The crucial role of NR2A mediating the activation of satellite glial cells in the trigeminal ganglion contributes to orofacial inflammatory pain during TMJ inflammation.

Temporomandibular joint inflammatory diseases are a significant subtype of temporomandibular disorders (TMD) characterized by inflammatory pain in the orofacial area. The N-methyl-D-aspartate receptor (NMDAR), specifically the NR2A subtype, was crucial in neuropathic pain. However, the exact role of NR2A in inflammatory pain in the TMJ and the molecular and cellular mechanisms mediating peripheral sensitization in the trigeminal ganglion (TG) remain unclear. This study utilized male and female mice to induce the TMJOA model by injecting Complete Freund's adjuvant (CFA) into the TMJ and achieve conditional knockout (CKO) of NR2A in the TG using Cre/Loxp technology. The Von-Frey filament test results showed that CFA-induced orofacial pain with reduced mechanical withdrawal threshold (MWT), which was not developed in NR2A CKO mice. Additionally, the up-regulation of interleukin (IL)-1ß, IL-6, and nerve growth factor (NGF) in the TG induced by CFA did not occur by NR2A deficiency. In vitro, NMDA activated satellite glial cells (SGCs) with high expression of glial fibrillary acidic protein (GFAP), and both NMDA and LPS led to increased IL-1ß, IL-6, and NGF in SGCs. NR2A deficiency reduced these stimulating effects of NMDA and LPS. The regulation of IL-1ß involved the p38, Protein Kinase A (PKA), and Protein Kinase C (PKC) pathways, while IL-6 signaling relied on PKA and PKC pathways. NGF regulation was primarily through the p38 pathway. This study highlighted NR2A's crucial role in the TG peripheral sensitization during TMJ inflammation by mediating ILs and NGF, suggesting potential targets for orofacial inflammatory pain management.

Adipose tissue-derived extracellular vesicles aggravate temporomandibular joint osteoarthritis associated with obesity.

INTRODUCTION: Temporomandibular joint osteoarthritis (TMJ OA) is a major disease that affects maxillofacial health and is characterised by cartilage degeneration and subchondral bone remodelling. Obesity is associated with the exacerbation of pathological manifestations of TMJ OA. However, the underlying mechanism between adipose tissue and the TMJ axis remains limited. OBJECTIVES: To evaluate the effects of obesity and the adipose tissue on the development of TMJ OA. METHODS: The obesity-related metabolic changes in TMJ OA patients were detected by physical signs and plasma metabolites. The effects of adipose tissue-derived EVs (Ad-EVs) on TMJ OA was investigated through histological and cytological experiments as well as gene editing technology. Alterations of Ad-EVs in obese state were identified by microRNA-seq analysis and the mechanism by which EVs affect TMJ OA was explored in vitro and in vivo. RESULTS: Obesity and the related metabolic changes were important influencing factors for TMJ OA. Ad-EVs from obese mice induced marked chondrocyte apoptosis, cartilage matrix degradation and subchondral bone remodelling, which exacerbated the development of TMJ OA. Depletion of Ad-EVs secretion by knocking out the geranylgeranyl diphosphate synthase (Ggpps) gene in adipose tissue significantly inhibited the obesity-induced aggravation of TMJ OA. MiR-3074-5p played an important role in this process . CONCLUSIONS: Our work unveils an unknown link between obese adipose tissue and TMJ OA. Targeting the Ad-EVs and the miR-3074-5p may represent a promising therapeutic strategy for obesity-related TMJ OA. KEY POINTS: High-fat-diet-induced obesity aggravate the progression of TMJ OA in mice. Obese adipose tissue participates in cartilage damage through the altered miRNA in extracellular vesicles. Inhibition of miR-3074-5p/SMAD4 pathway in chondrocyte alleviated the effect of HFD-EVs on TMJ OA.

Association between Estrogen Levels and Temporomandibular Disorders: An Updated Systematic Review.

