Hypoxia-inducible Factor Expression Is Related to Apoptosis and Cartilage Degradation in Temporomandibular Joint Osteoarthritis

Background: It remains unclear whether hypoxic conditions affect apoptosis and contribute to degradation of cartilaginous tissues in osteoarthritis (OA) lesions. In this study, we hypothesized that hypoxic conditions induced the accumulation of hypoxia-inducible factor (HIF) and activated apoptosis to contribute to OA cartilage degeneration in vivo.Methods: Malocclusion stress was applied for 2 weeks, 4 weeks and 8 weeks to induce an OA-like lesion animal model (OD) in rats. Histological analysis was performed by H&E staining and safranin O/fast green staining. The expression levels of protein in condylar cartilage were examined by immunostaining to evaluate cartilage degeneration.Results: We found apparent histological phenotypes associated with degeneration in the occlusion disorder stress (OD) group. The OD group at 4 weeks and 8 weeks had obviously reduced expression of Acan and Col II in cartilage. In contrast, the OD groups had higher levels of Col X, ADAMTS5 and MMP13 in the condylar cartilage than the control group. Moreover, the OD group cartilage had prominent degenerative changes with reduced levels of HIF1α and increased levels of HIF2α and the apoptosis factor Caspase3 in condylar cartilage at 8 weeks.Occlusion disorder stress results in cartilage degeneration. HIF1α and HIF2α are involved in temporomandibular joint (TMJ) cartilage homeostasis by regulating chondrocyte apoptosis, which contributes to TMJ cartilage degeneration. Conclusion: Thus, abnormal hypoxic conditions inducing opposite expression patterns of HIF1α and HIF2α could be involved in the pathogenesis of condylar cartilage degeneration. HIF2α may provide a potential negative feedback mechanism for HIF1α during cartilage damage.

The Attenuating Effect of Low-Intensity Pulsed Ultrasound on Hypoxia-Induced Rat Chondrocyte Damage in TMJ Osteoarthritis Based on TMT Labeling Quantitative Proteomic Analysis

Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease with a complex and multifactorial etiology. An increased intrajoint pressure or weakened penetration can exacerbate the hypoxic state of the condylar cartilage microenvironment. Our group previously simulated the hypoxic environment of TMJOA in vitro. Low-intensity pulsed ultrasound (LIPUS) stimulation attenuates chondrocyte matrix degradation via a hypoxia-inducible factor (HIF) pathway-associated mechanism, but the mode of action of LIPUS is currently poorly understood. Moreover, most recent studies investigated the pathological mechanisms of osteoarthritis, but no biomarkers have been established for assessing the therapeutic effect of LIPUS on TMJOA with high specificity, which results in a lack of guidance regarding clinical application. Here, tandem mass tag (TMT)-based quantitative proteomic technology was used to comprehensively screen the molecular targets and pathways affected by the action of LIPUS on chondrocytes under hypoxic conditions. A bioinformatic analysis identified 902 and 131 differentially expressed proteins (DEPs) in the <1% oxygen treatment group compared with the control group and in the <1% oxygen + LIPUS stimulation group compared with the <1% oxygen treatment group, respectively. The DEPs were analyzed by gene ontology (GO), KEGG pathway and protein-protein interaction (PPI) network analyses. By acting on extracellular matrix (ECM)-associated proteins, LIPUS increases energy production and activates the FAK signaling pathway to regulate cell biological behaviors. DEPs of interest were selected to verify the reliability of the proteomic results. In addition, this experiment demonstrated that LIPUS could upregulate chondrogenic factors (such as Sox9, Collagen Ⅱ and Aggrecan) and increase the mucin sulfate content. Moreover, LIPUS reduced the hydrolytic degradation of the ECM by decreasing the MMP3/TIMP1 ratio and vascularization by downregulating VEGF. Interestingly, LIPUS improved the migration ability of chondrocytes. In summary, LIPUS can regulate complex biological processes in chondrocytes under hypoxic conditions and alter the expression of many functional proteins, which results in reductions in hypoxia-induced chondrocyte damage. ECM proteins such as thrombospondin4, thrombospondin1, IL1RL1, and tissue inhibitors of metalloproteinase 1 play a central role and can be used as specific biomarkers determining the efficacy of LIPUS and viable clinical therapeutic targets of TMJOA.

