Progression Of Temporomandibular Joint Osteoarthritis Research Articles

FSTL1 Can Be a Promising Target in TMJ Osteoarthritis via Regulating Chondrocyte Mitophagy and Apoptosis.

Previous studies have shown that follistatin-like protein 1 (FSTL1) is elevated in the synovial fluid of osteoarthritis and whether it is associated with disease development progress in cartilage degeneration is still unclear. The experiment was performed to explore the effect and mechanism of FSTL1 on chondrocyte degeneration and its further impaction in osteoarthritis as well as its treatment method. The patients who were diagnosed with temporomandibular joint (TMJ) disc displacement and osteoarthritis (OA) group was divided into 2 groups, anterior disc displacement (ADD) without bone resorption and ADD with bone resorption group according to the radiologic examination. The ELISA kit was used to determine the expression level of FSTL1 in patients TMJ environment. The function of FSTL1 in promoting chondrocyte degeneration was tested by quantitative reverse transcription polymerase chain reaction (Rt-qPCR) and western blot. The chondrocyte apoptosis and mitophagy were further test by flow cytometry and mitosox staining by upregulating and downregulating of FSTL1. In the end, the effectiveness of regulating FSTL1 in OA procedure was further validated by hematoxylin-eosin (HE), safranin O, and immunohistochemical (IHC) staining in vivo. There were 56 samples collected from the patients were included into this study. According to the ELISA results, FSTL1 expression levels of ADD without bone resorption groups were significantly lower than that in ADD with bone resorption group. Furthermore, the rate of cell apoptosis cells and the mitophagy procedure were highly activated when FSTL1 was upregulated. The morphology analysis of mitochondria showed significant changes when FSTL1 was highly upregulated in vitro. The in vivo and in vitro experiments showed that suppressing FSTL1 could alleviate the cartilage degeneration in TMJ OA progression. To sum up, upregulated expression level of FSTL1 in synovial fluid promoted the progression of TMJ OA by upregulating accelerating the chondrocyte apoptosis and mitophagy, and suppressing the FSTL1 in TMJ can rescue the OA progression. Therefore, it may be a promising result to consider the FSTL1 as a therapeutic target in the future.

An "All-in-One" Strategy to Reconstruct Temporomandibular Joint Osteoarthritic Microenvironment Using γ-Fe2O3@TA@ALN Nanoparticles.

Current clinical strategies for the treatment of temporomandibular joint osteoarthritis (TMJOA) primarily target cartilage biology, overlooking the synergetic effect of various cells and inorganic components in shaping the arthritic microenvironment, thereby impeding the effectiveness of existing therapeutic options for TMJOA. Here, γ-Fe2O3@TA@ALN magnetic nanoparticles (γ-Fe2O3@TA@ALN MNPs) composed of γ-Fe2O3, tannic acid (TA), and alendronate sodium (ALN) are engineered to reconstruct the osteoarthritic microenvironment and mitigate TMJOA progression. γ-Fe2O3@TA@ALN MNPs can promote chondrocytes' proliferation, facilitate chondrogenesis and anisotropic organization, enhance lubrication and reduce cartilage wear, and encourage cell movement. Magnetic-responsive γ-Fe2O3@TA@ALN MNPs also exhibit pH sensitivity, which undergoes decomposition within acidic environment to release ALN on demand. Under a 0.2 T static magnetic field, γ-Fe2O3@TA@ALN MNPs accelerate the synthesis of cartilage-specific proteins, and suppress catabolic-related genes expression and reactive oxygen species generation, affording additional protection to TMJ cartilage. In TMJOA mouse models, articular injection of γ-Fe2O3@TA@ALN MNPs effectively alleviates cartilage degeneration and subchondral bone loss in short and long terms, offering promising avenues for the development of therapeutic interventions for TMJOA.

Exploring the therapeutic potential of urine-derived stem cell exosomes in temporomandibular joint osteoarthritis.

