Development of the synovial membrane in the rat temporomandibular joint as demonstrated by immunocytochemistry for heat shock protein 25

The synovial lining layer of the temporomandibular joint (TMJ) consists of macrophage-like type A cells and fibroblast-like type B cells. Until now, little information has been available on the development of the synovial membrane in TMJ. In the present study we examined the development of the synovial lining layer in the rat TMJ by light- and electron-microscopic immunocytochemistry for heat shock protein (Hsp) 25, which is a useful marker for type B cells. At embryonic day 19 (E19), a few Hsp25-positive cells first appeared in the upper portion of the developing condyle. During the formation of the upper articular cavity (E21 to postnatal day 1 (P1)), a few positive cells were arranged on its surface. Immunoelectron microscopy demonstrated that these cells had ultrastructural features of fibroblast-like type B cells. In addition, some Hsp25-positive cells moved to the deep portion by extending their cytoplasmic processes toward the articular cavity at P3. At that time, the presence of typical macrophage-like type A cells in the lining layer was confirmed by immunoelectron microscopy. The slender processes of Hsp25-positive cells showed a continuous covering with the synovial surface at P7, followed by a drastic increase in the Hsp25-positive cells at P15 and later, when active jaw movement occurred. These findings suggested that the arrangement and morphological maturation of type B cells are closely related to the formation of the articular cavity in the embryonic period and the commencement of active jaw movement after birth, respectively.     

Immunocytochemical localization of MAPKAPK-2 and Hsp25 in the rat temporomandibular joint

One series of our research has shown an intense expression of immunoreaction for heat shock protein 25 (Hsp25) in various cellular elements in the rat temporomandibular joint (TMJ). This protein is the major substrate of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2), which mediates an intracellular stress-activated signaling pathway to stimulate cytosolic actin reorganization under various stresses. The present study was undertaken to examine the localization of MAPKAPK-2 in the rat TMJ by immunocytochemical techniques. Furthermore, confocal microscopy with double staining was employed to demonstrate the colocalization of MAPKAPK-2 and Hsp25. Immunocytochemistry for MAPKAPK-2 showed an intense immunoreaction in the cytoplasm of the synovial lining cells, the endothelial cells, and the fibroblasts in the synovial membrane of the rat TMJ. Double immunostaining under a confocal microscope succeeded in demonstrating the colocalization of MAPKAPK-2 and Hsp25 immunoreactions in the cytoplasm of fibroblastic type B synoviocytes in the TMJ. On the other hand, the macrophage-like type A-cells expressed MAPKAPK-2 immunoreactions but lacked Hsp25 immunoreactivity. The cells in the articular disk and the chondrocytes in the maturative and hypertrophic layer of the mandibular cartilage also showed intense immunoreactions for MAPKAPK-2 and Hsp25. In addition to cytoplasmic localization, MAPKAPK-2 immunoreactions were found in the nucleus of some synovial lining cells, cells in the articular disk, and chondrocytes. Current observations imply the presence of the phosphorylation of Hsp25 via activated MAPKAPK-2 in the cytoplasm. MAPKAPK-2 and Hsp25 possibly participate in the induction of cytoskeletal changes to the various cellular elements in rat TMJ under normal conditions.     

Three-dimensional ultrastructure of synoviocytes in the horse joint as revealed by the scanning electron microscope.

The synovial membrane displays a superficial cellular lining composed of two types of synoviocytes: "absorptive" macrophages (type A cells) and "secretory" fibroblast-like cells (type B cells). The types are intermingled and extend a variety of processes, rendering the cellular architecture of the synovial membrane difficult to visualize. Previous electron microscopic and histochemical studies failed to demonstrate the entire shape of synoviocytes, except our immunohistochemical study for protein gene product 9.5 in the horse joint. The present SEM study is the first to demonstrate the three-dimensional ultrastructure of synoviocytes as well as their distribution in the synovial membrane, using macerated samples from the horse carpal joints. The equine synovial membrane was largely covered by conspicuously developed synovial villi. Type A synoviocytes were closely similar to macrophages in regard to surface structure, and showed uneven distribution with the densest occurrence around the tips of the synovial villi. In the basal half of villi, type B synoviocytes, which were situated in close proximity to the synovial cavity, projected thick processes horizontally and intertwined to form a regular network of processes on the synovial surface. Those in the upper half of the villi were located in the abluminal layers and protruded an antenna-like process into the joint cavity with tips covered with long microvilli, in addition to forming the superficial plexus of processes. Type B cells were also provided with fine, membranous extensions that tended to cover the surface of synovial intima. The meshwork of horizontal processes, the antenna-like processes, and the membranous processes imply advantages in not only secretion but also sensation and regulation of the barrier function in the synovial membrane.     

