Isolation and characterization of synovial cells from the human temporomandibular joint

BACKGROUND: The synovial tissues with temporomandibular disorders (TMDs) often show chronic inflammatory changes and the synovial cells participate in the pathogenic processes of TMDs. The synovial membrane is composed of a synovial lining layer and a connective sublining layer. The synovial lining layer is made up of two kinds of cells: macrophage-like type A and fibroblastic type B cells. The aim of this study was to isolate and characterize synovial cells from the human temporomandibular joint (TMJ). METHODS: Synovial cells were isolated using an explant culture method. Then, we characterized the cultured synovial cells (SGA2 cells) using immunocytochemistry. RESULTS: SGA2 cells expressed the fibroblastic markers vimentin and prolyl 4-hydroxylase; they also expressed laminin and heat shock protein 27, all of which are markers of type B cells. However, some cells expressed the macrophage marker CD68. These CD68-positive cells simultaneously expressed laminin. CONCLUSIONS: We isolated and cultured synovial type B cells from the human TMJ, and identified the presence of intermediate type synovial lining cells, having the phenotypic properties of both type A and type B cells, among the synovial lining cells.     

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.     

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.     

A fluid-phase endocytotic capacity and intracellular degradation of a foreign protein (horseradish peroxidase) by lysosomal cysteine proteinases in the rat junctional epithelium

We investigated the co-localization of lysosomal cathepsins B, H and L, and horseradish peroxidase (HRP) in junctional epithelial (JE) cells both as a fluid-phase endocytotic marker to demonstrate the fluid-phase endocytotic capacity of JE cells, and to understand the morphological relationships of the endocytosed foreign substances to lysosomal cathepsins in these cells. The diaminobenzidine (DAB) histochemical and cytochemical methods and immunohistochemical avidin-biotin-peroxidase complex and immunocytochemical post-embedding colloidal gold methods were used. Under light microscopy, DAB reaction products based on HRP were found in JE but were rare or absent in the oral sulcular epithelium and oral epithelium. Immunolabeling for cathepsins B and H was found in the granular structures of the cells, but no cathepsin L was identified. With electron microscopy, DAB reaction products, which indicated both HRP and the azurophil granules of neutrophils, were endocytosed into JE cells. Using a post-embedding technique, gold particles indicating HRP were present on the plasma membrane of JE cells, at the periphery of electronlucent vacuoles, and in the electrondense granules. Gold particles indicating cathepsin B or H were found in the electrondense granules. With different sizes of colloidal golds, the co-localization of cathepsin B or H with HRP was indicated only in the electrondense portion of the larger vacuoles consisting of electronlucent and -dense parts. This study provided the first morphological data which indicate that JE has a fluid phase endocytotic capacity, and which suggest that the lysosomal cathepsins B and H are involved in the intracellular degradation of foreign substances invading through the gingivl sulcus in JE cells.     

Cytochemical study of uptake of exogenous peroxidase by the rat submandibular salivary gland.

When horseradish peroxidase (HPO) was administered intravenously to rats, its reaction product was detected cytochemically in the basal lamella surrounding the submandibular gland cells and in the intercellular spaces between the gland cells in the first few minutes after administration. HPO was also seen in vesicles in the cells of both the acini and duct elements. Ten min to 6 h after administration, HPO was found only in large membrane-bound vacuoles (probably lysosomes). It is concluded that exogenous HPO is rapidly incorporated into the cells by pinocytosis through the basal and lateral cell membranes, and then the pinocytotic vesicles are fused with lysosomes, and that exogenous HPO is transported into the gland lumen by lysosomes rather than by penetration through tight junctions.     

Specific expression of inducible nitric oxide synthase in the synovium of the diseased temporomandibular joint

OBJECTIVE: The expression of inducible nitric oxide synthase (iNOS) in temporomandibular joint (TMJ) specimens obtained arthroscopically from diseased TMJs was investigated by using immunohistochemistry and compared with clinical, arthroscopic, and histologic findings. STUDY DESIGN: Synovial biopsies were obtained arthroscopically from 18 TMJs in 15 patients with symptomatic internal derangement (ID) or osteoarthritis (OA). We also obtained arthroscopic biopsies from 8 control TMJs (3 with habitual luxation of the mandible, one with ID with clicking, and 4 with mandibular condyle fractures). The expression of iNOS was examined by immunohistochemistry and was compared with clinical, arthroscopic, and histologic findings. RESULTS: Definite or intense iNOS immunoreactivity was observed in both the synovial lining cells and the endothelial cells of TMJs with symptomatic ID or OA. Weaker immunoreactivity was present in synovial fibroblasts. In contrast, in TMJs without synovitis (eg, those with habitual luxation of the mandible) the expression of iNOS was weak or marginal. The expression of iNOS correlated significantly with arthroscopic evidence of synovitis (r = 0.406, P <.05) but not with cartilaginous degeneration (P >.05). The expression of iNOS also correlated with the histologic grade of the synovial lining cell layers (r = 0.530, P <.05). However, in patients with ID or OA of the TMJ, there was no statistically significant correlation between the intensity of iNOS immunoreactivity and clinical, arthroscopic, or histologic findings (P >.05). CONCLUSION: These data clearly suggest that nitric oxide is locally produced in the synovial lining of the TMJ in ID and OA.     