Temporomandibular joint osteoarthritis (TMJOA) is a degenerative ailment that causes slow cartilage degeneration, aberrant bone remodeling, and persistent discomfort, leading to a considerable reduction in the patient's life quality. Current treatment options for TMJOA have limited efficacy. This investigation aimed to explore a potential strategy for halting or reversing the progression of TMJOA through the utilization of exosomes (EXOs) derived from urine-derived stem cells (USCs). The USC-EXOs were obtained through microfiltration and ultrafiltration techniques, followed by their characterization using particle size analysis, electron microscopy, and immunoblotting. Subsequently, an invivo model of TMJOA induced by mechanical force was established. To assess the changes in the cartilage of TMJOA treated with USC-EXOs, we performed histology analysis using hematoxylin-eosin staining, immunohistochemistry, and histological scoring. Our findings indicate that the utilization of USC-EXOs yields substantial reductions in TMJOA, while concurrently enhancing the structural integrity and smoothness of the compromised condylar cartilage surface. Additionally, USC-EXOs exhibit inhibitory effects on osteoclastogenic activity within the subchondral bone layer of the condylar cartilage, as well as attenuated apoptosis in the rat TMJ in response to mechanical injury. In conclusion, USC-EXOs hold considerable promise as a potential therapeutic intervention for TMJOA.

17β-estradiol promotes the progression of temporomandibular joint osteoarthritis by regulating the FTO/IGF2BP1/m6A-NLRC5 axis.

Temporomandibular joint osteoarthritis (TMJOA) is a degenerative cartilage disease. 17β-estradiol (E2) aggravates the pathological process of TMJOA; however, the mechanisms of its action have not been elucidated. Thus, we investigate the influence of E2 on the cellular biological behaviors of synoviocytes and the molecular mechanisms. Primary fibroblast-like synoviocytes (FLSs) isolated from rats were treated with TNF-α to establish cell model, and phenotypes were evaluated using cell counting kit-8, EdU, Tanswell, enzyme-linked immunosorbent assay, and quantitative real-time PCR (qPCR). The underlying mechanism of E2, FTO-mediated NLRC5 m6A methylation, was assessed using microarray, methylated RNA immunoprecipitation, qPCR, and western blot. Moreover, TMJOA-like rat model was established by intra-articular injection of monosodium iodoacetate (MIA), and bone morphology and pathology were assessed using micro-CT and H&E staining. The results illustrated that E2 facilitated the proliferation, migration, invasion, and inflammation of TNF-α-treated FLSs. FTO expression was downregulated in TMJOA and was reduced by E2 in FLSs. Knockdown of FTO promoted m6A methylation of NLRC5 and enhanced NLRC5 stability by IGF2BP1 recognition. Moreover, E2 promoted TMJ pathology and condyle remodeling, and increased bone mineral density and trabecular bone volume fraction, which was rescued by NLRC5 knockdown. E2 promoted the progression of TMJOA.

Glutathione peroxidase 4 restrains temporomandibular joint osteoarthritis progression by inhibiting ferroptosis.

There are few effective therapeutic strategies for temporomandibular joint osteoarthritis (TMJOA) due to the unclear pathology and mechanisms. We aimed to confirm the roles of GPX4 and ferroptosis in TMJOA progression. ELISA assay was hired to evaluate concentrations of ferroptosis-related markers. The qRT-PCR assay was hired to assess gene mRNA level. Western blot assay and immunohistochemistry were hired to verify the protein level. CCK-8 assay was hired to detect cell viability. Human fibroblast-like synoviocytes (FLSs) were cultured to confirm the effects of GPX4 and indicated inhibitors, and further verified the effects of GPX4 and ferroptosis inhibitors in TMJOA model rats. Markers of ferroptosis including 8-hidroxy-2-deoxyguanosine (8-OHdG) and iron were notably increased in TMJOA tissues and primary OA-FLSs. However, the activity of the antioxidant system including the glutathione peroxidase activity, glutathione (GSH) contents, and glutathione/oxidized glutathione (GSH/GSSG) ratio was notably inhibited in TMJOA tissues, and the primary OA-FLSs. Furthermore, the glutathione peroxidase 4 (GPX4) expression was down-regulated in TMJOA tissues and primary OA-FLSs. Animal and cell experiments have shown that ferroptosis inhibitors notably inhibited ferroptosis and promoted HLS survival as well as up-regulated GPX4 expression. Also, GPX4 knockdown promoted ferroptosis and GPX4 overexpression inhibited ferroptosis. GPX4 also positively regulated cell survival which was the opposite with ferroptosis. In conclusion, GPX4 and ferroptosis regulated the progression of TMJOA. Targeting ferroptosis might be an effective therapeutic strategy for TMJOA patients in the clinic.