Localization of CD44 and hyaluronan in the synovial membrane of the rat temporomandibular joint

Previous studies have pointed out a lack of adhesion structures in the synovial lining layer of the rat temporomandibular joint (TMJ) despite showing an epithelial arrangement. CD44, a major cell adhesion molecule, plays crucial roles as an anchor between cells and extracellular matrices by binding hyaluronan (HA) for the development of organs or the metastasis of tumors. The present study examined the localization of CD44 in the synovial membrane of the rat TMJ by immunocytochemistry for OX50, ED1, and Hsp25, which are markers for the rat CD44, macrophage-like type A, and fibroblast-like type B synoviocytes, respectively. Histochemistry for HA-binding protein (HABP) was also employed for the detection of HA. OX50 immunoreactions were found along the cell surface and, in particular, accumulated along the surface of the articular cavity. Observations by a double immunostaining and immunoelectron microscopy revealed that all the OX50-immunopositive cells were categorized as fibroblastic type B cells, which had many caveolae and a few vesicles reactive to intense OX50. However, the macrophage-like type A cells did not have any OX50 immunoreaction in the synovial lining layer. A strong HABP reaction was discernable in the extracellular matrix surrounding both OX50-positive and -negative cells in the synovial lining layers, exhibiting a meshwork distribution, but weak in its sublining layer. This localization pattern of CD44 and HABP might be involved in the formation of the epithelial arrangement of the synovial lining layer. Furthermore, OX50 immunonegativity in the type A cells suggests their low phagocytotic activity in the rat TMJ under normal conditions.     

Expression of decay-accelerating factor is reduced on hyperplastic synovial lining cells in rheumatoid synovitis.

Decay-accelerating factor (DAF), a membrane inhibitor of homologous complement activation, is present in synovial cells lining joint space and detected in synovial fluid. DAF is considered to protect synovial membrane from complement-mediated injury associated with articular inflammation. We studied the immunohistopathological features of DAF molecules in synovial membrane of rheumatoid synovitis using a DAF-specific monoclonal antibody, 1C6. Reacting molecules with the 1C6 antibodies in synovial tissue extracts formed a 70-kD band in Western blot analysis. DAF was strongly detected on the flat synovial lining cells, but weakly on the hyperplastic and multi-layered lining cells in rheumatoid synovitis. The latter cells reacted with anti-Leu-M3 antibodies specific for a cell surface marker of activated macrophages, sometimes accompanied by C3 and IgM deposition on the superficial synovial membrane. These results suggest that active rheumatoid synovitis characteristically with hyperplastic synovial lining cells is out of control by DAF, thereby permitting further complement-mediated injury.     

Expression of caveolin-1 in the rat temporomandibular joint

This immunocytochemical study revealed the expression of caveolin-1, a major protein of caveolae, in the rat temporomandibular joint. In the synovial lining layer, immunoreactive products for caveolin-1 were detected on the cell membrane of the fibroblast-like type B cells, as confirmed by immunocytochemistry for heat shock protein 25. The cells in the articular disk, the articular layer, and zone of proliferation of the mandibular condyle also showed intense immunoreactions for caveolin-1.     

