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.

Glycyrrhizin regulates antioxidation through Nrf2 signaling pathway in rat temporomandibular joint osteoarthritis.

BACKGROUND: Regulation of redox homeostasis could reduce osteoarthritis severity and limit disease progression, while glycyrrhizin (GL) shows great antioxidant and anti-inflammatory capacity. OBJECTIVE: The aim of this study was to investigate the role of GL on oxidative stress and the potential regulatory mechanism in rat temporomandibular joint (TMJ) chondrocytes under oxidative stress, and investigate the effect of GL in the rat temporomandibular joint osteoarthritis (TMJOA) model. METHODS: Rat TMJ chondrocytes were cultured in oxidative stress with different doses of GL. The effect of glycyrrhizin on the nuclear factor-erythroid 2-related factor 2 (Nrf2) in oxidative stress was evaluated by western blot and immunofluorescence staining. A rat model of TMJOA was treated with GL. Micro-computed tomography, histological and immunohistochemical analysis were used to assess the pathological change of TMJOA. RESULTS: The expression of superoxide dismutase 1 (SOD1), heme oxygenase-1 (HO-1), and peroxiredoxin 6 (PRDX6) were decreased, and intracellular Nrf2 signaling pathway was activated in chondrocytes in oxidative stress. GL upregulates the expression of antioxidants, especially PRDX6, as well as increases Nrf2 expression and nuclear translocation in rat condylar chondrocytes. Administration of GL attenuates condylar bone destruction, cartilage degeneration, and synovitis in rats TMJOA. Meanwhile, GL alleviated oxidative stress and enhanced the antioxidant capacity of TMJOA cartilage. CONCLUSION: This study suggested that GL alleviates rat TMJOA by regulating oxidative stress in condylar cartilage.

Transglutaminase 2 inhibitors attenuate osteoarthritic degeneration of TMJ-osteoarthritis by suppressing NF-κB activation.

BACKGROUND: The temporomandibular joint osteoarthritis (TMJ-OA) is characterized by progressive cartilage degradation, subchondral bone erosion, and chronic pain, leading to articular damage and chewing dysfunction. Studies have shown that interleukin-1ß (IL-1ß) plays a critical role in the development of TMJ-OA. Transglutaminase 2 (TG2) has been identified as a marker of chondrocyte hypertrophy and IL-1ß was able to increase TG2 expression in chondrocytes. Therefore, the aim of this study was to explore the ability of TG2 inhibitors to suppress TMJ-OA progression. METHODS: Firstly, toluidine blue staining, cell counting kit-8 assay, immunocytofluorescent staining and western blot were used to investigate the anti-inflammatory effects of TG2 inhibitors in IL-1ß-stimulated murine chondrocytes and the underlying mechanisms. Afterwards, micro-CT analysis, histological staining, immunohistochemical and immunohistofluorescent staining were used to evaluate the therapeutic efficacy of TG2 inhibitors in monosodium iodoacetate (MIA)-induced TMJ-OA in rats. RESULTS: TG2 inhibitors suppressed the IL-1ß-induced upregulation of COX-2, iNOS, MMP-13, and MMP-3 and reversed the IL-1ß-induced proteoglycan loss in chondrocytes through inhibiting NF-κB activation. Consistently, the MIA-induced upregulation of MMP-13 and MMP-3, and loss of structural integrity of the articular cartilage and subchondral bone were markedly reversed by TG2 inhibitors via inhibiting NF-κB activation. CONCLUSIONS: TG2 inhibitors demonstrated a potent therapeutic efficacy on cartilage and subchondral bone structures of TMJ-OA by reducing inflammation and cartilage degradation through suppressing NF-κB activation.

ALPK1 Accelerates the Pathogenesis of Osteoarthritis by Activating NLRP3 Signaling.