The aim of this systematic review is to evaluate the impact of estrogen levels on the occurrence of temporomandibular disorders (TMDs) in humans. Searches were conducted in the same databases as follows: PubMed, the Cochrane Collaboration database, and the Scopus database. In accordance with the MeSH database and previous work, the following keywords were used: 'estrogens' and 'temporomandibular joint disorders'. Twelve studies were included in the review and were assessed for the quality of evidence. Estrogen levels are associated with pain modulation in the temporomandibular joint and the entire orofacial region. There is insufficient evidence to either confirm or refute the influence of estrogen on the occurrence of TMDs. The study was registered under the identifier: 10.17605/OSF.IO/BC7QF.

Syndecan-4 inhibition attenuates cartilage degeneration in temporomandibular joint osteoarthritis.

BACKGROUND: Syndecan 4 (SDC4), a type I transmembrane proteoglycan, serves as a critical link between chondrocytes and the extracellular matrix. OBJECTIVE: This study aimed to explore the role of SDC4 in cartilage degeneration of temporomandibular joint osteoathritis (TMJOA). METHODS: Condylar chondrocytes were stimulated with varying concentrations of recombinant rat interleukin-1ß (rrIL-1ß) and SDC4 small interfering RNA (si-SDC4). Anti-SDC4 ectodomain-specific antibodies or IgG were intra-articularly administrated in a TMJOA model rats. SDC4 conditional knockout (SDC4-cKO) and Sdc4flox/flox mice were induced TMJOA. Cartilage degeneration was assessed using haematoxylin & eosin (H&E) and safranin O (SO) staining. Protein levels of SDC4, matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase with a thrombospondin motifs 5 (ADAMTS5), tumour necrosis factor α (TNFα), type II collagen (Col-II), aggrecan (ACAN), cleaved caspase 3 (CASP3), Ki67 and related pathways in condylar cartilage were evaluated by immunohistochemical (IHC) staining or western blot assays. RESULTS: SDC4 expression was evidently increased in MIA-model animals compared to control groups. rrIL-1ß stimulation increased the expression of SDC4, MMP3 and ADAMTS5 expression in chondrocytes, while decreasing the expression of Col-II. These effects were reversed by si-SDC4 in vitro. In vivo, SDC4 blockade reduced the death of chondrocytes and the loss of cartilage matrix, which was evidenced by increased expression of Col-II and ACAN, and a decrease in SDC4, MMP13 and cleaved-CASP3-positive cells. Furthermore, the protein levels of ACAN and Ki67 were elevated, and the ERK1/2 and P38 signalling pathways were activated following SDC4 inhibition. CONCLUSIONS: SDC4 inhibition significantly ameliorates condylar cartilage degeneration, which was mediated, at least partly, through P38 and ERK1/2 signalling. Inhibition of SDC4 may be of great value for the treatment of TMJOA.

Synovial microenvironment in temporomandibular joint osteoarthritis: crosstalk with chondrocytes and potential therapeutic targets.

Temporomandibular joint osteoarthritis (TMJOA) is considered to be a low-grade inflammatory disease involving multiple joint tissues. The crosstalk between synovium and cartilage plays an important role in TMJOA. Synovial cells are a group of heterogeneous cells and synovial microenvironment is mainly composed of synovial fibroblasts (SF) and synovial macrophages. In TMJOA, SF and synovial macrophages release a large number of inflammatory cytokines and extracellular vesicles and promote cartilage destruction. Cartilage wear particles stimulate SF proliferation and macrophages activation and exacerbate synovitis. In TMJOA, chondrocytes and synovial cells exhibit increased glycolytic activity and lactate secretion, leading to impaired chondrocyte matrix synthesis. Additionally, the synovium contains mesenchymal stem cells, which are the seed cells for cartilage repair in TMJOA. Co-culture of chondrocytes and synovial mesenchymal stem cells enhances the chondrogenic differentiation of stem cells. This review discusses the pathological changes of synovium in TMJOA, the means of crosstalk between synovium and cartilage, and their influence on each other. Based on the crosstalk between synovium and cartilage in TMJOA, we illustrate the treatment strategies for improving synovial microenvironment, including reducing cell adhesion, utilizing extracellular vesicles to deliver biomolecules, regulating cellular metabolism and targeting inflammatory cytokines.