Morphology of the synovium during its differentiation and development in the mouse knee joint

Summary The prenatal and postnatal development of the mouse knee joint was investigated by transmission and scanning electron microscopy. In the prenatal stage, following the appearance of a narrow intercellular cleft between two skeletal elements on the 16th fetal day, clefting extended into the lateral synovial mesenchyme. In some regions, the extension of the cleft was very rapid, but in a certain region (future fat pad region), it was somewhat slower. Macrophage-like cells appeared in the synovial mesenchyme on the 16th fetal day, and then increased in number, and were distributed as if they were clustering around the presumptive clefting zone in the future fat pad region on the 17th–18th fetal day. This suggests that macrophage-like cells may participate in joint development, as they phagocytize and remove some kinds of solid extracellular matrix, and facilitate the cleft extension. In the early postnatal stage, scanning electron microscopic observations showed that there were two different types of cell in the synovial lining. One of them exhibited a surface morphology corresponding to that of macrophages: a spherical cell body and numerous pseudopodia. The other type of cell exhibited various cell shapes with many cytoplasmic processes extending along the synovial surface.     

Expression of estrogen receptor alpha (ER alpha) in the rat temporomandibular joint

Numerous epidemiological studies have pointed out a higher frequency of temporomandibular disorder (TMD) in women than in men, which indicates the involvement of a sex hormone, such as estrogen, in the pathogenesis of TMD. Although estrogen is known to play pivotal roles in osteoarthrosis or rheumatoid arthritis in systemic joints, there have been few reports about the role of estrogen in the temporomandibular joint (TMJ). The effect of estrogen is generally mediated by the estrogen receptors (ERs) ER alpha (the predominant type) and ER beta. In this study we examined the expression of ER alpha protein and mRNA in the TMJ of adult male rats by immunocytochemistry and in situ hybridization histochemistry. Intense ER alpha immunoreactivity was localized in the synovial lining cells, stromal cells in the articular disc, and chondrocytes in the TMJ. These ER alpha-immunopositive synovial lining cells are characteristic of cytoplasmic processes identified with confocal and immunoelectron microscopy, which indicates that they are synovial type B cells. In situ hybridization histochemistry confirmed intense signals for ER alpha in the synovial lining cells and the sublining fibroblasts at mRNA levels. The nuclei of chondrocytes showed an intense immunoreaction for ER alpha in the maturative and hypertrophic layers of the articular cartilage. In addition to the nuclear localization of ER alpha, a weak immunoreaction appeared in the cytoplasm of some ER alpha-positive cells. These findings support the hypothesis that TMJ tissue-at least in the male rat-has the potential to be an estrogen target tissue.     

Histological studies on the inner surfaces of the articular cavities of human temporomandibular joints with special reference to arthroscopic observations

Histological findings relating to the arthroscopic anatomical features in the inner surfaces of human temporomandibular joints are described. Articular surface covering the bony surface is histologically fibrocartilage. The articular surface of the articular disk is histologically composed mainly of tightly packed collagen fibers, but partly of fibrocartilage which is considered to be developed in the area where presumably maximal stress is given during the joint movement. The synovial membrane consist of 2 layers. The superficial one is the lining cell layer formed by single or multiple rows of cells with from ovoid to cuboidal shapes. The deep one is a capillary rich loose connective tissue layer. The present study suggests that arthroscopic views well reflect histological features of the inner wall of the articular cavity of the temporomandibular joint.     

Synovial membrane in the temporomandibular joint--its morphology, function and development

This paper reviews recent findings of the synovial membrane, in particular the morphology, function and development of synovial lining cells, in the temporomandibular joint (TMJ). Electron microscopic studies have confirmed the synovial membrane in TMJ consists of macrophage-like type A cells and fibroblast-like type B cells identical to those in other systematic joints. The macrophage-like type A cells react with anti-macrophage and macrophage-derived substances including the major histocompatibility class II molecule, and show a drastic increase in their number in the inflamed synovial membrane. In addition, they have the ability to produce substances involved in the progression of TMJ inflammation such as nitric oxide and inducible nitric oxide synthase. Observation of osteopetrotic mice revealed that macrophage-like type A cells in TMJ are derived from monocyte lineage. Immunocytochemistry for 25kDa heat shock protein was able to depict the entire shape of fibroblast-like type B cells including their unique processes. The expression of an estrogen receptor alpha-immunoreaction in the fibroblast-like type B cells may explain the etiology of temporomandibular disorders at a higher frequency in females than in males, suggesting that TMJ is a target tissue for estrogen. Furthermore, fibroblast-like type B cells are equipped with a basement membrane to serve as an adhesion molecule for the fibroblast-like type B cells to keep their epithelial arrangement. A clear understanding of the morphology of the intact synovial membrane will serve to clarify the etiology and development of temporomandibular disorders.     

