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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.

Temporomandibular joint osteoarthritis (TMJOA) is one of the most common diseases causing chronic pain in the oral and maxillofacial region. So far, there are few ways to relieve the pain of TMJOA. Melatonin (MT) has a good analgesic effect in many diseases, including fibromyalgia, neuropathic pain, chronic headache, and burn pain, with very low acute toxicity and side effects. This study was to investigate the role and mechanism of MT in TMJOA chronic pain. In rats TMJOA chronic pain occurred at day 14 after an intra–temporomandibular joint injection of monosodium iodoacetate, which we previously reported. The enzyme-linked immunosorbent assay results showed that MT levels were higher in the synovial fluid from patients and rats with TMJOA as compared with those from control. Fluorescent retrograde tracing (Dil) identified that upregulation of MT type 2 receptor (MT2R) in trigeminal ganglion (TG) neurons innervating rat temporomandibular joints was accompanied by TMJOA chronic pain. Nociceptive behavior as assessed by von Frey and the Rat Grimace Scale demonstrated that exogenous administration of MT relieved chronic pain in TMJOA rats, whereas blocking MT2R with 4P-PDOT reversed the analgesic effect of MT. Immunofluorescence analysis also confirmed that MT inhibited CGRP and IB4 expression of TG neurons, and this inhibition was reversed by administering the MT2R antagonist in TMJOA rats. By using Fluo-3 AM-based calcium imaging in vitro, MT elicited calcium transients in Dil+ TG neurons, which were significantly abolished by 4P-PDOT. Collectively, this study suggested that MT relieves the TMJOA chronic pain of rats through downregulation of sensitized CGRP+ and IB4+ neurons in TG via MT2R. This will be helpful for health care professionals utilizing MT as an option against TMJOA chronic pain.

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‐1β–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‐1β–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.

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-1β (IL-1β) plays a critical role in the development of TMJ-OA. Transglutaminase 2 (TG2) has been identified as a marker of chondrocyte hypertrophy and IL-1β 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.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-1β-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.TG2 inhibitors suppressed the IL-1β-induced upregulation of COX-2, iNOS, MMP-13, and MMP-3 and reversed the IL-1β-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.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.

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 content of Netrin-1 in the synovial fluid of temporomandibular joint disorders (TMDs) patients was detected by ELISA. After injection of MIA into the TMJ animal model, hematoxylin & eosin (H&E) staining, toluidine blue (TB) staining and micro-CT were performed to detect the change of TMJ. Netrin-1 expression in the condyle was detected by immunohistochemical (IHC) staining. The pain experience was examined by the von Frey test in animal model. The concentration of Netrin-1 increased in the synovial fluid of TMJ OA patients with the VAS scores increased. In the TMJ OA model, the structural disorder of subchondral bone was the most obvious at the 2nd week. In addition, the head withdrawal threshold (HWT) was significantly decreased in the TMJ OA model. Netrin-1 expression increased in the subchondral bone at the 2nd week after MIA injection in the mouse TMJ. Cultivate raw264.7 cells and induce them to differentiate into osteoclasts. Netrin-1 and its receptor Unc5B are detected to be expressed on the osteoclast membrane. Thus, Netrin-1 might be a regulator during degeneration and pain in the process of TMJ OA.

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.

Background Chronic pain is the prevalent symptom that drives temporomandibular joint osteoarthritis (TMJOA) patients to ask for medical care, yet present alleviator remain less effective. This study aimed to investigate the actual TMJOA related chronic pain and the peripheral and central response in a TMJOA animal model. Methods This study firstly determined appropriate MIA dose based on pain behavior assessment with automated electronic von frey in rats. TMJOA pain correlated condylar structure alteration was evaluated by histological staining and Micro-CT. Then, the period of TMJOA chronic pain was further explored by assessing the alteration of glial fibrillary acidic protein (GFAP) positive astrocytes and ionized calcium binding adaptor molecule 1 (Iba1) positive microglia numbers in trigeminal spinal nucleus (TSN) and carrying out non-steroidal anti-inflammatory drugs (NSAIDS) pharmacological efficacy experiment. Finally, expression of neurofilament 200 (NF200), calcitonin gene-related peptide (CGRP), isolectin B4 (IB4) in trigeminal ganglion (TG) and TSN was detected by immunofluorescence. Results 1 mg/50 µl of MIA was considered as an appropriate dose. MIA induced gradual alteration of condylar structure correlated to TMJ mechanical allodynia. GFAP and IBA-1 positive cell numbers upregulated on 2, 3, 4 weeks after MIA injection. NSAIDS pharmacological efficacy disappeared on 10 days post MIA injection. Up-regulation of CGRP and IB4 was found in TG and TSN on 2 and 4 weeks, while expression of NF200 remained unchanged. Conclusion MIA induced TMJOA related chronic pain period emerges on 2 weeks after MIA injection. CGRP, IB4 positive afferent in both peripheral and central nervous system may involve in TMJOA related chronic pain in rats.

Synovitis contributes to temporomandibular joint (TMJ) pain, nevertheless, the detailed nociceptive mechanism remains unclear. In this study, a rat model of TMJ synovitis was induced by intra-articular injection with complete Freund’s adjuvant (CFA). After CFA-induced synovitis, pain behaviors were observed. Then, TMJ, trigeminal ganglion, and trigeminal nucleus caudalis (TNC) tissues were collected, and immunohistochemistry was used to detect the expression of substance P (SP) and protein gene product 9.5 (PGP9.5) in the synovium tissue. Furthermore, the gene expression level of SP and PGP9.5 in synovium was detected by reverse transcription-polymerase chain reaction (RT-PCR). Afterwards, the expression of SP in the trigeminal ganglion and TNC and c-fos in the TNC was detected by immunohistochemistry. Compared with the control group, the expression of SP and PGP9.5 nerve fibers density and gene levels of them in the synovium tissue were significantly increased in CFA-induced TMJ synovitis rats. Similarly, SP expression in the trigeminal ganglion and TNC, and c-fos expression in the TNC were also obviously increased in CFA-induced TMJ synovitis rats. Collectively, CFA-induced rat TMJ synovitis resulted in obvious pain. This nociceptive reaction could be attributed to the augmented quantity of SP and PGP9.5 positive-stained nerve fibers distributed in the inflammatory synovium as well as enhanced SP expression in the trigeminal ganglion and TNC tissue. c-fos expression in the rat TNC illustrates CFA-induced TMJ synovitis can evoke the acute pain.