Analysis of joint protein expression profile in anterior disc displacement of TMJ with or without OA.

OBJECTIVE: Anterior disc displacement (ADD) is a common clinical issue and may cause osteoarthritis (OA). However, the research of protein changes in synovial fluid as disease development marker and potential treatment clue is still insufficient. MATERIALS AND METHODS: We conducted the high-resolution mass spectrometry (MS) of synovial fluid collected from 60 patients with normal disk position to ADD and ADD with osteoarthritis (OA). The proteins with significant changes among the 3 groups were analyzed by biological information and further validated by in primary rat condyle chondrocytes and OA animal model. RESULTS: FGL2, THBS4, TNC, FN1, OMD etc. were significantly increased in ADD without OA (p < 0.05), which reflected the active extracellular matrix and collagen metabolism. FGFR1, FBLN2, GRB2 etc. were significantly increased in ADD with OA group (p < 0.05), which revealed an association with apoptosis and ferroptosis. Proteins such as P4HB, CBLN4, FHL1, VIM continuously increase in the whole disease progress (p < 0.05). Both the in vitro and in vivo results are consistent with protein changes detected in MS profile. CONCLUSION: This study firstly provides the expression changes of proteins from normal disc condyle relationship toward ADD with OA, which can be selected and studied further as disease progress marker and potential treatment targets.

Relationship between activity of gluthatione peroxidase and nitric oxide in synovial fluid and the progression of temporomandibular joint internal derangement.

The purposes of this study were to measure the activity of glutathione peroxidase (GPX) and nitric oxide (NO) in the synovial fluid of patients with temporomandibular joint (TMJ) internal derangement (ID) and to indicate the relationship between the activity of GPX and NO and the progress of the ID. Twenty-six patients with TMJ ID were identified and classified according to Wilkes staging through clinical and radiologic examinations. Levels of GPX were determined indirectly by a coupled reaction with glutathione reductase. Levels of NO were measured colorimetrically. The activity of GPX and NO was observed to be progressively increasing as the stage of the TMJ ID progressed. There were significant correlations between the 2 substances and the Wilkes stages. Oxidative stress may have a role in the pathogenesis of TMJ ID. In synovial fluid, GPX and NO activities are increased as the stage of the disease increased. Increase in the activities of GPX might not be enough to prevent progression of the TMJ ID.

Potential biomarkers of temporomandibular joint disorders.

PURPOSE: The purpose of this study was to identify protein markers present in subjects with temporomandibular joint disorders (TMDs) and clicking compared with the levels in controls. MATERIALS AND METHODS: This was a pilot case-control study, and we report the preliminary results. Samples of joint aspirate collected from patients with TMDs and controls who had undergone surgery for a problem other than TMDs were analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) and biotin-labeled-based protein arrays. The data obtained from these techniques were used to identify the proteins of interest, which were then quantitated using enzyme-linked immunosorbent assay (ELISA). The patient samples studied included joint aspirate collected clinically from the controls and patients and included samples from both the right and the left sides of each patient with a TMD. RESULTS: The 8 TMJ aspirate samples from 6 subjects included 5 aspirate samples from 4 patients and 3 from 2 controls. The greatest standardized protein concentration of endocrine gland-derived vascular endothelial growth factor/prokineticin-1 (EG-VEGF/PK1) and D6 was found in both joints of the controls compared with the levels from the joints of the patients. With 1 exception, the standardized protein concentration was significantly lower in the patients than in the controls. The lower levels of EG-VEGF/PK1 and D6 in the patients compared with the controls suggest that these cytokines might be possible biomarkers for TMDs. CONCLUSION: In the present pilot study, greater levels of EG-VEGF/PK1 and D6 were found in the controls than in the patients with TMDs. Proteomic analysis of the proteins present in the diseased joints compared with those in the controls might help to identify proteins present when pain or degeneration of the joint occurs. The proteomic information might be useful in the development of future therapies.

Phenotypic features of carbohydrate sulfotransferase 3 (CHST3) deficiency in 24 patients: congenital dislocations and vertebral changes as principal diagnostic features.