Contribution of synovial lining cells to synovial vascularization of the rat temporomandibular joint

The lining layer of the synovial membrane in the temporomandibular joint (TMJ) contains two types of lining cells: macrophage‐like type A and fibroblast‐like type B cells. The type B cells are particularly heterogeneous in their morphology and immunoreactivity, so that details of their functions remain unclear. Some of the type B cells exhibit certain resemblances in their ultrastructure to those of an activated capillary pericyte at the initial stage of the angiogenesis. The articular surface, composed of cartilage and the disc in the TMJ, has few vasculatures, whereas the synovial lining layer is richly equipped with blood capillaries to produce the constituent of synovial fluid. The present study investigated at both the light and electron microscopic levels the immunocytochemical characteristics of the synovial lining cells in the adult rat TMJ, focusing on their contribution to the synovial vascularization. It also employed an intravascular perfusion with Lycopersicon esculentum (tomato) lectin to identify functional vessels in vivo. Results showed that several type B cells expressed desmin, a muscle‐specific intermediate filament which is known as the earliest protein to appear during myogenesis as well as being a marker for the immature capillary pericyte. These desmin‐positive type B cells showed immunoreactions for vimentin and pericyte markers (neuron‐glial 2; NG2 and PDGFRβ) but not for the other markers of myogenic cells (MyoD and myogenin) or a contractile apparatus (αSMA and caldesmon). Immunoreactivity for RECA‐1, an endothelial marker, was observed in the macrophage‐like type A cells. The arterioles and venules inside the synovial folds extended numerous capillaries with RECA‐1‐positive endothelial cells and desmin‐positive pericytes to distribute densely in the lining layer. The distal portion of these capillaries showing RECA‐1‐immunoreactivity lacked lectin‐staining, indicating a loss of blood‐circulation due to sprouting or termination in the lining layer. The desmin‐positive type B and RECA‐1‐positive type A cells attached to this portion of the capillaries. Some capillaries in the lining layer also expressed ninein, a marker for sprouting endothelial cells, called tip cells. Since an activated pericyte, macrophage and tip cell are known to act together at the forefront of the vessel sprout during angiogenesis, the desmin‐positive type B cell and RECA‐1‐positive type A cell might serve as these angiogenic cells in the synovial lining layer. Tomato lectin perfusion following decalcification would be a highly useful tool for research on the vasculature of the mineralized tissue. Use of this technique combined with immunohistochemistry should permit future extensive investigations on the presence of the physiological angiogenesis and on the function of the lining cells in the synovial membrane.     

A developmental study of the synovial membrane of the rat temporomandibular joint: changes in the three-dimensional configuration during postnatal development

The development of synovial membranes in the posterior synovial portion of the rat temporomandibular joint was studied and the three-dimensional structure of the posterior synovial portion reconstructed from sagittal semithin sections. Reconstructions showed that the synovial membrane expanded and that synovial folds increased in number and became complicated in shape with the growth of the joint. Using transmission-electron microscopy, it was observed that the synovial lining cells degenerated, that the synovial membrane split to make further synovial folds, and that the folded-end structures consisted of synovial lining cells that extended into the subsynovial connective tissue. It is suggested that in the development of the three-dimensional configuration of the synovial membrane, several processes proceed simultaneously to form the synovial folds: a splitting of the synovial membrane, infolding of the synovial membrane into the subsynovial connective tissue, and outgrowth of the synovial folds towards the synovial cavity.     

Ultrastructure of Human Tendon Sheath and Synovium: Implications for Tumor Histogenesis

Normal human tendon sheath and synovium were studied by scanning and transmission electron microscopy. The lining cells of the two tissues appear to be identical ultrastructurally. The most superficial cells (B-cells) possess long cytoplasmic extensions that clothe the membrane surface. Intermingled with deeper B-cells are the so-called A-cells, which have similar cytoplasmic features but lack long processes and instead have many filopodia. The frequent occurrence of intermediate forms indicates that the two cells form part of a morphologic spectrum. Comparison with cells of tumors that have been ascribed to synovium or tendon sheath (synovial sarcoma, epithelioid sarcoma, clear cell sarcoma) do not reveal any close similarities that might support a histogenetic relationship.     

