Regulation of HAS expression in human synovial lining cells of TMJ by IL-1beta

Hyaluronan (HA), a major glycosaminoglycan of synovial fluid, is synthesised by a class of membrane-bound HA synthase (HAS) proteins. In the present study, we investigated the regulatory roles of IL-1beta on HAS gene expression and HA production by the fibroblastic synovial lining cells. The synovial lining cells from synovial membrane in human temporomandibular joint (TMJ) were cultured and characterised using immunocytochemistry with CD14, CD44, and vimentin monoclonal antibodies. With or without treatment with IL-1beta, the production of HA was detected with radiometric assay and the expression of HAS mRNAs were analysed with a semi-quantitative reverse transcribed polymerase chain reaction (RT-PCR). HA synthesis was significantly augmented with 1ng/ml of IL-1beta for both 24 and 48h stimulation, however the production of HA declined if stimulated with 10ng/ml of IL-1beta. The expression of HAS2 and 3 mRNA were enhanced about 4.2- and 7.2-fold after 4h stimulation with 1ng/ml of IL-1beta, respectively. From these results, it is concluded that IL-1beta functions on regulating HAS expression and consequently promoting the secretion of HA in synovial lining cells from TMJ.     

Interleukin-1β induces interleukin-6 mRNA expression and protein production in synovial cells from human temporomandibular joint

BACKGROUND: Interleukin (IL)-1 beta and IL-6 were found to be elevated in fluid from the temporomandibular joint (TMJ), although the source of these cytokines was not elucidated. There is little known about the function and response of synovial cells in the TMJ. The purpose of this study was to prepare cultured human synovial cells (HTS cells) from the TMJ and to investigate IL-6 production in HTS cells incubated with IL-1 beta. METHODS: HTS cells were isolated from temporomandibular joint synovial tissue using an outgrowth method and then cultured in Ham's F12 medium containing 10% fetal bovine serum. The HTS cells were treated with or without IL-1 beta for 3, 6, 9 and 24 h. IL-6 and soluble IL-6 receptor (sIL-6R) levels in cultured supernatant were measured by ELISA. IL-6 mRNA expression was investigated using immunocytochemistry and RT-PCR. RESULTS: HTS cells were morphologically heterogeneous. IL-1 beta increased IL-6 production in HTS cells. In those treated with IL-1 beta, several cells were strongly stained in the cytoplasm around the nucleus, while several cells were weakly stained in this area. IL-1 beta also stimulated IL-6 mRNA expression. In contrast, sIL-6R could not be detected in cells treated with or without IL-1 beta. CONCLUSIONS: IL-1 beta increased IL-6 production in synovial cells resulting from an increase in IL-6 mRNA expression. Enhanced production of IL-6, which is associated with bone resorption and inflammatory response, seems to be related to the progression of TMJ disorders.     

Immunocytochemical demonstration of laminin in the synovial lining layer of the rat temporomandibular joint.

This immunocytochemical study describes the distribution of laminin in the synovial lining of the rat temporomandibular joint. Laminin immunostaining was present around some synovial lining cells and blood vessels. Ultrastructurally, immunoreactive products for laminin were deposited around cells with a well-developed rough endoplasmic reticulum and secretory granules, suggesting that they were type B synovial lining cells. The localization of laminin immunoreactivity was not uniform around the cell membrane, the most intense immunoreaction being present on the basal aspect membrane as is seen in the basement membrane of epithelia. In contrast, macrophage-like synovial lining type A cells did not show laminin immunoreactivity. This different immunostaining pattern suggests that laminin acts as an adhesion molecule for the type B cells in their epithelial-like arrangement.     

Inducible nitric oxide synthase and apoptosis-related factors in the synovial tissues of temporomandibular joints with internal derangement and osteoarthritis.

PURPOSE: In this study, we investigated the relationship between oxidative stress and apoptosis in synovial tissues in temporomandibular joint diseases (TMDs), including internal derangement (ID) and osteoarthritis (OA), comparing immunohistochemical, arthroscopic, and histologic findings. MATERIALS AND METHODS: Synovial specimens obtained from patients with ID (31 patients), osteoarthritis (11 patients), and condylar fractures of the mandible (5 patients) during arthroscopy were examined immunohistochemically using antibodies against CD68, inducible nitric oxide synthase (iNOS), Fas, and single-stranded DNA (ssDNA). RESULTS: CD68 and iNOS immunoreactivity were detected mainly in synovial lining cells and subintimal macrophages, and tended to increase with synovial hyperplasia. Fas and ssDNA immunoreactivity was detected mainly in synovial lining cells, and Fas-positive regions exhibited a number of ssDNA-positive cells. Fas expression was significantly greater in fractures than in OA, and ssDNA expression was significantly greater in OA than in ID. Fas expression was significantly greater in iNOS-positive versus iNOS-negative TMJs, and ssDNA expression tended to increase with iNOS expression. CONCLUSION: These immunohistochemical findings suggest that oxidative stress and apoptosis in synovial tissues are involved in the onset and progression of TMDs.     