We recently reported on the deficiency of carbohydrate sulfotransferase 3 (CHST3; chondroitin-6-sulfotransferase) in six subjects diagnosed with recessive Larsen syndrome or humero-spinal dysostosis [Hermanns et al. (2008); Am J Hum Genet 82:1368-1374]. Since then, we have identified 17 additional families with CHST3 mutations and we report here on a series of 24 patients in 23 families. The diagnostic hypothesis prior to molecular analysis had been: Larsen syndrome (15 families), humero-spinal dysostosis (four cases), chondrodysplasia with multiple dislocations (CDMD "Megarbane type"; two cases), Desbuquois syndrome (one case), and spondylo-epiphyseal dysplasia (one case). In spite of the different diagnostic labels, the clinical features in these patients were similar and included dislocation of the knees and/or hips at birth, clubfoot, elbow joint dysplasia with subluxation and limited extension, short stature, and progressive kyphosis developing in late childhood. The most useful radiographic clues were the changes of the lumbar vertebrae. Twenty-four different CHST3 mutations were identified; 16 patients had homozygous mutations. We conclude that CHST3 deficiency presents at birth with congenital dislocations of knees, hips, and elbows, and is often diagnosed initially as Larsen syndrome, humero-spinal dysostosis, or chondrodysplasia with dislocations. The incidence of CHST3 deficiency seems to be higher than assumed so far. The clinical and radiographic pattern (joint dislocations, vertebral changes, normal carpal age, lack of facial flattening, and recessive inheritance) is characteristic and distinguishes CHST3 deficiency from other disorders with congenital dislocations such as filamin B-associated dominant Larsen syndrome and Desbuquois syndrome.

Anterior fracture-dislocation is more severe than lateral: a biomechanical and neuropathological comparison in rat thoracolumbar spine.

ABSTRACT Fracture-dislocation is one of the most common causes of spinal cord injury (SCI) in human adults, yet it is not widely studied experimentally. Clinical studies have found that anterior fracture-dislocation occurs more commonly and produces greater neurological deficit than lateral fracture-dislocation. However, the effect of loading direction on SCI neuropathology has not been investigated experimentally and the reasons behind these clinical differences are not known. Thoracolumbar vertebrae T12-L1 of anaesthetized rats were dislocated anteriorly or laterally by 9 mm at 220 mm/sec. Spinal cord sections from animals euthanized at 1, 3, and 6 h post-injury, were stained with hematoxylin and eosin (H&E) to detect hemorrhage, the pathologic accumulation of beta-amyloid precursor protein (betaAPP) in white matter axons, and degenerating neurons (Fluoro-Jade and loss of NeuN) in the gray matter. The vertebral fracture load and maximum load were similar for both directions of dislocation; however, vertebral fracture occurred at 4.3 mm (+/-1.5 mm SD) during anterior dislocation compared to 1.1 mm (+/-0.7 mm SD) during lateral dislocation (p < 0.001). betaAPP accumulation and reduction of NeuN immunoreactivity (IR) were greatest along a diagonal band across the spinal cord angled at 45 degrees to the direction of loading (in different planes for each loading direction). Hemorrhage volume (p < 0.05), betaAPP-IR, and reduction of NeuN-IR (p < 0.05 in ventral horns) were more pronounced following anterior dislocation. In addition, there was a different spatial distribution of axonal damage for each direction of dislocation. The findings of this study may explain the greater severity of anterior fracture-dislocation observed clinically and reinforces the need to experimentally model differing human SCIs.

Specific expression of substance P in synovial tissues of patients with symptomatic, non-reducing internal derangement of the temporomandibular joint: comparison with clinical findings.