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.     

Ultrastructure and cytochemistry of the Golgi apparatus and related organelles of the secretory ameloblasts of the rat incisor

Glutaraldehyde-fixed rat incisors were either post-fixed in ferrocyanide-reduced osmium impregnated with the Ur-Pb-Cu technique or incubated in the medium for acid-phosphatase (AcPase) reaction. The Golgi apparatus of the secretory ameloblast was composed of 4-7 cisternae, small vesicles and condensing vacuoles. It formed an elongated, continuous membrane system over from one- to two-thirds of the supranuclear cytoplasm. Condensing vacuoles seemed to be produced from the dilated margins of both Golgi cisternae and GERL. AcPase activity was demonstrated in the inner 2 or 3 Golgi cisternae, in GERL and in some condensing vacuoles and secretory granules within the Tomes process. It thus seemed that primary lysosomes originate from both the Golgi apparatus and GERL. Whereas many lysosomal bodies appeared in the supranuclear cytoplasm, autophagic vacuoles were rare. A well-developed Golgi apparatus and GERL were, therefore, considered to be involved in the digestion of exogeneous materials as well as in the formation of the precursor of enamel matrix. The simple Golgi apparatus consisting of only compactly stacked cisternae and small vesicles, were sometimes observed in the supranuclear cytoplasm and having no clear relationship with rough-surfaced endoplasmic reticula or GERL, may serve as a source of the plasma membranes necessary for continuous renewal of the cisternae of the well-developed Golgi apparatus.     

Ultrastructural study on the developmental process of the dentin bridge following direct capping using hydroxyapatite ceramic

Hydroxyapatite ceramics (HAP), which have been used in the endodontics or periodontics because of their bioafinitive characteristics, were applied directly as the capping of the wound surface after the experimental exposure of pulp in versional teeth to be extracted on orthodontics ground. In order to investigate the developmental process of dentine bridge induced by HAP capping, the pulp was studied ultrastructurally, 1, 3, 6 months after the treatment. One month after the capping, the multi-nuclear giant cells being rich in mitochondria and lysosomes were in contact with the HAP granules directly by extending their projections into the granules, which appeared to absorb the HAP granules. At the same time, the osteoblast-like cells with remarked rough endoplasmic reticulum (rER) were also observed just behind the giant cells. Only a few matrix vesicles and fibers were recognized around these osteoblastic cells. The HAP granules embeded in the pulp for 3 months were surrounded by fine matrix fibers which seemed to be produced by adjacent osteoblastic cells. The surrounding of these cells were filled with abundant matrix vesicles and collagen fibers, which would show the production of osteodentin matrix and the minerilization were active in this region. Six months after the treatment, the osteodentin matrix in upper dentin bridge became compact; matrix fiber surrounding the HAP granules became thick and a part of fibers inserted into the granules, which should mean that the HAP granules had bonded to the osteodentin matrix. In the deeper region of the dentin bridge, tubular dentin was formed newly. Both osteodentin and tubular dentin fused tightly without any organelles in the boader between them. The covering of hard tissue of dentin bridge then bonded to primary and reparative dentin closed the wound surface to protect the inner pulp.     

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.     

Ultrastructural observation on Langerhans cells in the rat gingival epithelium

The present study was carried out to investigate ultrastructurally Langerhans cells in the rat gingival epithelium. The gingivae of lower incisors of 15 Wistar rats were examined by electron microscopy. The results were as follows: Langerhans cells were observed mainly in the lower prickle-cell layer of the gingival epithelium. On rare occasions Langerhans cells were also found in the basal and granular layers. The average number of Langerhans cells per 100 cells in the prickle-cell layer was 1.0 cell. Usually Langerhans cells had clear cytoplasms and convoluted or indented nuclei, although sometimes the cells exhibited round nuclei. The clear cytoplasm contained a moderately developed Golgi apparatus and a small number of rough surfaced endoplasmic reticulum, free ribosomes, mitochondria, and lysosomes, but it lacked tonofilaments. Birbeck granules were often found in close vicinity of the Golgi apparatus. The average number of Birbeck granules per one Langerhans cell was 4.3 granules. The cell membrane of Langerhans cells had no junctional complexes like desmosomes. The degeneration of keratinocytes adjacent to Langerhans cells was observed in a few specimens.     