TRIM52 knockdown inhibits proliferation, inflammatory responses and oxidative stress in IL-1β-induced synovial fibroblasts to alleviate temporomandibular joint osteoarthritis.

To explore the mechanism of tripartite motif 52 (TRIM52) in the progression of temporomandibular joint osteoarthritis (TMJOA). Gene and protein expression were tested by quantitative real-time polymerase chain reaction and western blot, respectively. The levels of pro-inflammatory cytokines and oxidative stress factors were evaluated using enzyme-linked immunosorbent assay and biochemical kit, respectively. Cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays were carried out to assess cell proliferation. Immunofluorescence was used to detect the expression of CD68 and Vimentin in primary synovial fibroblasts (SFs). Haematoxylin and eosin staining and Safranin O/Fast green were used to evaluate the pathological damage of synovial and cartilage tissue in rats. TRIM52 was upregulated in the synovial tissue and SFs in patients with TMJOA. Interleukin (IL)-1β treatment upregulated TRIM52 expression in TMJOA SFs and normal SF (NSF), promoting cell proliferation, inflammatory response and oxidative stress in NSF, SFs. Silence of TRIM52 relieved the cell proliferation, inflammatory response and oxidative stress induced by IL-1β in SFs, while overexpression of TRIM52 enhanced IL-1β induction. Meanwhile, IL-1β induction activated toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB pathway, which was augmented by upregulation of TRIM52 in NSF, and was attenuated by TRIM52 knockdown in SFs. Besides, pyrrolidinedithiocarbamic acid ameliorated IL-1β-induced proliferation and inflammatory response by inhibiting TLR4/NF-κB signalling. Meanwhile, TRIM52 knockdown inhibited cell proliferation, oxidative stress and inflammatory response in IL-1β-induced SFs through downregulation of TLR4. TRIM52 promoted cell proliferation, inflammatory response, and oxidative stress in IL-1β-induced SFs. The above functions were mediated by the activation of TLR4/NF- κB signal pathway.

Macrophage Activation in Synovitis and Osteoarthritis of Temporomandibular Joint and Its Relationship with the Progression of Synovitis and Bone Remodeling

This study investigates the regulatory mechanisms of synovial macrophages and their polarization in the progression of temporomandibular joint osteoarthritis (TMJOA). Macrophage depletion models were established by intra-articular injection of clodronate liposomes and unloaded liposomes. TMJOA was induced by intra-articular injection of 50 μL Complete Freund's Adjuvant and the surgery of disc perforation. The contralateral joint was used as the control group. The expression of F4/80, CD86, and CD206 in the synovium was detected by immunofluorescence staining analysis. Hematoxylin and eosin staining and TMJOA synovial score were detected to show the synovial changes in rat joints after TMJOA induction and macrophage depletion. Changes in rat cartilage after TMJOA induction and macrophage depletion were shown by safranin fast green staining. The bone-related parameters of rats' joints were evaluated by micro-computed tomography analysis. The TMJOA model induced by Complete Freund's Adjuvant injection and disc perforation aggravated synovial hyperplasia and showed a significant up-regulation of expression of F4/80-, CD86-, and CD206-positive cells. F4/80, CD86, and CD206 staining levels were significantly decreased in macrophage depletion rats, whereas the synovitis score further increased and cartilage and subchondral bone destruction was slightly aggravated. Macrophages were crucially involved in the progression of TMJOA, and macrophage depletion in TMJOA synoviocytes promoted synovitis and cartilage destruction.

Circular RNA CircACAP2 regulates temporomandibular joint osteoarthritis via miR-21-5p/PLAG1 axis.