Our aim was to find out the extent of expression of substance P in synovial tissue from the human temporomandibular joints (TMJ) with symptomatic, non-reducing internal derangement, and to investigate the relationship between substance P and clinical findings. Fifty-four joints in 54 patients were examined immunohistochemically. Specimens of synovial tissue from 10 joints in 8 subjects with habitual dislocation of the TMJ with no pain were examined as controls. Cells that stained for substance P were found mainly among the endothelial cells in the blood vessels beneath the lining cells in synovial tissues from 47 of the 54 joints (87%) with internal derangement and from 5 of the 10 control joints. The extent score of cells that stained for substance P in joints with internal derangement was significantly higher than that in controls (p=0.02). The extent score of these cells did not correlate with pain in the joint or the degree of synovitis. These results suggest that substance P may have some roles in both the physiological and pathological conditions in patients with symptomatic internal derangement of the TMJ.

Increase in RANKL: OPG ratio in synovia of patients with temporomandibular joint disorder.

Although a recent study suggested the involvement of RANKL and osteoprotegerin (OPG) in the pathogenesis of bone-destructive disease, no study has focused on the RANKL:OPG ratio in the synovial fluid of patients with temporomandibular joint (TMJ) disorder. This communication reports on the concentrations of RANKL and OPG in synovial fluid from TMJ patients and healthy control individuals. In contrast to an unchanged concentration of RANKL, a strong decrease in the concentration of OPG was detected in the synovial fluid from patients with TMJ internal derangement. Treatment with the synovial fluid of osteoarthritis (OA) patients resulted in the high production of osteoclast-like cells from blood mononuclear cells in vitro, as well as in pit formation in dentin slices. The addition of anti-RANKL IgG or OPG attenuated OA-synovial fluid-induced osteoclast formation, suggesting that the increase in the RANKL:OPG ratio in the microenvironment of the joint has the potential to induce osteoclastogenesis in TMJ osteoarthritis.

The pathogenesis of congenital radial head dislocation/subluxation.

The pathogenesis of congenital radial head dislocation/subluxation is unknown and has not been previously investigated. In this review, we explore the pathogenesis and define five different primary insults: collagen abnormalities, abnormal endochondral ossification of the developing growth plate, abnormalities of forearm ossification outside the growth plate, disproportionate growth of the radius and ulna, and altered HOX D expression/activity. Finally, the clinical relevance of our review is discussed.

The roles of Tenascin C and Fibronectin 1 in adhesive capsulitis: a pilot gene expression study.

OBJECTIVES: We evaluated mRNA expression levels of genes that encode TGF-ß1; the TGF-ß1 receptor; the collagen-modifying enzymes LOX, PLOD1, and PLOD2; and the extracellular matrix proteins COMP, FN1, TNC and TNXB in synovial/capsule specimens from patients with idiopathic adhesive capsulitis. Possible associations between the measured mRNA levels and clinical parameters were also investigated. METHODS: We obtained glenohumeral joint synovium/capsule specimens from 9 patients with idiopathic adhesive capsulitis who had not shown improvement in symptoms after 5 months of physiotherapy. Adhesive capsulitis was confirmed in all patients by magnetic resonance imaging. We also obtained specimens from 8 control patients who had underwent surgery for acute acromioclavicular joint dislocation and who had radiological indication of glenohumeral capsule alteration based on arthroscopic evaluation. mRNA expression in the synovium/capsule specimens was analyzed by quantitative reverse transcription PCR. The B2M and HPRT1 genes were used as references to normalize target gene expression in the shoulder tissue samples. RESULTS: The synovium/capsule samples from the patients with adhesive capsulitis had significantly higher TNC and FN1 expression than those from the controls. Additionally, symptom duration directly correlated with expression of TGFß1 receptor I. CONCLUSION: Elevated levels of TNC and FN1 expression may be a marker of capsule injury. Upregulation of TGFß1 receptor I seems to be dependent on symptom duration; therefore, TGFß signaling may be involved in adhesive capsulitis. As such, TNC, FN1 and TGFß1 receptor I may also play roles in adhesive capsulitis by contributing to capsule inflammation and fibrosis.

Elevated levels of beta-endorphin in temporomandibular joint synovial lavage fluid of patients with closed lock.