Phenytoin and cyclosporin A suppress the expression of MMP-1, TIMP-1, and cathepsin L, but not cathepsin B in cultured gingival fibroblasts

BACKGROUND: Fibroblasts are known not only to synthesize and secrete extracellular matrix proteins, but also to degrade them for connective tissue remodeling. Drug-induced gingival overgrowth is characterized by a massive accumulation of extracellular matrix components in gingival connective tissues. Although some previous reports suggested that causative drugs stimulated the fibroblast proliferation, the results are not conclusive yet. In this study, we hypothesized that drug-induced gingival overgrowth could be a consequence of impaired ability of matrix degradation rather than an enhanced proliferation of gingival fibroblasts induced by these drugs. METHODS: Normal human gingival fibroblasts were cultured with or without either 20 microg/ml of phenytoin or 200 ng/ml of cyclosporin A. Total RNA and cellular proteins were collected every day for RT-PCR analyses and for measuring lysosomal enzyme activity. In addition, an immunohistochemical study was performed to detect lysosomal enzymes in cells from enlarged gingiva of the patients with phenytoin-induced gingival overgrowth. RESULTS: RT-PCR analyses revealed that these drugs suppressed the expression of MMP-1, TIMP-1, and cathepsin L, but not that of cathepsin B in a time-dependent manner. Then, we measured the activity of lysosomal enzymes and cathepsin B and L. The results indicated that although cathepsin B activity was not observed to be impaired, regardless of the drugs used in these cells, both total and active forms of combined activity of cathepsins B and L were suppressed in a time-dependent manner. CONCLUSIONS: The results indicate that, besides suggested effects of these drugs on gingival fibroblasts and/or on accumulated cells in the gingival tissues, extracellular matrix-degrading ability, particularly that by cathepsin L, is also suppressed by cyclosporin A and phenytoin in gingival fibroblasts, and that lysosomal enzyme plays an important role in the pathogenesis of drug-induced gingival hyperplasia.     

Collagen degradation by interleukin-1beta-stimulated gingival fibroblasts is accompanied by release and activation of multiple matrix metalloproteinases and cysteine proteinases

Objective: Several collagenolytic matrix metalloproteinases (MMPs) have recently been identified in gingival fibroblasts, while secreted cysteine proteinases could also participate in connective tissue destruction in periodontitis. To clarify their involvement, we examined enzyme release during collagen breakdown by cultured cytokine-stimulated fibroblasts. Materials and methods: Gingival fibroblasts were derived from four chronic periodontitis patients and cultured on collagen gels in serum-free medium for 1-4 days. Collagenolysis was measured by hydroxyproline release into the medium. Proteinases were assessed by electrophoresis and immunoblotting. Results: Adding interleukin-1beta resulted in progressive gel breakdown. This was associated particularly with a shift in MMP-1 band position from proenzyme to active enzyme and the appearance of active as well as proenzyme forms of cathepsin B. There was also partial processing of pro-MMP-13 and increased immunoreactivity for active cathepsin L. In addition, both pro-forms and active forms of MMP-8, membrane-type-1-MMP and MMP-2 were present in control and treated cultures. Conclusions: Fibroblast MMP-1 was most likely responsible for collagen dissolution in the culture model, while cathepsin B may have been part of an activation pathway. All studied proteinases contribute to extracellular matrix destruction in inflamed gingival tissue, where they probably activate each other in proteolytic cascades.     

Penetration pathway of a topically applied foreign protein into rat gingiva

Topically applied horseradish peroxidase (HRPO) failed to penetrate through keratinized rat gingiva. It rapidly entered the intercellular spaces of the non-keratinized epithelial attachment and within 10 minutes it spread as far as the attachment's apical limit. Widening of the intercellular spaces occurred. Spread into the underlying connective tissue was associated with dilation of venules and, after 30 minutes, emigration of neutrophils into the epithelial attachment. Macrophages and to a lesser extent, fibroblasts showed uptake of HRPO into vesicles and lysosomes. Some macrophages containing HRPO were located close to the crest of the alveolus. A few cells of theepithelial attachment also showed the presence of HRPO in lysosomes.     

Zinc iodide-osmium staining of membrane-coating granules in keratinized and non-keratinized mammalian oral epithelium

Specimens of keratinized and non-keratinized oral epithelium were examined in the electron microscope after being stained with zinc iodide-osmium. In both types of tissue, reaction was seen in unmyelinated nerves, in the specific granules of epithelial Langerhans cells and within lysosome-like organelles and small vesicles associated with Golgi systems. In keratinized epithelia, the reaction was also present in the membrane-coating granules and between the deepest cells of the keratinized layer. In contrast, the membrane-coating granules of non-keratinized epithelia lacked Zn iodide-osmium staining despite the presence of reaction in adjacent Golgi systems. It is suggested that Zn iodide-osmium stains glycolipid or glycoprotein material in the cell. This material is elaborated in the Golgi systems from which lysosomes and the membrane-coating granules of keratinized tissues are probably derived.     