Alpha-kinase 1 (ALPK1), a member of the alpha-kinase family, has been shown to be involved in mediating inflammatory responses and is strongly associated with gout; however, its modulatory role in osteoarthritis (OA) remains unclear. Here, we uncovered elevation of ALPK1 in degraded cartilage of destabilized medial meniscus (DMM) and collagenase-induced osteoarthritis (CIOA), two different mouse OA models induced by mechanical stress or synovitis. Intraarticular administration of recombinant human ALPK1 (rhALPK1) in vivo exacerbated OA pathogenesis in both DMM and CIOA mice, whereas ALPK1 knockout reversed this process. In vitro study demonstrated that ALPK1 aggravates metabolic disturbances in chondrocytes by enhancing the production of NOD-like receptor protein 3 (NLRP3), an inflammasome sensors driving interlukin-1ß (IL-1ß)-mediated inflammatory conditions. Furthermore, the selective inhibition of nuclear factor-κB (NF-κB) or NLRP3 indicates that NLRP3 is a downstream signaling governed by NF-κB in ALPK1-activated chondrocytes. Collectively, these results establish ALPK1 as a novel catabolic regulator of OA pathogenesis, and targeting this signaling may be a promising treatment strategy for OA. © 2022 American Society for Bone and Mineral Research (ASBMR).

Chronic Pain Causes Peripheral and Central Responses in MIA-Induced TMJOA Rats.

Chronic pain is the predominant symptom that drives temporomandibular joint osteoarthritis (TMJOA) patients to seek medical care; however, currently used treatment modalities remain less effective. This study aimed to investigate chronic pain and the peripheral and central responses in monoiodoacetate (MIA)-induced TMJOA rats. First, the appropriate dose of MIA was determined based on pain behavior assessment in rats. Alterations of the condylar structure in TMJOA rats were evaluated by histological staining and micro-computed tomography (micro-CT). Second, the period of TMJOA chronic pain was further explored by assessing the numbers of glial fibrillary acidic protein (GFAP)-positive astrocytes and ionized calcium-binding adaptor molecule 1 (IBA-1)-positive microglia in the trigeminal spinal nucleus (TSN) and performing nonsteroidal anti-inflammatory drug (NSAID) efficacy experiments. Finally, the expression of neurofilament 200 (NF200), calcitonin gene-related peptide (CGRP), and isolectin B4 (IB4) in the trigeminal ganglion (TG) and TSN was assessed by immunofluorescence. MIA at 4 mg/kg was considered an appropriate dose. Gradual MIA-induced alterations of the condylar structure were correlated with temporomandibular joint (TMJ) pain. The numbers of GFAP- and IBA-1-positive cells were increased at 2, 3, and 4 weeks after MIA injection. NSAIDs failed to alleviate pain behavior 10 days after MIA injection. CGRP and IB4 levels in the TG and TSN were upregulated at 2 and 4 weeks. These results suggest that TMJOA-related chronic pain emerged 2 weeks after MIA injection. CGRP- and IB4-positive afferents in both the peripheral and central nervous systems may be involved in MIA-induced TMJOA-related chronic pain in rats.

The expression of Netrin-1 in the MIA-induced osteoarthritic temporomandibular joint in mice.

Subchondral bone degeneration is the main pathological change during temporomandibular joint (TMJ) osteoarthritis (OA) development. Netrin-1, an axon-guiding factor, might play roles in OA development and pain. The purpose of this study was to investigate the expression of Netrin-1 in TMJ OA and its possible role in the progression of TMJ OA and pain. The synovial fluids of temporomandibular joint disorders (TMDs) patients were collected for Netrin-1 by enzyme linked immunosorbent assay (ELISA). TMJ OA model was built by MIA joint injection, and then the von Frey test, hematoxylin & eosin (H&E) staining, toluidine blue (TB) staining, immunohistochemical (IHC) staining and micro-CT were performed. After induction of osteoclast differentiation of raw264.7 cells, immunofluorescence (IF) was used to detect the Netrin-1 and its receptors on osteoclast membrane. The concentration of Netrin-1 increased in the synovial fluid of TMJ OA patients. After MIA injection to TMJ, the head withdrawal threshold (HWT) was significantly decreased. Microscopically, the structural disorder of subchondral bone was the most obvious at the 2nd week after MIA injection. In addition, Netrin-1 expression increased in the subchondral bone at the 2nd week after MIA injection. In vitro, the expressions of Netrin-1 and its receptor Unc5B were upregulated on the osteoclast membrane. Netrin-1 might be an important regulator during bone degeneration and pain in the process of TMJ OA.