AIMS: To investigate the presence of endogenous beta-endorphin, an opioid, in the synovial lavage fluid of the temporomandibular joint (TMJ), and to compare the concentration of 3-endorphin in patients with closed lock with that in symptom-free subjects. METHODS: Thirty-eight patients (38 joints) with closed lock diagnosed on the basis of the results of clinical examination and magnetic resonance imaging (MRI) and 11 healthy volunteers (19 joints) were examined. Samples of lavage fluid were obtained prior to arthrocentesis by washing the joint with saline. Samples were assayed for beta-endorphin by an enzyme immunoassay, and concentrations of protein were measured by a bicinchoninic acid assay. Subjective pain was assessed by patients using a visual analog scale. Bone changes in the condyle were assessed by MRI, and synovitis was assessed on the basis of arthroscopic findings. RESULTS: beta-endorphin was present in the synovial fluid of the TMJ, and the concentration was significantly higher in patients with closed lock of the TMJ compared to symptom-free volunteers. The beta-endorphin levels were not, however, significantly correlated with clinical parameters in the patients. CONCLUSION: The study results support recent findings that some opioids and their receptors exist not only within the central nervous system but also in the TMJ region, and that opioid concentrations are higher in patients with pain and dysfunction of the TMJ.

Levels of soluble cytokine factors in temporomandibular joint effusions seen on magnetic resonance images.

OBJECTIVE: To elucidate the correlations between joint effusion (JE) on T2-weighted magnetic resonance images (MRI) of the temporomandibular joint (TMJ) and the levels of various cytokine receptors, cytokine antagonists, and protein in the synovial fluid of patients with temporomandibular joint disorders (TMD). STUDY DESIGN: Fifty-five TMJs of 55 patients with TMD were scanned by MRI, and synovial fluid samples were obtained on the same day. The grade of JE was evaluated on a scale of 0 to 3: Grades 0 and 1 indicated absence, and grades 2 and 3 indicated the presence of JE. Correlations were evaluated between JE and the concentrations of soluble tumor necrosis factor receptors I and II (sTNFR-I and sTNFR-II, respectively), IL-6 soluble receptor (IL-6sR), IL-1 soluble receptor type II, and IL-1 receptor antagonist and protein in the synovial fluid of patients with TMD. RESULTS: The concentrations of sTNFR-I and protein in the group with JE (18 joints) were significantly higher than in the group without JE (37 joints). In addition, there were significant positive correlations between the grade of JE and the levels of sTNFR-I, sTNFR-II, and protein. CONCLUSIONS: sTNFRs and protein may play important roles in the pathogenesis of TMD. These mediators seem to influence the expression of JE, which may reflect synovial inflammation of the TMJ.

Gene expression of fibrinolytic factors urokinase plasminogen activator and plasminogen activator inhibitor-1 in rabbit temporo-mandibular joint cartilage with disc displacement.

BACKGROUND: The urokinase plasminogen activator system is believed to play an important role in degradation of the extracellular matrix associated with cartilage and bone destruction; however its precise roles in temporomandibular disorders have not yet been clarified. The aims of this study were to investigate the gene expression of fibrinolytic factors urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in the articular cartilage of rabbit temporomandibular joint (TMJ) with disc displacement (DD) and to probe the relationship between fibrinolytic activity and cartilage remodeling. METHODS: Disc displacement of right joints was performed in 36 of 78 rabbits under investigation. The animals were sacrificed at 4 days and 1, 2, 4, 8 and 12 weeks after surgery, respectively. The right joints of these animals were harvested and processed for the examination of mRNA expression of uPA and PAI-1 in articular cartilage using in situ hybridization techniques. RESULTS: The expression of uPA and PAI-1 was co-expressed weakly in the chondrocytes from transitive zone to hypertrophic zone and mineralized zone, while no hybridizing signals were shown in proliferative zone and superficial zone in control rabbits. The most striking was the up-regulation of uPA and PAI-1 mRNA in 4-day rabbits postoperatively at the onset of cartilage degeneration. The strongest hybridizing signals for uPA and PAI-1 were seen in 2-week rabbits postoperatively. After 2 weeks, the expression of uPA and PAI-1 began to decrease and reached nearly normal level at 12 weeks. CONCLUSIONS: The expression of the uPA/PAI-1 system coincides with the pathological changes in condylar cartilage after DD. The uPA/PAI-1 system may be one of the essential mediators in articular cartilage remodeling.