Immunocytochemical localization of cathepsin L in the synovial lining cells of the rat temporomandibular joint

Localization of cathepsin L in the synovial lining cells of the normal rat temporomandibular joint was investigated by the avidin-biotin-peroxidase complex method for semithin (1 μm) cryosections and the colloidal gold-labelled IgG method for ultrathin sections of LR gold resin. At the light-microscopic level, type A (macrophage-like) and B (fibroblast-like) cells formed the synovial lining layer. Extensive immunoreactivity for cathepsin L was observed in many granules and vacuoles of type A cells, while in the type B cells, immunoreactivity was found in very few granules. In the sublining layer, macrophages and a few fibroblasts were positive for cathepsin L. By electron microscopy, at the peripheral cytoplasm of the type A cells close to the lateral intercellular spaces and joint cavity, numerous coated vesicles and vacuoles (probably early endosomes) indicating endocytotic function were found. Gold particles indicating cathepsin L were localized in the vesicles (primary lysosomes) in the perinuclear cytoplasm and in the larger amorphous vacuoles (1 μm dia) as phagolysosomes. In type B cells, gold particles were limited to the vesicles only (primary lysosomes). The cathepsin L-positive primary lysosomes were numerous in a few fibroblasts in the sublining layer. These results indicate that type A cells contain a large amount of cathepsin L, and suggest that these cells endocytose surplus substances such as collagen and proteoglycan fragments in normal rat TMJ, effecting their digestion and degradation by the action of this proteolytic cathepsin.     

Immunohistochemical localization of inducible nitric oxide synthase in synovial tissue of human temporomandibular joints with internal derangement

The expression and distribution of inducible nitric oxide synthase (iNOS) was examined in 12 samples of human temporomandibular joint (TMJ) with internal derangement (ID) and four control specimens. In the diseased joints, strong or definite iNOS reactivity was expressed in synovial lining and endothelial cells; weaker activity was present in synovial fibroblasts. In contrast, although there was weak expression of iNOS in synovial fibroblasts and endothelial cells in the two control specimens, there was no iNOS staining in the synovial lining cell layers. This original report that iNOS is expressed in the synovial tissue of the temporomandibular joint indicates that nitric oxide is produced locally at least in the synovial lining in these joints when affected by internal derangement.     

Ultrastructural study of synovitis induced by trauma to the rat temporomandibular joint (TMJ)

BACKGROUND: Electron microscopy was used to examine the histologic effect of trauma on the rat temporomandibular joint synovial membrane. METHODS: Trauma to the TMJ in male Wister rats (100-200 g) was introduced through repeated forced condylar hypermobility. Ultrastructural observations were made 5 days and 6 weeks after the trauma. RESULTS: The early response of the synovial membrane was synovial hyperplasia, type A synovial cell loss, dilation of the r-ER in the type B synovial cells and fibrin deposition on the synovial surfaces. The late response included degeneration of synovial cells with swollen mitochondria and cell projections, and cell fragmentation. Large amount of fibrin deposition on opposing surface layers was also noticed. CONCLUSION: The type A cell loss and fibrin deposition followed by the occurrence of fibrinous materials at opposing surface layers of the synovial membrane suggest that traumatic synovitis causes synovial adhesions.     

The expression of tenascin mRNA in human temporomandibular joint specimens

The expression of mRNA of tenascin in the temporomandibular joint (TMJ) disc and synovial membrane was examined in 20 human TMJ samples from patients with internal derangement of the TMJ and 10 control specimens by in situ hybridization technique using paraffine-embedded tissue, and antisense and sense cRNA probes. In control specimens, tenascin mRNA was not expressed. However, we were able to find tenascin mRNA expression in the surgical specimens. In 15 of 20 samples, ranging numbers of synovial cells expressed tenascin mRNA in the hypertrophic synovial membranes. Also, in 6 of 20 samples, tenascin mRNA was identified in fibroblasts. In four specimens, vascular endothelial cells were positive for the mRNA. In internal derangement cases, histopathological findings are often found such as synovitis, new capillary growth and fibrosis. The present study demonstrates that tenascin is produced specifically in synovial cells, vascular endothelial cells and fibroblasts affected in the portion of TMJ with internal derangement.     

