF-1 and IGFBP, which may be linked to chondrocyte proliferation and differentiation in the mandibular condylar cartilage that showed progressive degeneration. [ABSTRACT FROM AUTHOR]

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 (E 2 ) 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. E 2 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. [ABSTRACT FROM AUTHOR]

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. • Negative pressure is beneficial for superficial chondrocytes of TMJ condyle to proliferate. • HMGB2 activated chondrocyte proliferation under negative pressure through the activation of AKT. • The effective activation of chondrocyte proliferation could be a promising way to treat cartilage degeneration diseases. [ABSTRACT FROM AUTHOR]

• Fluid flow shear stress decreased Derlin-3 expression in chondrocytes. • Derlin-3 overexpression protected chondrocytes from biomechanical apoptosis. • Unilateral anterior crossbite promoted apoptosis of condylar chondrocytes. • Unilateral anterior crossbite reduced the expression of Derlin-3 in cartilage. • Removal of unilateral anterior crossbite reversed the reduction in Derlin-3. 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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

• Two-week dental stimulation induced masseter and lateral pterygoid muscle injuries. • Two-week dental stimulation caused limited inflammatory and fibrosis adaptation. • Two-week dental stimulation induced protective responses in jaw muscles. • Early cessation of dental stimulation promoted jaw muscle recovery from injury. Dental occlusion are frequently changed in clinic. Molecular responses in jaw muscles to aberrant dental occlusion are changes are attractive, yet remain are obscure. 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. 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. 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. [ABSTRACT FROM AUTHOR]

• Obvious OA-like lesion can be induced by unilateral anterior crossbite (UAC). • High level of oestrogen could aggravate the degenerative changes of condylar cartilage in TMJ OA. • The effect of oestrogen on condylar cartilage is at least partially via oestrogen-ERβ-HIF2α pathway. 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. [ABSTRACT FROM AUTHOR]