The pathogenesis of temporomandibular joint osteoarthritis (TMJOA) remains not fully understood. Our previous studies demonstrated that miR-21-5p may participate in the TMJOA development and the interaction between circRNA-ACAP2 (CircACAP2) and miR-21-5p. Our present study aimed to explore the biological functions and regulatory mechanisms of CircACAP2 in TMJOA. The differential expression pattern of CircACAP2 in OA and normal tissues or cells was detected. CircACAP2 biological functions experiments were performed in chondrocytes by overexpression and interference techniques. The interaction of CircACAP2 with miR-21-5p and downstream target mRNA, polymorphic adenoma gene 1 (PLAG1), was predicted by bioinformatic databases and then demonstrated by dual-luciferase reporter assay. The biological role of CircACAP2 in TMJOA was investigated and validated in a mouse model. The expression level of CircACAP2 was markedly reduced in OA cartilage and directly related to chondrocyte proliferation and apoptosis as well as ECM metabolism in the cartilage. CircACAP2 functioned in chondrocytes via targeting miR-21-5p and PLAG1. Overexpressing of CircACAP2 alleviated TMJOA in mouse models. The present study unveiled that CircACAP2/miR-21-5p/PLAG1 axis may play an important regulatory role in TMJOA progression, which may highlight a potentially effective intervention and therapeutic strategy for the treatment of TMJOA.

Plumbagin alleviates temporomandibular joint osteoarthritis progression by inhibiting chondrocyte ferroptosis via the MAPK signaling pathways.

The acceleration of osteoarthritis (OA) development by chondrocytes undergoing ferroptosis has been observed. Plumbagin (PLB), known for its potent antioxidant and anti-inflammatory properties, has demonstrated promising potential in the treatment of OA. However, it remains unclear whether PLB can impede the progression of temporomandibular joint osteoarthritis (TMJOA) through the regulation of ferroptosis. The study aims to investigate the impact of ferroptosis on TMJOA and assess the ability of PLB to modulate the inhibitory effects of ferroptosis on TMJOA. The study utilized an in vivo rat model of unilateral anterior crossbite (UAC)-induced TMJOA and an in vitro study of chondrocytes exposed to H2O2 to create an OA microenvironment. Various experiments including cell viability assessment, quantitative RT-PCR, western blot analysis, histology, and immunofluorescence were conducted to examine the impact of ferroptosis on TMJOA and evaluate the potential of PLB to mitigate the inhibitory effects of ferroptosis on TMJOA. Additionally, RNA-seq and bioinformatics analysis were performed to investigate the underlying mechanism by which PLB regulates ferroptosis in TMJOA. Fer-1 demonstrated its potential in mitigating the advancement of TMJOA through its inhibitory effects on ferroptosis and matrix degradation in chondrocytes, thereby substantiating the role of ferroptosis in the pathogenesis of TMJOA. Furthermore, the observed protective impact of PLB on cartilage implied that PLB can modulate the inhibition of ferroptosis in TMJOA by regulating the MAPK signaling pathways. PLB alleviates TMJOA progression by suppressing chondrocyte ferroptosis via MAPK pathways, indicating PLB to be a potential therapeutic strategy for TMJOA.

Abnormal mechanical stress induced chondrocyte senescence by YAP loss-mediated METTL3 upregulation.

Abnormal mechanical stress is the pivotal risk factor of temporomandibular joint osteoarthritis (TMJOA). This study investigated the pathogenic mechanism by which abnormal mechanical stress induced chondrocyte senescence. Cellular senescence was investigated in the rodent model of unilateral anterior crossbite and in the chondrocytes subjected to mechanical overloading invitro. The effects of Yes-associated protein (YAP) in chondrocyte senescence and its correlation with methyltransferase-like 3 (METTL3) and N6-methyladenosine (m6A) modification were evaluated. The role of m6A modification in chondrocyte senescence was determined. The therapeutic effects of m6A inhibition in TMJOA were investigated. Senescent chondrocytes were accumulated in the mechanically induced TMJOA lesions in rats and mechanical overloading could trigger chondrocyte senescence invitro. This mechanical stress-induced cellular senescence was revealed to be mediated by YAP deficiency that promoted METTL3-dependent m6A modification. Moreover, inhibition of m6A modification rescued chondrocyte senescence invitro and invivo, and suppressed TMJOA progression in rats. This study uncovered the underlying mechanism of mechanically induced senescence in TMJOA from the perspective of epitranscriptomics and revealed the therapeutic potential of m6A inhibition in TMJOA.