Co-expression of interleukin-1β and tumor necrosis factor α in synovial tissues and synovial fluids of temporomandibular joint with internal derangement: comparision with histological grading of synovial inflammation

BACKGROUND: It has been clarified that interleukin-1 (IL-1)beta and tumor necrosis factor (TNF)alpha play an important role in pathogenesis of various joint disease. The purpose of this study was to investigate the cellular source of IL-1beta and TNFalpha in temporomandibular joint (TMJ), and to analyze the relation between the expression of these cytokines and the intensity of TMJ synovial inflammation. METHODS: We examined 33 synovial biopsy specimens from patients with internal derangement of the TMJ by an immunohistochemical technique using specific antibodies to IL-1beta and TNFalpha. We also studied 20 synovial fluids from the patients by enzyme-linked immunosorbent assay method. These data are compared with histological grading of synovial inflammation by Gynther's system. RESULTS: Both IL-1beta and TNFalpha were predominantly localized in the synovial lining cell layer and the blood vessels of synovial biopsy specimens obtained from patients with TMJ internal derangement. A statistically significant correlation was found between the intensity of IL-1beta expression and that of TNFalpha. Additionally, the intensity of TNFalpha expression was statistically correlated with histological grading by Gynther's system. CONCLUSION: These results supported that IL-1beta and TNFalpha may be involved in the occurrence of TMJ internal derangement and that they coordinately play an role in pathogenesis of TMJ internal derangement.     

Up-regulation of vascular endothelial growth factor in synovial fibroblasts from human temporomandibular joint by hypoxia

INTRODUCTION: Enhanced expression of vascular endothelial growth factor (VEGF) has been described in patients with internal derangement (ID). Herein, we examined the expression of VEGF in synovial fibroblasts from temporomandibular joint (TMJ) under hypoxia and investigated the regulation of hypoxia-inducible factor-1alpha (HIF-1alpha) involved in the expression of VEGF. METHODS: Synovial fibroblasts were prepared from human TMJ. These cells were incubated under hypoxia or normoxia for the indicated time periods. VEGF levels in cultured supernatant were measured by an ELISA. VEGF mRNA isoforms and stability were assessed using RT-PCR and Northern blot analysis respectively. HIF-1alpha accumulation was evaluated by Western blotting and immunofluorescence. RESULTS: VEGF were significantly induced by hypoxia in synovial fibroblasts. In response to hypoxia, VEGF121 and VEGF165 mRNA were both remarkably increased, while there was no change in VEGF mRNA stability. The accumulation and nuclear translocation of HIF-1alpha occurred under hypoxia. CONCLUSIONS: Hypoxia may mainly induce the expression of VEGF121 and VEGF165 in synovial fibroblasts to promote inflamed angiogenesis of TMJ. HIF-1alpha, which is clearly activated in response to hypoxia, may control the expression of VEGF in synovial fibroblasts from TMJ.     

肿瘤坏死因子对颞下颌关节滑膜影响的超微结构观察

本文采用透谢电镜和图像分析法，对12侧家兔颞下颌关节内一次性注射肿瘤坏死因子（TNF）后滑膜变化进行了观察。结果表明：颞下颌关节腔内注射肿瘤坏死因子后可引起下列三种变化：①额下颌关节滑膜细胞性内膜层增厚和血管增生；②B型滑膜细胞大量增殖及细胞内结构改变；③对A型滑膜细胞无影响。实验结果提示：肿瘤坏死因子可导致颞下颌关节沿膜结构变化，该变化类似关节滑膜早期退行性变。     

The immunohistochemical distribution of vimentin in human temporomandibular joint samples

The aim of this investigation was to evaluate the immunohistochemical distribution of vimentin in the temporomandibular joint (TMJ) and to compare it with the control specimens. Immunohistochemical distribution in the disc and synovial membrane in 30 human TMJ (internal derangement of TMJ, n = 20; and control, n = 10) was studied immunohistologically using paraffin-embedded tissue and specific anti-human vimentin monoclonal antibody. Vimentin expression was distributed in chondrocyte-like cells, synovial cells and endothelial cells. There was an obvious distinction of vimentin immunoreactivity between the control specimens and internal derangement cases, in the posterior and/or anterior loose connective tissues. In particular, intensive vimentin expression was detected in the hypertrophic synovial membrane of internal derangement cases. The findings of the present study suggest that vimentin might be an important marker of pathological hypertrophy of the synovial membrane and/or connective tissue with internal derangement of TMJ.     