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 in vivo 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.

Deletion of vitamin D receptor exacerbated temporomandibular joint pathological changes under abnormal mechanical stimulation.

AIMS: Temporomandibular disorder can cause degenerative pathological changes by aseptic inflammation in the temporomandibular joint (TMJ). Vitamin D (VD) is known for maintaining calcium homeostasis, and recent studies indicated that VD and the vitamin D receptor (VDR) are important in inflammatory responses. In this study, we explored the anti-inflammatory effect of VD-VDR signaling axis in TMJ pathological degeneration. MAIN METHODS: Mice ablated for Vdr (Vdr-/-res) were fed with a rescue diet to avoid hypocalcemia. With abnormal mechanical stimulation, unilateral anterior crossbite (UAC) induced temporomandibular disorders in mice. Histological staining, immunohistochemistry staining, and micro-CT analysis were performed to evaluate TMJ pathological changes. To identify the mechanisms in the aseptic inflammatory process, in vitro experiments were conducted on wild-type (WT) and Vdr-/- chondrocytes with compressive mechanical stress loading, and the related inflammatory markers were examined. KEY FINDINGS: Vdr-/-res mice did not develop rickets with a high calcium rescue diet. The TMJ cartilage thickness in Vdr-/-res mice was significantly decreased with mechanical stress stimulation compared to WT mice. UAC-induced bone resorption was obvious, and the number of osteoclasts significantly increased in Vdr-/-res mice. The proliferation was inhibited and the gene expression of Il1b, Mmp3, and Mmp13 was significantly increased in Vdr-/- chondrocytes. However, WT chondrocytes showed significantly increased Tnfa gene expression as a response to mechanical stress but not in Vdr-/- chondrocytes. SIGNIFICANCE: VD-VDR is crucial in TMJ pathological changes under abnormal mechanical stimulation. Deletion of Vdr exacerbated inflammatory response excluding TNFα, inhibited chondrocyte proliferation, and promoted bone resorption in TMJ.

Early changes in asporin levels in osteoarthritis of the temporomandibular joint.

OBJECTIVES: The present study aimed to elucidate the pathogenesis of temporomandibular joint (TMJ) osteoarthritis (TMJ-OA) in a mouse model. We investigated morphological and histological changes in the head of mandible cartilage and early immunohistochemical (IHC) changes in transforming growth factor (TGF)-ß, phosphorylated Smad-2/3 (p-Smad2/3), a TGF-ß signaling molecule, and asporin. METHODS: TMJ-OA was induced in a mouse model through unilateral partial discectomy. Micro-computed tomography (micro-CT) and safranin-O staining were performed to morphologically and histologically evaluate the degeneration of the head of mandible caused by TMJ-OA. IHC staining for TGF-ß, p-Smad2/3, and asporin was performed to evaluate the changes in protein expression. RESULTS: In the experimental group, three-dimensional (3D) morphometry revealed an enlarged head of mandible and safranin-O staining showed degeneration of cartilage tissue in the early stages of TMJ-OA compared to the control group. IHC staining revealed that TGF-ß, p-Smad2/3, and asporin expression increased in the head of mandible cartilage before the degeneration of cartilage tissue, and subsequently decreased for a short period. CONCLUSION: The findings suggested a negative feedback relationship between the expression of asporin and the TGF-ß/Smad transduction pathway, which may be involved in the degeneration of the head of mandible in the early stages of TMJ-OA. Asporin is a potential biomarker of the early stages of TMJ-OA, which ultimately leads to the irreversible degeneration of TMJ tissues.

Enkephalin Rescues Temporomandibular Joint Pain-Related Behavior in Rats.