Pyroptosis of chondrocytes activated by synovial inflammation accelerates TMJ osteoarthritis cartilage degeneration via ROS/NLRP3 signaling

ObjectivesSynovial inflammation and chondrocyte death have been widely acknowledged as key contributors to the pathological progression of temporomandibular joint osteoarthritis (TMJ-OA), a degenerative joint disease currently lacking definitive treatments. This study aims to understand the regulatory role of chondrocyte pyroptosis in condylar cartilage degradation during TMJ-OA. MethodsThe levels of cytokines, cartilage degeneration markers, and pyroptotic biomarkers in the synovium and synovial fluid of temporomandibular disorders (TMD) patients were examined. The synovitis, cartilage degradation, and chondrocyte pyroptosis in wild-type and alpha-kinase 1 (ALPK1)-deficient TMJ-OA mice were then compared following monosodium iodoacetate (MIA) induction. Subsequently, we investigated the downstream mechanisms of cytokines- or macrophage supernatants-induced metabolic disorders and pyroptosis in chondrocytes using primary TMJ chondrocytes and ATDC5 chondrocyte cultures. ResultsWe found a positive correlation between pyroptotic biomarkers and cartilage degradation mediators and cytokines in the synovial fluid of TMD patients. MIA-induced TMJ-OA mice demonstrated significant synovitis, cartilage degradation, and chondrocyte pyroptosis, which were mitigated in ALPK1-deficient TMJ-OA mice, inflammation-restrained mice. Ex-vivo study revealed the contribution of reactive oxygen species (ROS) to inflammation-irritated macrophage supernatants-induced pyroptosis and metabolic disorders in chondrocytes. Targeting NOD-like receptor protein 3 (NLRP3) alleviated cytokines- or ROS-induced pyroptosis and metabolic disorders in chondrocytes by inhibiting caspase-1 activation and interleukin-1β (IL-1β) secretion. ConclusionOur findings offer novel insight into the role of synovial inflammation-induced chondrocyte pyroptosis in promoting cartilage degradation during TMJ-OA via the ROS and NLRP3 signaling pathway.

MMP-13 Regulates Matrix Binding and Internalization of NG2/CSGP4 in Mandibular Fibrochondrocytes

Objective: Post-traumatic osteoarthritis (OA) is associated with cartilage degradation resulting in changes in cell-matrix mediated signaling after injury. Matrix metalloproteinase-13 (MMP13) is a critical enzyme in this process, degrading the extracellular matrix and modifying receptors. One of the receptors altered by MMP13 is a transmembrane proteoglycan, Neuron Glial antigen 2 (NG2/CSPG4). In the temporomandibular joint (TMJ), NG2/CSPG4 is membrane bound and binds with type VI collagen. During OA, the NG2/CSPG4 intracellular domain is internalized into the cytosol. While NG2/CSPG4 is a known substrate for MMP13, there are important gaps in knowledge related to how MMP-13 modifies NG2/CSPG4 after injury. The hypothesis of this study is that MMP13 regulates NG2/CSPG4 interactions with the extracellular matrix, the internalization of the intracellular domain, and key cartilage signaling pathways during mechanical loading and OA progression. Methods: To characterize MMP13 and NG2/CSPG4 levels during the progression of TMJ OA, we used a surgical destabilization preclinical murine model (approved under UIC ACC #20-068) and compared them with clinical samples from human patients receiving total joint replacement for advanced stage TMJ OA (approved under UIC IRB #2017-0033). To mechanistically interrogate MMP-13-NG2/CSPG4, we cultured primary mandibular fibrochondrocytes and treated them with an MMP13 inhibitor (CAS 544678-85-5) at 15 nM for 24 hours or vehicle control. To evaluate matrix binding, we performed immunocytochemistry using an antibody against the NG2/CSPG4 ectodomain on decellularized coverslips. To mechanically load samples, cell-agarose-collagen scaffolds received a constrained static compression treatment for 2 hours at 2.5 N with and without the MMP13. Gene expression changes were quantified using RT-qPCR and protein changes were quantified with western blot. Data were evaluated using a one-way ANOVA. Results: Our results illustrate that MMP13 is present in preclinical and clinical samples a spatiotemporally consistent pattern with NG2/CSPG4 internalization. In vitro, the inhibition of MMP13 impairs the ability of the NG2/CSPG4 ectodomain to be retained in the extracellular matrix. Mechanical loading of the cells activates MMP13, leading to clearance of membrane associated NG2/CSPG4. Inhibition of MMP13 during mechanical loading promotes the accumulation of membrane associated NG2/CSPG4 and a decrease in internalized NG2/CSPG4 (n = 4; p < 0.05). MMP13 did not cause the complete proteolytic degradation of the ectodomain. This mechanically sensitive MMP-13-NG2/CSPG4 axis regulates the expression of key mineralization and OA genes including BMP2 and PTHrP (n = 4; p < 0.05). Conclusions: Together, these findings indicate that MMP13 mediated cleavage of NG2/CSPG4 potentiates the internalization of the intracellular domain and regulates genes implicated in the mineralization signaling axis during growth and degenerative diseases such as OA. Funding support from NIH/NIDCR 1R01DE029835-01. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Loss of RAP2A Aggravates Cartilage Degradation in TMJOA via YAP Signaling