Comparative histochemical, biochemical and immunocytochemical studies of cathepsin B in human gingival

Cathepsin B activity was demonstrated histochemically in unfixed cryostat sections of inflamed human gingiva using the 2-methoxy-4-naphthylamide (MNA) substrates Z-Val-Lys-Lys-Arg-MNA and Z-Ala-Arg-Arg-MNA with a post-azo-coupling technique. Enzyme localisation was confirmed by immunocyto-chemistry with polyclonal sheep anti-human cathepsin B. In both cases, staining was found in connective tissue fibroblasts and also in cells varying in shape from rounded to more irregular forms. The latter were present both in areas of cellular infiltration and in the oral and pocket epithelium. Examination of adjacent sections with monoclonal antibodies directed against leukocyte differentiation antigens showed that the rounded to irregular cells were CD68 positive macrophages and monocytes. The histochemical staining had the form of fine cytoplasmic particles consistent with the known lysosomal occurrence of cathepsin B. Cells stained by the post-coupling method using the tryptase substrates Z-Ala-Ala-Lys-MNA and D-Val-Leu-Arg-MNA showed a different distribution and morphology, with reaction product confined to mast cell granules. The differences between the cathepsin B and tryptase staining patterns were confirmed by differential extraction from cryostat sections with salt-free and high-salt buffers respectively. Biochemical characterisation of activities in the extracts with the 7-amino-4-trifluoromethyl coumarin (AFC) substrates Z-Val-Lys-Lys-Arg-AFC and Z-Ala-Ala-Lys-AFC and protease inhibitors confirmed the identity of the two enzymes. Selective inhibitors could also be used in histochemical incubations to distinguish between cathepsin B and tryptase staining.     

Oxidative stress-induced DNA damage in the synovial cells of the temporomandibular joint in the rat

Synovial hyperplasia is a feature of degenerative temporomandibular joint (TMJ) disease. However, the mechanism by which hyperplasia progresses in the TMJ is unknown. Based on the hypothesis that the oxidative stress generated by mechanical loading causes degenerative changes in the TMJ synovium, we investigated the generation of the highly reactive species, peroxynitrite, and the occurrence of DNA damage in the synovium. After condylar hypermobility of rat TMJs, a marker of peroxynitrite, nitrotyrosine, was localized to the nuclei and cytoplasm of the synovial lining cells and fibroblasts in synovitis-induced TMJ. DNA single-strand breaks were found in the nuclei of the synovial cells only after enzyme treatment, whereas DNA double-strand breaks were not detected. These findings indicate that condylar hypermovement induces the proliferation of synovial cells, and suggest that oxidative stress leads to the progression of synovial hyperplasia via DNA damage of the synovial cells in TMJs after mechanical loading.     

Human gingival fibroblast cell lines in vitro

Eight human gingival fibroblast cell lines were cultivated in glass cover slip chambers and analyzed by electron microscopy for their capacity to synthesize an extracellular fibrillar matrix. Both microfibrils and amorphous components of elastic tissue and several forms of banded and unbanded collagen fibrils were elaborated by the fibroblasts. Banding periods of 64 nm and 100 nm were observed. Collagenases lysed collagen fibrils and in some cases altered them to display prominently banded FLS images. Numerous matrix vesicles were observed among the extracellular collagen fibrils and some were observed intracellularly either isolated in pinocytotic-like vesicles or as clusters in larger vacuoles. Matrix granules also were observed among the extracellular fibrils. The Golgi sacs of synthesizing fibroblasts contained fine thread-like parallel filaments with a banding period of 220 nm. The presence of annulate lamellae was related to culture aging and the appearance of large mitochondrial matrix granules was related to the effect of small increments of vitamin C. Two cells were shown to have phagocytosed collagen elaborated by other cells earlier, one contained an interiorized fibril with a 64 nm banding period and the other an interiorized fibril with a 100 nm banding period. A turnover of collagen in vitro was thus established.     

口腔粘膜下纤维化组织中内皮素1的免疫电镜研究

目的 观察内皮素（ＥＴ－１）在人口腔粘膜下纤维（ＯＳＦ）组织中的形态学定位，并探讨ＥＴ－１与ＯＳＦ病理学的关系。方法 用免疫电镜技术检测ＥＴ－１在ＯＳＦ组织中的超微结构定位。结果 ＥＴ－１免疫阳性颗粒主要存在于ＯＳＦ组织的上皮细胞、成纤维细胞及内皮细胞的胞浆内。正常口腔粘膜组织中未发现ＥＴ－１免疫阳性颗粒。ＯＳＦ组织中，上皮细胞内的ＥＴ－１阳性颗粒定位于细胞膜上，胞浆内的阳性颗粒可能定位于粗面内质