Changes in nitric oxide levels in striated muscles of rats following different types of death.

BACKGROUND: Nitric oxide is a signal molecule regulating the organism functions in living bodies. The aim of this study was to investigate the NO levels of striated muscles after different types of death in rats. METHODS: Nitric oxide levels in the muscles of masseter, triceps, and quadriceps obtained from right and left sides of 24 Spraque-Dawley rats following death were investigated. The rats were divided into three groups as cervical dislocation (control) group, electric shock group, and drowning group. After applying a light anesthesia, the rats were killed by cervical dislocation, electric shock and drowning. The samples were taken immediately and 120 minutes after death. RESULTS: In all muscle types of all groups, NO concentrations were lower in samples obtained 120 minutes after death than in those obtained immediately after death. NO concentrations were lower in the electric shock and drowning group than in the control group for both times. CONCLUSION: We can conclude that the type of death may affect the occurrence of rigor mortis and NO measurement may give an important clue in evaluation the mode of death.

[Experimental research on substance P content of hypothalamus and dorsal root ganglia in rats with lumbar vertebrae Gucuofeng model].

OBJECTIVE: To detect the effects of lumbar vertebrae Gucuofeng on the substance P content of hypothalamus and dorsal root ganglia in rat models. METHODS: A hundred and twenty SPF level SD male rats with the weight of 350 to 450 g were randomly divided into rotary fixation group (RF group), simple fixation group (SF group) and sham-operation group (Sham group). The external link fixation system was implanted into the L4-L6 of rats in RF group and SF group; and in RF group, that the L5 spinous process was rotated to the right resulted in L4, L5, L6 spinous process not collinear; in SF group, the external link fixation system was simply implanted and not rotated. The rats of Sham group were not implanted the external link fixation system and only open and suture. The substance P content of hypothalamus and dorsal root ganglia were detected at 1, 4, 8, 12 weeks after operation. RESULTS: Substance P content of hypothalamus in RF group and SF group was lower than Sham group at 1, 4, 8 weeks after operation (P<0.05). Substance P content of dorsal root ganglia was higher than Sham group at 1, 4, 8, 12 weeks after operation (P<0.05). There was no significant differences in the substance P content of hypothalamus among three groups at 12 weeks after operation (P>0.05). CONCLUSION: Lumbar vertebrae Gucuofeng can inhibit the analgesic activity of substance P in hypothalamus and promote the synthesis and transmission of substance P in dorsal root ganglia, so as to cause or aggravate the pain.

Antioxidant capacity of synovial fluid in the temporomandibular joint correlated with radiological morphology of temporomandibular disorders.

We investigated the correlation between the antioxidant capacity of synovial fluid and radiological findings of intra-articular structures in patients with disorders of the temporomandibular joint (TMJ). We recruited 21 patients (9 men and 12 women, aged 18-84 years of age) with such disorders, excluding myofascial pain and dysfunction syndrome, or other muscular disorders. The clinical variables recorded included age, sex, interincisal distance, and visual analogue pain scores (VAS). Radiological findings were obtained from diagnostic arthrogram and cone-beam computed tomography (CT). The antioxidant capacity of the synovial fluid was measured by chemiluminescence. Eleven patients were radiologically diagnosed with closed lock, and the remaining 10 with no closed lock. An anchored intra-articular disc was most often seen on cone-beam CT (n=19) followed by perforated disc (n=7), osteoarthrosis (n=7), and anterior disc displacement without reduction (n=5). Although there were no significant differences between antioxidant capacity and age, sex, VAS, or any findings on cone-beam CT, antioxidant capacity was significantly decreased in the patients with closed lock compared with those who did not have closed lock (p=0.02). The results suggest an association between the oxidative stress of the synovial fluid and closed-lock in disorders of the TMJ.