Abnormal stress loading has been considered a major contributor to the initiation of temporomandibular joint osteoarthritis (TMJOA), but studies to date have not identified a functional molecule that transforms physical stress into biological or biochemical signaling in chondrocytes in response to excessive mechanical stress. Ras-related protein Rap-2a (RAP2A) is reportedly a molecular switch that relays extracellular matrix rigidity signals via the Hippo/Yes-associated protein (YAP) pathway. In the present study, RAP2A diminished with cartilage degradation in unilateral anterior crossbite-induced TMJOA mice, as well as severe cartilage matrix degeneration and TMJOA formation in Cre-loxP–mediated conditional RAP2A knockout mice. RAP2A in chondrocytes regulated the Hippo/YAP pathway directly in response to matrix stiffness, and RAP2A/Hippo/YAP was critical for a chondrocyte phenotype switch and matrix synthesis function. Loss of RAP2A impaired cartilage homeostasis and altered chondrocyte phenotype via Hippo/YAP/SRY-box transcription factor 9 signaling. It may be possible to generate therapeutic strategies using RAP2A or YAP to attenuate the TMJOA pathological process at an early stage. This is the first study to reveal the molecular function of RAP2A in TMJOA progression as a mechanotransduction molecule in condylar chondrocytes.

NG2/CSPG4 regulates cartilage degeneration during TMJ osteoarthritis.

Changes in the mechanical homeostasis of the temporomandibular joint (TMJ) can lead to the initiation and progression of degenerative arthropathies such as osteoarthritis (OA). Cells sense and engage with their mechanical microenvironment through interactions with the extracellular matrix. In the mandibular condylar cartilage, the pericellular microenvironment is composed of type VI collagen. NG2/CSPG4 is a transmembrane proteoglycan that binds with type VI collagen, and has been implicated in the cell stress response through mechanical loading-sensitive signaling networks including ERK 1/2. The objective of this study is to define the role of NG2/CSPG4 in the initiation and progression of TMJ OA and to determine if NG2/CSPG4 engages ERK 1/2 in a mechanical loading dependent manner. In vivo, we induced TMJ OA in control and NG2/CSPG4 knockout mice using a surgical destabilization approach. In control mice, NG2/CSPG4 is depleted during the early stages of TMJ OA and NG2/CSPG4 knockout mice have more severe cartilage degeneration, elevated expression of key OA proteases, and suppression of OA matrix synthesis genes. In vitro, we characterized the transcriptome and protein from control and NG2/CSPG4 knockout cells and found significant dysregulation of the ERK 1/2 signaling axis. To characterize the mechanobiological response of NG2/CSPG4, we applied mechanical loads on cell-agarose-collagen scaffolds using a compression bioreactor and illustrate that NG2/CSPG4 knockout cells fail to mechanically activate ERK 1/2 and are associated with changes in the expression of the same key OA biomarkers measured in vivo. Together, these findings implicate NG2/CSPG4 in the mechanical homeostasis of TMJ cartilage and in the progression of degenerative arthropathies including OA.