Morphology and functional roles of synoviocytes in the joint

The joint capsule exhibits a unique cellular lining in the luminal surface of the synovial membrane. The synovial intimal cells, termed synoviocytes, are believed to be responsible for the production of synovial fluid components, for absorption from the joint cavity, and for blood/synovial fluid exchanges, but their detailed structure and function as well as pathological changes remain unclear. Two types of synoviocytes, macrophagic cells (type A cells) and fibroblast-like cells (type B cells) have been identified. Type A synoviocytes are non-fixed cells that can phagocytose actively cell debris and wastes in the joint cavity, and possess an antigen-presenting ability. These type A cells, derived from blood-borne mononuclear cells, can be considered resident macrophages (tissue macrophages) like hepatic Kupffer cells. Type B synoviocytes are characterized by the rich existence of rough endoplasmic reticulum, and dendritic processes which form a regular network in the luminal surface of the synovial membrane. Their complex three-dimensional architecture was first revealed by our recent scanning electron microscopy of macerated samples. The type B cells, which are proper synoviocytes, are involved in production of specialized matrix constituents including hyaluronan, collagens and fibronectin for the intimal interstitium and synovial fluid. The proliferative potentials of type B cells in loco are much higher than type A cells, although the transformation of subintimal fibroblasts into type B cells can not be excluded. In some mammals, type B cells show features suggesting endocrine and sensory functions, but these are not recognized in other species. The synoviocytes, which form a discontinuous cell layer, develop both fragmented basement membranes around the cells and junctional apparatus such as desmosomes and gap junctions. For an exact understanding of the mechanism of arthritis, we need to establish the morphological background of synoviocytes as well as their functions under normal conditions.     

Pigmented villonodular synovitis. A clinicopathologic study of 52 cases

Clinicopathologic, enzyme histochemical and electron microscopic findings in 52 patients with pigmented villonodular synovitis (PVS) are reported. The lesion was by far the most common in the knee joint (48%), followed by the ankle joint (25%). As to sex incidence, there seemed to be no predilection (46% in men, 54% in women). Microscopically, the PVS showed thin or thick villous projections of the involved synovial membrane, associated with or without nodular formation. The nodule of PVS consisted essentially of a proliferation of histiocyte-like cells with phagocytic activities. Another characteristic feature was large clefts and pseudoglandular or alveolar spaces lined by synovial cells. Enzyme histochemical studies revealed that the lesional cells had functional properties of macrophages. Electron microscopically, the lesion consisted essentially of histiocyte-like and fibroblast-like cells, together with intermediate cells and myofibroblasts.     

PIGMENTED VILLONODULAR SYNOVITIS A Clinicopathologic Study of 52 Cases

Clinicopathologic, enzyme histochemical and electron microscopic findings in 52 patients with pigmented villonodular synovitis (PVS) are reported. The lesion was by far the most common in the knee joint (48%), followed by the ankle joint (25%). As to sex incidence, there seemed to be no predilection (46% in men, 54% in women). Microscopically, the PVS showed thin or thick villous projections of the involved synovial membrane, associated with or without nodular formation. The nodule of PVS consisted essentially of a proliferation of histiocyte-like cells with phagocytic activities. Another characteristic feature was large clefts and pseudoglandular or alveolar spaces lined by synovial cells. Enzyme histochemical studies revealed that the lesional cells had functional properties of macrophages. Electron microscopically, the lesion consisted essentially of histiocyte-like and flbroblast-like cells, together with intermediate cells and myofibroblasts.     

Synovial sarcoma--a misnomer.