Role of osteoclast differentiation in the occurrence of osteoarthritis of temporomandibular joint.

OBJECTIVES: This study aimed to explore the role of osteoclast differentiation in the occurrence of temporomandibular joint osteoarthritis (TMJOA). METHODS: A mouse TMJOA model was constructed. Micro-CT was used to observe the changes in condylar bone during the development of TMJOA. Hematoxylin-eosin (HE) staining was used to observe the histological structure changes of the condyle of TMJOA mice. Tartrate resistant acid phosphatase (TRAP) staining was used to observe the presence of osteoclasts in TMJOA joint tissue. The synovial fluid of patients with TMJ-OA was collected to determine the effect on osteoclast differentiation. RESULTS: Micro-CT revealed that the condyle of the TMJOA group had the most obvious damage in the second and third weeks, and the shape of the condyles also changed in a beak-like manner. HE staining showed that the condyle cartilage and subchondral bone structure of TMJOA mice were disordered in the second week. TRAP tissue staining showed that the number of osteoclasts of the TMJOA group obviously increased in the second week. Results of cell experiments showed that the number of osteoclast differentiation significantly increased after stimulation of synovial fluid from TMJOA patients, and the cell volume increased. CONCLUSIONS: TMJOA animal models and TMJOA patient synovial cell experiments could induce osteoclast differentiation, indicating that osteoclast differentiation plays an important role in TMJOA occurrence.

TLR4 contributes to the damage of cartilage and subchondral bone in discectomy-induced TMJOA mice.

The abundance of inflammatory mediators in injured joint indicates innate immune reactions activated during temporomandibular joint osteoarthritis (TMJOA) progression. Toll-like receptor 4 (TLR4) can mediate innate immune reaction. Herein, we aimed to investigate the expression profile and effect of TLR4 in the cartilage and subchondral bone of the discectomy-induced TMJOA mice. The expression of TLR4 and NFκB p65 in the synovium of TMJOA patients was measured by immunohistochemistry, Western blotting and RT-PCR. H&E and Masson staining were utilized to assess the damage of cartilage and subchondral bone of the discectomy-induced TMJOA mice. A TLR4 inhibitor, TAK-242, was used to assess the effect of TLR4 in the cartilage and subchondral bone of the discectomy-induced TMJOA mice by Safranin O, micro-CT, immunofluorescence and immunohistochemistry. Western blotting was used to quantify the expression and effect of TLR4 in IL-1ß-induced chondrocytes. The expression of TLR4 and NFκB p65 was elevated in the synovium of TMJOA patients, compared with the normal synovium. TLR4 elevated in the damaged cartilage and subchondral bone of discectomy-induced TMJOA mice, and the rate of TLR4 expressing chondrocytes positively correlated with OA score. Intraperitoneal injections of TAK-242 ameliorate the extent of TMJOA. Furthermore, TLR4 promotes the expression of MyD88/NFκB, pro-inflammatory and catabolic mediators in cartilage of discectomy-induced TMJOA. Besides, TLR4 participates in the production of MyD88/NFκB, pro-inflammatory and catabolic mediators in IL-1ß-induced chondrocytes. TLR4 contributes to the damage of cartilage and subchondral bone in discectomy-induced TMJOA mice through activation of MyD88/NFκB and release of pro-inflammatory and catabolic mediators.