Microarray analysis of IL-1β-stimulated chemokine genes in synovial fibroblasts from human TMJ

BACKGROUND: Interleukin (IL)-1beta is thought to play a key role in several pathologic conditions of the temporomandibular joint (TMJ). Gene expression profile of synovial fibroblasts stimulated with IL-1beta was studied by oligonucleotide microarray analysis to elucidate candidate genes associated with intracapsular pathologic conditions of TMJ. METHODS: RNA was isolated from synovial fibroblasts from five patients after IL-1beta treatment. Gene expression profiling was performed with a GeneChip. Changes in gene expression were determined by comparing IL-1beta-treated cells with untreated cells. RESULTS: A total of 121 genes showed a greater than threefold difference in average intensity between untreated and IL-1beta-treated synovial fibroblasts in five experiments. Five chemokines were among the 10 most upregulated genes, and the most upregulated gene was CCL20. The 121 IL-1beta-responsive genes included 12 chemokines whose mRNA levels were confirmed by real-time PCR. CONCLUSION: These data should provided useful information about the pathologic conditions of TMJ, especially in support of diagnosis and therapeutic approaches to TMJ.     

Quantitative histological changes of repeated antigen-induced arthritis in the temporomandibular joints of rabbits treated with intra-articular corticosteroid

Background:  To compare the inflammatory changes of antigen-induced temporomandibular joint (TMJ) arthritis in rabbits by different histological methods and to evaluate the immunomodulatory effect of intra-articular corticosteroid injections histologically.
Methods:  35 rabbits (10 weeks old) pre-sensibilized with ovalbumin were divided into three groups: a placebo group of five (saline), an arthritis group of 15 (ovalbumin) and a steroid-treated group of 15 (ovalbumin + corticosteroid). Additionally, a group of seven rabbits receiving no sensibilization with ovalbumin and no intra-articular injections served as controls. Histomorphometry of the inflammatory changes in the subsynovial connective tissue (SSCT) of the TMJ included: (i) semi-quantitative (S-Q) scoring of inflammation and synovial proliferation, (ii) thickness measurements and fractional surface and (iii) stereological quantitative assessment of volume and plasma cells in thick sections of the SSCT by an optical fractionator.
Results:  The histomorphometry showed synovial proliferation in both the arthritis and the steroid groups. The plasma cell count obtained by the optical fractionator was significantly reduced when treating the TMJ with corticosteroids. However, the thickness of the synovial lining and volume of the SSCT as well as S-Q scoring of inflammation showed no difference between the arthritis and the steroid-treated groups. The optical fractionator proved a superior tool compared to S-Q assessments.
Conclusion:  Counting of plasma cells in the SSCT showed that corticosteroids reduced the inflammation, but did not eliminate it. Semiquantitative scoring of synovial proliferation and inflammation demonstrated low sensitivity regarding changes in immunomodulation in antigen-induced arthritis compared to stereological quantitative estimations using an optical fractionator.     

Expression of 25 kDa heat shock protein by synovial type B cells of the mouse temporomandibular joint.

Earlier studies have demonstrated immunoreactivity for heat shock protein 25 (Hsp25) in type B synovial lining cells of the rat temporomandibular joint, and also the presence of characteristic cytoplasmic processes in these cells, but it is unclear whether or not the type B cells in other animals possess such elaborate cytoplasmic projections and as there is as yet no evidence for the synthesis of this protein by these cells. For these reasons, the expression of Hsp25 was investigated in the synovial membrane of the mouse temporomandibular joint by immunocytochemistry and by in situ hybridization using a specific cRNA probe. Intense immunoreaction for Hsp25 was found in the cytoplasm of certain synovial lining cells that were identified as type B by immunoelectron-microscopy. These Hsp25-positive cells had slender cytoplasmic processes, either projecting towards or covering the synovial surface. Morphological differences between cytoplasmic processes seemed to depend on the location of the type B cell bodies. In situ hybridization showed intense signals for Hsp25 mRNA in the synovial lining cells, suggesting that the type B cells produce, rather than resorb, Hsp25. These findings indicate that Hsp25 is a useful marker for the identification of the synovial type B cells in the temporomandibular joint. It is further hypothesized that Hsp25 in type B cells is involved in maintaining their specific profile and epithelial-like arrangement, and in protecting against mechanical stress.     