Chondrocyte Adipogenic Differentiation in Softening Osteoarthritic Cartilage

A chondrocyte-to-osteoblast lineage continuum exists in the growth plate. Adipogenic differentiation of chondrocytes in vivo should be investigated. Here, unilateral anterior crossbite (UAC), which can induce osteoarthritic lesions in the temporomandibular joint (TMJ), was applied to 6-wk-old C57BL/6 mice. Matrix loss in TMJ cartilage was obvious, as demonstrated by safranin O staining, and the condylar cartilage elastic modulus values, detected by using atomic force microscopy (AFM), were reduced, indicating cartilage softening that might be linked with loss of the highly charged proteoglycan. By crossing the Rosa26/tdTomato (TdT) mice with Sox9;CreERT2 mice or with Col10;CreERT2 mice, we obtained the Sox9-TdT and Col10-TdT strains, respectively, in which the Sox9- or Col10-expressing cells, accordingly, were labeled by TdT. A few TdT-labeled cells in both strains expressed AdipoQ or DMP-1. The Sox9-TdT+AdipoQ+ cells were primarily located in the deep zone cartilage and then in the whole cartilage. Col10-TdT+AdipoQ+ cells, Sox9-TdT+DMP-1+ cells, and Col10-TdT+DMP-1+ cells were located in the deep zone region. UAC promoted AdipoQ and DMP-1 expression in cartilage. The percentages of Sox9-TdT+AdipoQ+ and Col10-TdT+AdipoQ+ cells to Sox9-TdT+ and Col10-TdT+ cells, respectively, were increased (both P < 0.05), implying that more chondrocytes were undergoing adipogenic differentiation in the UAC group, the cartilage of which was softened. The percentages of Sox9-TdT+DMP-1+ and Col10-TdT+DMP-1+ cells to Sox9-TdT+ cells and Col10-TdT+ cells, respectively, were increased (both P < 0.05), consistent with our report that UAC enhanced deep zone cartilage calcification, causing stiffening of the deep zone cartilage. Our present data demonstrated that TMJ chondrocyte descendants can become adipogenic in vivo in addition to becoming osteogenic. This potential was promoted in osteoarthritic cartilage, in which deep zone cartilage calcification-associated cartilage stiffening and proteoglycan loss-associated cartilage softening were both stimulated.

Elder Mice Exhibit More Severe Degeneration and Milder Regeneration in Temporomandibular Joints Subjected to Bilateral Anterior Crossbite

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

Static Magnetic Fields Enhance the Chondrogenesis of Mandibular Bone Marrow Mesenchymal Stem Cells in Coculture Systems

Objectives. Combining the advantages of static magnetic fields (SMF) and coculture systems, we investigated the effect of moderate-intensity SMF on the chondrogenesis and proliferation of mandibular bone marrow mesenchymal stem cells (MBMSCs) in the MBMSC/mandibular condylar chondrocyte (MCC) coculture system. The main aim of the present study was to provide an experimental basis for obtaining better cartilage tissue engineering seed cells for the effective repair of condylar cartilage defects in clinical practice. Methods. MBMSCs and MCCs were isolated from SD (Sprague Dawley) rats. Flow cytometry, three-lineage differentiation, colony-forming assays, immunocytochemistry, and toluidine blue staining were used for the identification of MBMSCs and MCCs. MBMSCs and MCCs were seeded into the lower and upper Transwell chambers, respectively, at a ratio of 1 : 2, and exposed to a 280 mT SMF. MBMSCs were harvested after 3, 7, or 14 days for analysis. CCK-8 was used to detect cell proliferation, Alcian blue staining was utilized to evaluate glycosaminoglycan (GAG), and western blotting and real-time quantitative polymerase chain reaction (RT-qPCR) detected protein and gene expression levels of SOX9, Col2A1 (Collagen Type II Alpha 1), and Aggrecan (ACAN). Results. The proliferation of MBMSCs was significantly enhanced in the experimental group with MBMSCs cocultured with MCCs under SMF stimulation relative to controls ( P < 0.05 ). GAG content was increased, and SOX9, Col2A1, and ACAN were also increased at the mRNA and protein levels ( P < 0.05 ). Conclusions. Moderate-intensity SMF improved the chondrogenesis and proliferation of MBMSCs in the coculture system, and it might be a promising approach to repair condylar cartilage defects in the clinical setting.

Skeletal Class II Malocclusion Treatment with Combined Twin Block and Lip Bumper Appliances: A Case Report

Functional appliances have been used over a century in clinical orthodontic treatments for skeletal Class II malocclusion patients. Its popularity is attributed to its high patient adaptability and ability to produce rapid treatment changes. The twin block and lip bumper can be combined depending on the patient’s cases. The purpose of therapy with twin block is effective in mandibular growth deficiencies to induce supplementary lengthening of mandibular by stimulating increased growth at the condylar cartilage. The patient was a ten-year-old male patient with skeletal Class II malocclusion. He had a convex facial profile, SNA (sella, nasion, A point) angle of 77.5°, SNB (sella, nasion, B point) angle of 73.0°, ANB (A point, nasion, B point) angle of 4.5°, overjet of 6.5 mm, overbite of 11/41 = 5.0 mm, 21/31 = 4.5 mm, abnormal upper labial frenulum, crossbite in the second left premolar of maxilla, crowded anterior teeth of mandibular, deficiency of mandibular growth, lower lip sucking habit, anterior teeth of maxilla with diastema and proclination. Orthodontic treatment for patient is a combination of twin block and lip bumper appliances. After seven months, frenectomy is used to eliminate and correct the spacing in the frenulum. After 10 months, the patient’s skeletal and profile had improved to skeletal Class I malocclusion, SNA angle of 78.0°, SNB angle of 75.0°, ANB angle of 3.0°, overbite and overjet of 4.0 mm, and the lower lip sucking habit had stopped. Twin block and lip bumper appliances are particularly good alternative treatment in managing selected cases of skeletal Class II malocclusion.