Patient with multiple congenital anomalies and decreased production and processing of procollagen in cultured fibroblasts.

We report on a patient with hip and elbow dislocations, joint hyperextensibility, peculiar facial appearance, torticollis, cryptorchidism, unilateral hexadactyly, and other minor anomalies. Cultured cells from this patient produce less type I procollagen and have a slower rate of processing of type I procollagen to collagen in the culture medium. We think that the pattern of clinical anomalies constitutes a previously unreported syndrome with type I procollagen defect as a manifestation of the syndrome.

Correlations of the expression of fibroblast growth factor-2, vascular endothelial growth factor, and their receptors with angiogenesis in synovial tissues from patients with internal derangement of the temporomandibular joint.

Synovitis in internal derangement of the temporomandibular joint (TMJ) is accompanied by the growth of new blood vessels. Fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) are well-characterized angiogenic factors. The objective of this study was to elucidate the correlation between the expression of FGF-2, VEGF, and their receptors-FGF receptor-1 (FGFR-1) and VEGF receptor-1 (Flt-1)-with microvessel density in synovial tissues of the TMJ. Using an immunohistochemical technique, we examined 47 joints (45 patients) with internal derangement. Individual microvessel density was evaluated by means of the CD34 antibody, a specific endothelial marker. The correlation between the percentage of immuno-positive cells and microvessel density was evaluated. In multiple logistic regression analysis, the correlation between the percentage of Flt-1-positive cells and microvessel density was significant [p = 0.005, odds ratio = 1.071, 95% confidence interval = 1.021-1.124]. These results suggest that the expression of the VEGF/Flt-1 system is involved in angiogenesis in inflamed synovial tissue in the TMJ.

Type II collagen and aggrecan mRNA expression by in situ hybridization in rabbit temporomandibular joint posterior attachment following disc displacement.

Pathological changes and mRNA expression were studied in the posterior attachment of 40 adult Japanese white rabbits. The right temporomandibular joints of 28 rabbits were subjected to surgical disc displacement. Joints were studied by histochemistry and in situ hybridization. The collagen in the posterior attachment became dense, especially near the posterior band of the disc. Chondrocytes were found not only in the disc but also in the posterior attachment. Sometimes cartilage formation was seen. Type II collagen mRNA expression was first detected in the posterior attachment 4 days postoperatively and became progressively stronger with time. Aggrecan expression in the posterior attachment decreased at first, then increased gradually. It was concluded that, in the temporomandibular joint, chondrocytes appear in the posterior attachment as a result of biomechanical stimuli and the attachment becomes fibrocartilaginous following disc displacement.

An immunohistochemical study of the effects of surgical induction of anterior disc displacement in the rabbit craniomandibular joint on type I and type II collagens.

The right craniomandibular joint (CMJ) was exposed surgically and all the discal attachments severed except for the posterior one. The disc was then repositioned anteriorly and sutured to the zygomatic arch. The left joint served as a sham-operated control; 10 other joints were used as non-operated controls. Deeply anaesthetized rabbits were perfused with 2% buffered formalin 2 weeks (10 rabbits) or 6 weeks (10 rabbits) after the induction of the anterior disc displacement (ADD). The articular disc, bilaminar zone, mandibular condyle and articular eminence were excised. The condyles and the articular eminences were demineralized in EDTA. All tissues were then sectioned at 10 microns in a cryostat. Sections were incubated with polyclonal antibodies directed against type I or type II collagens. Following incubation in the appropriate fluorescein isothiocyanate-labelled secondary antibodies, these specimens were studied under the fluorescence microscope. At 2 weeks there was a reduction in type II collagen immunostaining; some areas of the experimental condylar cartilage showed a switch from type II to type I collagen. However, at 6 weeks there was an increase in type II collagen immunostaining and a decrease in type I compared to the 2-week group. It is concluded that surgical induction of ADD in the rabbit CMJ leads to alteration in the condylar cartilage collagen phenotype similar to that reported for osteoarthritic cartilage of other synovial joints.