Bone morphogenetic protein-2 in temporomandibular joints with internal derangement

OBJECTIVE: The purpose of this study was to analyze the expression of bone morphogenetic protein-2 (BMP-2) in patients with internal derangement of the temporomandibular joint. STUDY DESIGN: Twenty-one human temporomandibular joint samples (5 extirpated disks and 16 biopsy specimens of synovitis area from patients with internal derangement of the TMJ) and 2 control temporomandibular joint specimens (2 normal disks obtained by autopsy) were analyzed with specific antibodies through use of an immunohistochemical technique. RESULTS: BMP-2 was predominantly localized in chondrocytes around the damaged areas of the articular disks. BMP-2 expression was also found in synovial cells and endothelial cells of blood vessels. Control specimens demonstrated BMP-2 staining in synovial lining cells and endothelial cells of blood vessels. However, the chondrocytes in the normal cartilage layers of the control specimens showed no staining. CONCLUSIONS: These findings suggest that BMP-2 may be involved in the pathogenesis of osteoarthritic changes or the repair process of temporomandibular joint internal derangement.     

Development of temporomandibular disorder symptoms: a 3-year cohort study of university students

The aims of this study were to examine the incidence of temporomandibular disorders (TMDs) over a 3-year period and to evaluate the risk of self-reported TMDs among university students in Japan. The study population comprised 2374 university students examined at the start of their undergraduate course and 492 students re-examined after 3 years using questionnaires on symptoms of TMD and experiences of jaw injury, stress, orthodontic treatment and parafunctional habits. Cumulative incidence (%) and relative risks were calculated overall. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to determine the degree of risks of these variables for symptoms of TMDs using logistic regression. Results of logistic regression analysis showed that male subjects with experience of jaw injury had a 3·54 (CI=1·45-8·68, P<0·01)-fold higher risk of temporomandibular joint (TMJ) pain than that for those who did not. Female subjects who reported experiencing stress and bruxism had 10·56 (CI=1·28-87·54, P<0·05)- and 5·00 (CI=1·21-20·71, P<0·05)-fold higher risks of TMJ sound, respectively, than the risk for female subjects who had not experienced stress or bruxism. The results indicated that experiences of jaw injury, stress and bruxism were significantly associated with increased risks of development of TMJ disorders in a 3-year cohort.     

关节镜上腔灌洗术治疗中老年颞下颌关节紊乱病的临床研究

目的：评价应用颞下颌关节镜上腔灌洗术治疗临床表现为张口受限合并关节区疼痛的中老年颞颌关节紊乱病患者的临床疗效。方法：对保守治疗无效的16例颞下颌关节紊乱病引起张口受限合并关节疼痛的中老年患者，行颞下颌关节镜上腔灌洗术，分析治疗前后不同时期患者的疼痛值（疼痛直观模拟标尺VAS）、张口度和健侧侧向运动度变化，并通过MRI检测治疗前后关节盘位置的变化。结果：治疗后张口度35mm、健侧侧向运动≥6mm的患者占87．5％（14／16），不同时期的张口度均较治疗前有显著差异（P〈0．001），特别在治疗后1个月内增加明显，疼痛亦有显著缓解（P〈0．001），无并发症的发生。MRI显示，有1例患者的关节盘部分复位。结论：颞下颌关节内窥镜下的上腔灌洗术直视下操作准确，能有效治疗颞下颌关节紊乱病的患者，明显改善张口度和缓解疼痛。颞下颌关节镜治疗技术安全有效，有临床应用价值。     

Expression of interleukin 6 in synovial tissues in patients with internal derangement of the temporomandibular joint

Using an immunohistochemical technique, we examined synovial tissue from 46 temporomandibular joints (TMJ) with internal derangement in 44 patients. As controls, we examined synovial tissue specimens from 7 joints with habitual dislocation without pain. In synovial tissues from 21 of the 46 joints with internal derangement, interleukin 6 (IL-6) was expressed in the synovial lining cells and in the mononuclear cells infiltrating the periphery of the blood vessels. The density of IL-6-stained cells in specimens with internal derangement correlated significantly with the grade of joint effusion shown by magnetic resonance imaging (P=0.01, r=0.32).