Temporomandibular joint disorders include a variety of clinical syndromes that are difficult to manage if associated with debilitating severe jaw pain. Thus, seeking additional experimental therapies for temporomandibular joint pain reduction is warranted. Targeted enkephalin gene therapy approaches provide clear promise for pain control. The studies detailed here indicate significant analgesia and protection of joint tissue are provided after injection of an overexpression viral vector gene therapy near the joint. The viral vector gene therapy described provides overexpression of naturally occurring opioid peptides after its uptake by trigeminal nerve endings. The viral vectors act as independent "minipump" sources for the opioid peptide synthesis in the neuronal cytoplasm producing the intended biological function, reduction of pain, and tissue repair. The antinociceptive effects provided with this delivery method of opioid expression persist for over 4 weeks. This is coincident with the expected time frame for the duration of the transgene overproduction of the endogenous opioid peptide before its diminution due to dormancy of the virus. These experimental studies establish a basis for the use of replication-defective herpes simplex type 1-based gene therapy for severe chronic inflammatory temporomandibular joint destruction and pain. As innovative means of significantly reducing joint inflammation and preserving tissue architecture, gene therapies may extend their clinical usefulness for patients with temporomandibular joint disorders.

Biomechanism of abnormal stress on promoting osteoarthritis of temporomandibular joint through Piezo1 ion channel.

OBJECTIVE: This study aimed to investigate whether flow fluid shear stress (FFSS)-mediated signal transduction affects the function of Piezo1 ion channel in chondrocyte and to further explore the role of mechanical overloading in development of temporomandibular joint osteoarthritis (TMJ OA). METHODS: Immunohistochemical staining was used to determine the expression of Piezo1 in TMJ OA tissue collected from rat unilateral anterior crossbite (UAC) models. Chondrocytes harvested from normal adult SD rats were treated with FFSS (0, 4, 8, 12 dyn/cm2) in vitro. Immunofluorescent staining, real-time polymerase chain reaction, western blotting, flow cytometry and phalloidin assay were performed to detect the changes of cellular morphology as well as the expression of Piezo1 and certain pro-inflammatory and degradative factors in chondrocyte. RESULTS: Immunohistochemical analysis revealed that significantly increased Piezo1 expression was associated with UAC stimulation (p < .05). As applied FFSS escalated (4, 8 and 12 dyn/cm2), the expression levels of Piezo1, ADAMTS-5, MMP-13 and Col-X gradually increased, compared with the non-FFSS group (p < .05). Administering Piezo1 ion channel inhibitor to chondrocytes beforehand, it was observed that expression of ADAMTS-5, MMP-13 and Col-X was substantially decreased following FFSS treatment (p < .05) and the effect of cytoskeletal thinning was counteracted. The activated Piezo1 ion channel enhanced intracellular Ca2+ excess in chondrocytes during abnormal mechanical stimulation and the increased intracellular Ca2+ thinned the cytoskeleton of F-actin. CONCLUSIONS: Mechanical overloading activates Piezo1 ion channel to promote pro-inflammation and degradation and to increase Ca2+ concentration in chondrocyte, which may eventually result in TMJ OA.

Mesenchymal stem cells-derived exosomes alleviate temporomandibular joint disc degeneration in temporomandibular joint disorder.

Temporomandibular joint (TMJ) disorder (TMD) is a chronic progressive disease that is commonly seen in clinical settings. TMJ disc degeneration is an important manifestation of TMD, and further aggravates the progression of TMD. However, treatments on TMJ disc degeneration are very limited till now. In this study, we first observed the effects of bone marrow stem cells (BMSC) conditioned medium on functions of TMJ disc fibroblasts. Then BMSC-derived small extracellular vesicles (BMSC-EVs) were isolated and exposed to TMJ disc fibroblasts. RNA-sequencing was used to further investigate the mechanisms. BMSC-EVs were finally injected into a rat model with TMD. Results showed that in the transwell co-culture system, the medium derived from BMSC reduced inflammation and enhanced chondrogenesis in TMJ disc fibroblasts. BMSC-EVs promoted proliferation, migration, and chondrogenic differentiation of TMJ disc fibroblasts, and inhibited apoptosis and inflammatory responses. Local injection of BMSC-EVs into the TMD model alleviated TMJ disc degeneration. Therefore, BMSC-EVs were a potentially effective, sustainable and clinically translational-promising option for TMJ disc degeneration, and further reduce the progression of TMD.