Wnt/β-catenin signaling pathway is activated in the progress of mandibular condylar cartilage degeneration and subchondral bone loss induced by overloaded functional orthopedic force (OFOF)

ObjectiveTo explore the role of Wnt/β-catenin signaling pathway in the pathogenesis and progression of temporomandibular joint osteoarthritis (TMJ OA) caused by overloaded force. Materials and methodsWe generated a rat model of forward mandibular extension device to induce TMJ OA by overloaded force. Condylar cartilage samples were collected at 2wk, 4wk, and 8wk after appliances were installed. Changes of the condylar cartilage and subchondral bone were evaluated by hematoxylin and eosin (HE), Safranin O and Fast Green staining (SO&FG), micro-CT, tartrate resistant acid phosphatase (TRAP) staining. The expression levels of β-catenin, COL-2, MMP3 and sclerostin (SOST) were detected by immunohistochemistry (IHC) and PCR. ResultsHE, SO&FG, micro-CT, OARSI and Mankin scores showed that the condyle cartilage layer was significantly thinner and proteoglycan loss in the overloded group. TRAP staining exhibited that the number of positive osteoclasts increased and OPG level decreased in the overload group. IHC, PCR showed that the expression of COL2 and SOST decreased, while MMP3 and β-catenin increased in the overload group. ConclusionWnt/β-catenin signaling pathway is activated in the progress of mandibular condylar cartilage degeneration and subchondral bone loss induced by overloaded functional orthopedic force (OFOF)

MicroRNA-26b regulates BMSC osteogenic differentiation of TMJ subchondral bone through β-catenin in osteoarthritis.

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.

Expressions of parathyroid hormone-related protein (PTHrP) and parathyroid hormone receptor-1 (PTH1R) in the condylar cartilage of temporomandibular joint modulated by occlusal elevation

Parathyroid hormone-related protein (PTHrP) is an important regulatory factor in the growth, development and remodeling of bone or cartilage, and acts through its sole receptor, parathyroid hormone receptor-1 (PTH1R). The present study aimed to research the expression changes of PTHrP, PTH1R and other relevant factors in condylar cartilage during the progress of temporomandibular joint osteoarthritis (TMJOA). The animal model of TMJOA was constructed by the "resin-modified method", and Sprague Dawley (SD) rats were euthanized at 2 weeks, 4 weeks, 6 weeks and 8 weeks after occlusal elevation. The histological changes of condylar cartilage were observed by X-ray, hematoxylin-eosin (HE) and safranine O-fast green (SO-FG) staining. The expressions of PTHrP, PTH1R, Ki67, Collagen II (Col II), Collagen X (Col X) and Caspase 3 in each group were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). TMJOA progression was time-dependent. In the experimental group, PTHrP expression was unimodal with a peak at 4 weeks, but PTH1R expression showed a decreasing trend. From 2 weeks to 8 weeks in the experimental group, Col X expression rather than Caspase 3 expression was negatively related to PTHrP's, which has no positive relation to Ki67 or Col II. These results demonstrated abnormal occlusal load may be an important pathogenic factor of TMJOA. It may be one of the reasons of TMJOA progression that PTHrP can't play an effective role due to the low expression of PTH1R. PTHrP may be a direct factor regulating the hypertrophic differentiation of chondrocytes, but it does not directly regulate the proliferation and apoptosis of chondrocytes, and the realization of both regulatory effects may depend on the inhibition of hypertrophic differentiation.

Mechanical stress reduces secreted frizzled-related protein expression and promotes temporomandibular joint osteoarthritis via Wnt/β-catenin signaling.