Implications of zonal architecture on differential gene expression profiling and altered pathway expressions in mandibular condylar cartilage

Mandibular condylar cartilage (MCC) is a multi-zonal heterogeneous fibrocartilage containing different types of cells, but the factors/mechanisms governing the phenotypic transition across the zones have not been fully understood. The reliability of molecular studies heavily rely on the procurement of pure cell populations from the heterogeneous tissue. We used a combined laser-capture microdissection and microarray analysis approach which allowed identification of differential zone-specific gene expression profiling and altered pathways in the MCC of 5-week-old rats. The bioinformatics analysis demonstrated that the MCC cells clearly exhibited distinguishable phenotypes from the articular chondrocytes. Additionally, a set of genes has been determined as potential markers to identify each MCC zone individually; Crab1 gene showed the highest enrichment while Clec3a was the most downregulated gene at the superficial layer, which consists of fibrous (FZ) and proliferative zones (PZ). Ingenuity Pathway Analysis revealed numerous altered signaling pathways; Leukocyte extravasation signaling pathway was predicted to be activated at all MCC zones, in particular mature and hypertrophic chondrocytes zones (MZ&HZ), when compared with femoral condylar cartilage (FCC). Whereas Superpathway of Cholesterol Biosynthesis showed predicted activation in both FZ and PZ as compared with deep MCC zones and FCC. Determining novel zone-specific differences of large group of potential genes, upstream regulators and pathways in healthy MCC would improve our understanding of molecular mechanisms on regional (zonal) basis, and provide new insights for future therapeutic strategies.

Temporomandibular joint and orthodontics –A contemporary review

The Temporomandibular joint is an atypical synovial joint that is dynamic in structure. A delicate balance between the musculature, the condylar cartilage and the bony structures is maintained for harmonious functioning of the joint. Over the years the role of occlusion in temporomandibular disorders (TMD) has been extensively debated, leading to many opinions and much controversy. The changes in the joint caused in response to orthodontic therapy as well the adverse reactions caused have been studied. The present article is a review on the effects of Orthodontic appliance therapy on the joint and the present modalities opted to treat the joint disorders.

Effect of Mechanical Stress on Rheumatoid Arthritis of the Temporomandibular Joint: a Morphological and Histological Evaluation

Background Rheumatoid arthritis of the temporomandibular joint (TMJ-RA) has been reported to have a larger incidence range than systemic rheumatoid arthritis (RA). The presence or absence of mechanical stress (MS) is considered a factor in this. In this study, we hypothesized that TMJ-RA develops or worsens when excessive MS is applied to the temporomandibular joint of RA mouse models. We aimed to clarify the relationship between TMJ-RA and MS through morphological and histological evaluation. Methods Collagen antibody-induced arthritis (CAIA) was induced in male DBA/1JNCrlj 9–12 weeks old mice by administering Type II collagen antibody and lipopolysaccharide to produce RA model mice. MS was applied to the mandibular condyle. The group was separated into non-RA (control group (N = 5) and MS group (N = 5)), and RA group (CAIA group (N = 5)and CAIA MS group (N = 5)). To confirm the morphological changes in the mandibular condyle, micro-CT imaging was performed. Histological evaluation of the TMJ was performed by hematoxylin and eosin staining for condylar cartilage cell layer thickness, Safranin O staining for proteoglycans, and tartrate-resistant acidic phosphatase staining for osteoclast count. Immunohistochemical evaluation was performed to assess the localization of cartilage destruction enzymes using ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs) antibody. Additionally, CD3 (cluster of differentiation), CD45, and γδ TCR (T cell receptor) antibodies were used to localize and identify the type of lymphocytes. Results In the CAIA MS model, a three-dimensional analysis of the temporomandibular joint by microcomputer tomography showed a crude change in the surface of the mandibular condyle. Histological examination revealed a decrease in the chondrocyte layer width and an increase in the number of osteoclasts in the mandibular condyle. T cell accumulation was observed, and γδ T cell involvement was confirmed. Conclusions In the CAIA model, the TMJ was less sensitive to the initiation of RA. However, the results suggested that it was exacerbated by MS, and that γδ T cells may be involved in TMJ-RA.