Loss of ß-arrestin2 aggravated condylar cartilage degeneration at the early stage of temporomandibular joint osteoarthritis.

OBJECTIVE: Temporomandibular joint osteoarthritis (TMJOA) is a chronic degenerative joint disorder characterized by extracellular matrix degeneration and inflammatory response of condylar cartilage. ß-arrestin2 is an important regulator of inflammation response, while its role in TMJOA remains unknown. The objective of this study was to investigate the role of ß-arrestin2 in the development of TMJOA at the early stage and the underlying mechanism. METHODS: A unilateral anterior crossbite (UAC) model was established on eight-week-old wild-type (WT) and ß-arrestin2 deficiency mice to simulate the progression of TMJOA. Hematoxylin-eosin (HE) staining and microcomputed tomography (micro-CT) analysis were used for histological and radiographic assessment. Immunohistochemistry was performed to detect the expression of inflammatory and degradative cytokines, as well as autophagy related factors. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay was carried out to assess chondrocyte apoptosis. RESULTS: The loss of ß-arrestin2 aggravated cartilage degeneration and subchondral bone destruction in the model of TMJOA at the early stage. Furthermore, in UAC groups, the expressions of degradative (Col-X) and inflammatory (TNF-α and IL-1ß) factors in condylar cartilage were increased in ß-arrestin2 null mice compared with WT mice. Moreover, the loss of ß-arrestin2 promoted apoptosis and autophagic process of chondrocytes at the early stage of TMJOA. CONCLUSION: In conclusion, we demonstrated for the first time that ß-arrestin2 plays a protective role in the development of TMJOA at the early stage, probably by inhibiting apoptosis and autophagic process of chondrocytes. Therefore, ß-arrestin2 might be a potential therapeutic target for TMJOA, providing a new insight for the treatment of TMJOA at the early stage.

Dietary supplementation with grape seed extract from Vitus vinifera prevents suppression of GABAergic protein expression in female Sprague Dawley trigeminal ganglion in a model of chronic temporomandibular joint disorder.

OBJECTIVE: To investigate cellular changes in protein expression in the trigeminal ganglion in an established preclinical chronic model of temporomandibular joint disorder (TMD) in response to grape seed extract (GSE) supplementation based on its beneficial use in preclinical chronic orofacial pain models. DESIGN: Three experimental conditions included female Sprague-Dawley rats as naïve controls, and animals subjected to neck muscle inflammation and prolonged jaw opening with and without daily supplementation of GSE in the drinking water prior to inflammation. Changes were evaluated in mechanical sensitivity to von Frey filaments and protein expression in the trigeminal ganglion of animals 14 days post jaw opening. RESULTS: Calcitonin-gene related peptide and protein kinase A, proteins positively associated with peripheral sensitization and enhanced nociception, did not show elevated expression at day 14 in the model compared to naïve or GSE supplemented animals. However, neuronal levels of glutamate decarboxylase (GAD) 65/67, which are enzymes responsible for the synthesis of the inhibitory neurotransmitter GABA that functions to suppress neuronal excitability, were significantly decreased on day 14 post jaw opening. Similarly, a significant decrease in neuronal expression of the GABA receptor subunits GABAB1 and GABAB2, but not GABAA, was observed in the TMD model. Importantly, GSE prevented suppression of GAD 65/67 and GABAB subunits, maintaining levels similar to naïve animals. CONCLUSION: Results from our study provide evidence of the downregulation of inhibitory GABAergic proteins in trigeminal ganglion neurons in a preclinical chronic TMD model and the benefits of GSE supplementation in preventing their suppression and maintaining normal levels.

YAP promotes the early development of temporomandibular joint bony ankylosis by regulating mesenchymal stem cell function.