Mechanical stress overload in the temporomandibular joint (TMJ) is an important cause of TMJ osteoarthritis (TMJOA). Whether secreted frizzled-related proteins (SFRPs) play important roles in the development of mechanical stress-induced TMJOA remains controversial. In this study, we investigated the roles of the Wnt/β-catenin signaling and SFRPs in the progression of mechanical stress-induced TMJOA. We investigated the progression of mechanical stress-induced TMJOA using an in vivo model via modified increased occlusal vertical dimension (iOVD) malocclusion and an in vitro model in which isolated chondrocytes were subjected to mechanical stress. The effects of inhibition of Wnt/β-catenin signal on TMJOA induced by mechanical stress were studied by in vitro drug added and in vivo intra-articular injection of XAV-939. TMJOA progression, Wnt/β-catenin signaling and SFRPs was assessed by Cone beam computed tomography (CBCT) analysis, histochemical and immunohistochemical (IHC) staining, quantitative real-time PCR (qRT-PCR), Western blotting (WB), and immunofluorescence (IF) staining. Our in vivo results showed that iOVD-induced mechanical stress in the TMJ disrupted mandible growth, induced OA-like changes in TMJ cartilage, and increased OA-related cytokine expression. In addition, iOVD activated Wnt/β-catenin signaling and suppressed Sfrp1, Sfrp3, and Sfrp4 expression in condylar cartilage. Moreover, our in vitro study showed that stress disrupted homeostasis, activated Wnt/β-catenin signaling and inhibited SFRP3 and SFRP4 expression in chondrocytes. Suppression of Wnt/β-catenin signaling with XAV-939 promoted SFRP3 and SFRP4 expression and rescued mechanical stress-induced cartilage degeneration in vivo and in vitro. Our work suggests that mechanical stress reduces SFRPs expression both in vivo and in vitro and promotes TMJOA via Wnt/β-catenin signaling. Suppression of Wnt/β-catenin signaling promotes SFRPs expression, especially SFRP3 and SFRP4 expression, and rescues mechanical stress-induced cartilage degeneration. Wnt/β-catenin signaling and SFRPs may represent potential therapeutic targets for TMJOA.

MiR-132-3p participates in the pathological mechanism of temporomandibular joint osteoarthritis by targeting PTEN

ObjectiveThis study aimed to investigate the role of miR-132-3p in the progression of temporomandibular joint osteoarthritis (TMJOA) and its potential pathological mechanism. DesignA TMJOA model was established using six rats via the unilateral anterior crossbite method. The differential expression of miR-132-3p in the TMJOA (n = 6) and control groups (n = 6) was detected via miRNA sequencing and verified via PCR. The chondrocytes in the condylar cartilage of the temporomandibular joint were cultured and stimulated with IL-1β to simulate TMJOA in vitro. The changes in the proliferation, apoptosis, inflammation and extracellular matrix of these chondrocytes were detected after the upregulation of miR-132-3p expression. The targeted relationship of miR-132-3p and PTEN in TMJOA was verified, and rescue experiments were conducted via co-upregulation of the expression of both miR-132-3p and PTEN. ResultsCompared with that in the control group, miR-132-3p expression was lower in the cartilage tissues of TMJOA rats and IL-1β-induced TMJ chondrocytes. After upregulating the expression of miR-132-3p, the cell proliferation activity and expression levels of aggrecan and type II collagen of IL-1β-induced TMJ chondrocytes were increased, and the apoptosis rate and levels of inflammatory factors were decreased. miR-132-3p can regulate PTEN expression in a targeted manner, and upregulating PTEN expression could reverse the influences of the upregulation of miR-132-3p expression on TMJOA cells. ConclusionmiR-132-3p is less expressed in TMJOA, and it regulates the proliferation, extracellular matrix, and inflammatory response of TMJOA chondrocytes and participates in TMJOA progression by targeting PTEN.

Antisense Oligonucleotide-Based Therapy on miR-181a-5p Alleviates Cartilage Degradation of Temporomandibular Joint Osteoarthritis via Promoting SIRT1.

Temporomandibular joint osteoarthritis (TMJOA) condylar cartilage degeneration and abnormal subchondral bone pathological remodeling induce pain and joint dysfunction, and cartilage degeneration is considered irreversible. Very few therapeutic approaches are administrated in practice. Nucleotides have demonstrated considerable potential as a next-generation medication, and they have been applied in several models of osteoarthritis. There is a need to establish an effective protocol for TMJOA gene therapy. In the current study unilateral anterior crossbite (UAC) surgery was used to simulate mechanical stress-induced TMJOA in mice. Degeneration of condylar cartilage and destruction of subchondral bone were observed in damaged joints, and miR-181a-5p was elevated in chondrocytes. Intra-articular injection of miR-181a-5p antisense oligonucleotide (ASO) could reduce the cartilage damage and alleviate UAC-induced TMJOA progression, but it did not restore injured subchondral bone. Mechanically, miR-181a-5p evidently targeted the 3’ untranslated region of Sirt1 directly, resulting in inhibition of silent information regulator 1 expression and promoting apoptosis by elevating p53-dependent signaling, indicating that miR181a-5p ASO promoted chondrocyte survival. The present study suggests that ASO-based gene therapy may be an effective TMJOA treatment.