To explore the role of YAP, a key effector of the Hippo pathway, in temporomandibular joint (TMJ) ankylosis. The temporal and spatial expression of YAP was detected via immunohistochemistry and multiplex immunohistochemistry on postoperative Days 1, 4, 7, 9, 11, 14 and 28 in a sheep model. Isolated mesenchymal stem cells (MSCs) from samples of the Day 14. The relative mRNA expression of YAP was examined before and after the osteogenic induction of MSCs. A YAP-silenced MSC model was constructed, and the effect of YAP knockdown on MSC function was examined. YAP is expressed in the nucleus of the key sites that determine the ankylosis formation, indicating that YAP is activated in a physiological state. The expression of YAP increased gradually over time. Moreover, the number of cells coexpressing of RUNX2 and YAP-with the osteogenic active zone labelled by RUNX2-tended to increase after Day 9. After the osteogenic induction of MSCs, the expression of YAP increased. After silencing YAP, the osteogenic, proliferative and migratory abilities of the MSCs were inhibited. YAP is involved in the early development of TMJ bony ankylosis. Inhibition of YAP using shRNA might be a promising way to prevent or treat TMJ ankylosis.

Integration of metabolomics and transcriptomics provides insights into the molecular mechanism of temporomandibular joint osteoarthritis.

The deficiency of clinically specific biomarkers has made it difficult to achieve an accurate diagnosis of temporomandibular joint osteoarthritis (TMJ-OA) and the insufficient comprehension of the pathogenesis of the pathogenesis of TMJ-OA has posed challenges in advancing therapeutic measures. The combined use of metabolomics and transcriptomics technologies presents a highly effective method for identifying vital metabolic pathways and key genes in TMJ-OA patients. In this study, an analysis of synovial fluid untargeted metabolomics of 6 TMJ-OA groups and 6 temporomandibular joint reducible anterior disc displacement (TMJ-DD) groups was conducted using liquid and gas chromatography mass spectrometry (LC/GC-MS). The differential metabolites (DMs) between TMJ-OA and TMJ-DD groups were analyzed through multivariate analysis. Meanwhile, a transcriptomic dataset (GSE205389) was obtained from the GEO database to analyze the differential metabolism-related genes (DE-MTGs) between TMJ-OA and TMJ-DD groups. Finally, an integrated analysis of DMs and DE-MTGs was carried out to investigate the molecular mechanisms associated with TMJ-OA. The analysis revealed significant differences in the levels of 46 DMs between TMJ-OA and TMJ-DD groups, of which 3 metabolites (L-carnitine, taurine, and adenosine) were identified as potential biomarkers for TMJ-OA. Collectively, differential expression analysis identified 20 DE-MTGs. Furthermore, the integration of metabolomics and transcriptomics analysis revealed that the tricarboxylic acid (TCA) cycle, alanine, aspartate and glutamate metabolism, ferroptosis were significantly enriched. This study provides valuable insights into the metabolic abnormalities and associated pathogenic mechanisms, improving our understanding of TMJOA etiopathogenesis and facilitating potential target screening for therapeutic intervention.

DDIT3 aggravates TMJOA cartilage degradation via Nrf2/HO-1/NLRP3-mediated autophagy.

OBJECTIVE: DNA damage-inducible transcript 3 (DDIT3), as a downstream transcription factor of endoplasmic reticulum stress, is reported to regulate chondrogenic differentiation under physiological and pathological state. However, the specific involvement of DDIT3 in the degradation of condylar cartilage of temporomandibular joint osteoarthritis (TMJOA) is unclarified. DESIGN: The expression patterns of DDIT3 in condylar cartilage from monosodium iodoacetate-induced TMJOA mice were examined to uncover the potential role of DDIT3 in TMJOA. The Ddit3 knockout (Ddit3-/-) mice and their wildtype littermates (Ddit3+/+) were used to clarify the effect of DDIT3 on cartilage degradation. Primary condylar chondrocytes and ATDC5 cells were applied to explore the mechanisms of DDIT3 on autophagy and extracellular matrix (ECM) degradation in chondrocytes. The autophagy inhibitor chloroquine (CQ) was used to determine the effect of DDIT3-inhibited autophagy in vivo. RESULTS: DDIT3 were highly expressed in condylar cartilage from TMJOA mice. Ddit3 knockout alleviated condylar cartilage degradation and subchondral bone loss, compared with their wildtype littermates. In vitro study demonstrated that DDIT3 exacerbated ECM degradation in chondrocytes induced by TNF-α through inhibiting autophagy. The intraperitoneal injection of CQ further confirmed that Ddit3 knockout alleviated cartilage degradation in TMJOA through activating autophagy in vivo. CONCLUSIONS: Our findings identified the crucial role of DDIT3-inhibited autophagy in condylar cartilage degradation during the development of TMJOA.

Inflammation Triggers Chondrocyte Ferroptosis in TMJOA via HIF-1α/TFRC.

Inflammation and loss of articular cartilage are considered the major cause of temporomandibular joint osteoarthritis (TMJOA), a painful condition of the temporomandibular joint (TMJ). To determine the cause of TMJ osteoarthritis in these patients, synovial fluid of TMJOA patients was compared prior to and after hyaluronic lavage, revealing substantially elevated levels of interleukin (IL) 1ß, reactive oxidative stress (ROS), and an overload of Fe3+ and Fe2+ prior to lavage, indicative of ferroptosis as a mode of chondrocyte cell death. To ask whether prolonged inflammatory conditions resulted in ferroptosis-like transformation in vitro, we subjected TMJ chondrocytes to IL-1ß treatment, resulting in a shift in messenger RNA sequencing gene ontologies related to iron homeostasis and oxidative stress-related cell death. Exposure to rat unilateral anterior crossbite conditions resulted in reduced COL2A1 expression, fewer chondrocytes, glutathione peroxidase 4 (GPX4) downregulation, and 4-hydroxynonenal (4-HNE) upregulation, an effect that was reversed after intra-articular injections of the ferroptosis inhibitor ferrostatin 1 (Fer-1). Our study demonstrated that ferroptosis conditions affected mitochondrial structure and function, while the inhibitor Fer-1 restored mitochondrial structure and the inhibition of hypoxia-inducible factor 1α (HIF-1α) or the transferrin receptor 1 (TFRC) rescued IL-1ß-induced loss of mitochondrial membrane potential. Inhibition of HIF-1α downregulated IL-1ß-induced TFRC expression, while inhibition of TFRC did not downregulate IL-1ß-induced HIF-1α expression in chondrocytes. Moreover, inhibition of HIF-1α or TFRC downregulated the IL-1ß-induced MMP13 expression in chondrocytes, while inhibition of HIF-1α or TFRC rescued IL-1ß-inhibited COL2A1 expression in chondrocytes. Furthermore, upregulation of TFRC promoted Fe2+ entry into chondrocytes, inducing the Fenton reaction and lipid peroxidation, which in turn caused ferroptosis, a disruption in chondrocyte functions, and an exacerbation of condylar cartilage degeneration. Together, these findings illustrate the far-reaching effects of chondrocyte ferroptosis in TMJOA as a mechanism causing chondrocyte death through iron overload, oxidative stress, and articular cartilage degeneration and a potential major cause of TMJOA.

A loss of primary cilia by a reduction in mTOR signaling correlates with age-related deteriorations in condylar cartilage.

Age-related deterioration of condylar cartilage is an etiological factor in temporomandibular joint-osteoarthritis (TMJ-OA). However, its underlying mechanism remains unknown. Therefore, we examined age-related changes and the relationship between mTOR signaling and primary cilia in condylar cartilage to determine the intrinsic mechanisms of age-related TMJ-OA. Age-related morphological changes were analyzed using micro-computed tomography and safranin O-stained histological samples of the mandibular condyle of C57BL/6J mice (up to 78 weeks old). Immunohistochemistry was used to assess the activity of mTOR signaling, primary cilia frequency, and Golgi size of condylar chondrocytes. Four-week-old mice receiving an 11-week series of intraperitoneal injections of rapamycin, a potent mTOR signaling inhibitor, were used for the histological evaluation of the condylar cartilage. The condylar cartilage demonstrated an age-related reduction in cartilage area, including chondrocyte size, cell density, and cell size distribution. The Golgi size, primary cilia frequency, and mTOR signaling also decreased with age. Rapamycin injections resulted in both diminished cartilage area and cell size, resembling the phenotypes observed in aged mice. Rapamycin-injected mice also exhibited a smaller Golgi size and lower primary cilia frequency in condylar cartilage. We demonstrated that a loss of primary cilia due to a decline in mTOR signaling was correlated with age-related deteriorations in condylar cartilage. Our findings provide new insights into the tissue homeostasis of condylar cartilage, contributing to understanding the etiology of age-related TMJ-OA.

Pain and salivary biomarkers of stress in temperomandibular disorders were affected by maxillary splints.

BACKGROUND: Longitudinal intervention studies on treatment options in temporomandibular dysfunction (TMD) including self reports and salivary biomarkers of stress are rare and the exact therapeutic function of occlusal splints widely unknown. METHODS: We examined the therapeutic effects of a Michigan splint with occlusal relevance in patients with TMD using a placebo-controlled, delayed-start design. Two intervention groups received a Michigan splint, while one of them had a placebo palatine splint for the first 3 weeks. We collected pain intensities (at rest and after five occlusal movements), salivary measures associated with stress (cortisol and alpha-amylase) and self-reported psychological distress (stress, anxiety, catastrophizing) at baseline and 3 and 7 weeks after onset of intervention. RESULTS: At baseline, we observed increased pain intensity and psychological distress in TMD patients compared to 11 matched healthy controls. Baseline anxiety was linked to movement pain intensity through stress. Over therapy reductions in pain intensity and morning cortisol were more pronounced in those patients starting immediately with the Michigan splint, while psychological distress decreased similarly in both groups. CONCLUSION: Our results suggest that perceived stress plays a role for the association between anxiety and TMD pain and underlines the need for an interdisciplinary perspective on the pathogenesis and therapy of TMD in a setting where psychotherapeutic knowledge is still scarce or rarely applied.

Metabolic profiling of synovial fluid in human temporomandibular joint osteoarthritis.

Introduction: Temporomandibular joint (TMJ) osteoarthritis (OA) is a common TMJ degenerative disease with an unclear mechanism. Synovial fluid (SF), an important component of TMJ, contains various proteins and metabolites that may directly contribute to OA. The present study aimed to investigate the influence of SF in TMJOA at the metabolite level. Methods: Untargeted and widely targeted metabolic profiling were employed to identify metabolic changes in SF of 90 patients with different TMJOA grades according to TMJ magnetic resonance imaging. Results: A total 1498 metabolites were detected. Most of the metabolites were amino acids and associated metabolites, benzene and substituted derivatives, and lipids. Among patients with mild, moderate and severe TMJOA, 164 gradually increasing and 176 gradually decreasing metabolites were identified, indicating that biosynthesis of cofactors, choline metabolism, mineral absorption and selenocompound metabolism are closely related to TMJOA grade. Combined metabolomics and clinical examination revealed 37 upregulated metabolites and 16 downregulated metabolites in patients with pain, of which 19 and 26 metabolites were positively and negatively correlated, respectively, with maximum interincisal opening. A model was constructed to diagnose TMJOA grade and nine biomarkers were identified. The identified metabolites are key to exploring the mechanism of TMJOA. Discussion: In the present study, a metabolic profile was constructed and assessed using a much larger number of human SF samples from patients with TMJOA, and a model was established to contribute to the diagnosis of TMJOA grade. The findings expand our knowledge of metabolites in human SF of TMJOA patients, and provide an important basis for further research on the pathogenesis and treatment of TMJOA.