For an evaluation of the putative histogenetic relationship of synovia and synovial sarcomas, normal synovia, villonodular synovitis, and synovial sarcomas were compared for their patterns of expression of intermediate filaments of keratin and vimentin type and epithelial membrane antigen and for lectin binding sites. The lining cells in both normal synovia and villonodular synovitis reacted with anti-vimentin antibodies, but not with antibodies to different types of keratins or epithelial membrane antigen. The cleft-lining cells in synovial sarcoma, on the other hand, showed only keratin positivity, and epithelial membrane antigen could also be detected in these cells. Nonneoplastic synovial lining cells bound peanut agglutinin (PNA), Ricinus communis agglutinin (RCA), soybean agglutinin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA) conjugates, but not Ulex europaeus I lectin (UEA I). In contrast, the epithelial-like cleft lining cells in synovial sarcomas showed an apical cytoplasmic binding of PNA, UEA I, RCA, and SBA, and binding of WGA to the whole cytoplasm but did not bind Con A. The distinct differences between synovial lining cells and synovial sarcoma cells speak against synovial cell features in synovial sarcoma. These results indicate that synovial sarcoma is a carcinosarcomalike tumor with true epithelial differentiation, and the term "synovial sarcoma" apparently is a misnomer that should be abandoned.     

Immunocytochemical Localization of Caveolin‐3 in the Synoviocytes of the Rat Temporomandibular Joint During Development

Caveolins—caveolin‐1, ‐2, ‐3 (Cav1, 2, 3)—are major components of caveolae, which have diverse functions. Our recent study on the temporomandibular joint (TMJ) revealed expressions of Cav1 and muscle‐specific Cav3 in some synovial fibroblast‐like type B cells with well‐developed caveolae. However, the involvement of Cav3 expression in the differentiation and maturation of type B cells remains unclear. The present study therefore examined the chronological alterations in the localization of Cav3 in the synovial lining cells of the rat TMJ during postnatal development by immunocytochemical techniques. Observations showed immature type B cells possessed a few caveolae with Cav1 but lacked Cav3 protein at postnatal day 5 (P5). At P14, Cav3‐immunopositive type B cells first appeared in the synovial lining layer. They increased in number and immunointensity from P14 to P21 as occlusion became active. In immunoelectron microscopy and double immunolabeling with heat shock protein 25 (Hsp25) and Cav3, coexpressed type B cells developed rough endoplasmic reticulum and numerous caveolae, while the Cav3‐immunonegative type B cell with Hsp25 immunoreaction possessed few of these. Results suggest that Cav3 expression, which is closely related to added functional stimuli, reflects the differentiation of the type B synoviocytes. Anat Rec, 291:233–241, 2008. © 2008 Wiley‐Liss, Inc.     

Fine-structural changes of the synovial membrane in arthrosis deformans

Summary The known light microscopic findings in the inner part of joint capsules in arthrosis have largely been verified and in part supplemented by scanning and transmission electron microscopic examinations. Whereas in arthrosis of the hip joint sclerosis of the synovial membrane predominates with atrophy of the layer of lining cells, in arthrosis of the knee joint more polymorphic structures of the surface occur with variable but moderate hyperplasia and hypertrophy of the synovial cells. Inflammatory cell proliferations and infiltrates of the same degree as in non-specific arthritis and rheumatoid arthritis were not observed. Thus, the absence both of characteristic changes of the individual synovial cells and of more extensive inflammatory infiltrates in association with atrophy and sclerosis of the synovial folds may be regarded as an indirect indication of the synovial reaction in arthrosis deformans.

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Zusammenfassung Durch raster- und transmissionselektronenmikroskopische Untersuchungen ausgedehnten Synovektomie-Materials wurden die bekannten lichtmikroskopischen Befunde an der inneren Gelenkkapsel weitgehend bestätigt und teilweise ergänzt. Während bei chronischer Coxarthrose die Sklerose der Synovialmembran mit Atrophie der inneren gelenkauskleidenden Zellschicht im Vordergrund steht, kommen bei Gonarthrose polymorphere Oberflächenstrukturen mit wechselnder, im ganzen aber mäßiger Hyperplasie und Hypertrophie der Synovialzellschicht vor. Entzündlich-hyperplastische überschießende Zellproliferationen und Infiltrate wie bei unspezifischer Arthritis und rheumatoider Arthritis werden bei Arthrosis deformans nicht beobachtet. Das Fehlen charakteristischer Veränderungen der einzelnen Synovialzellen und das Fehlen eindeutiger entzündlicher Infiltrate werden zusammen mit der Sklerose und Atrophie der Synovialfalten als indirekter Hinweis auf eine synoviale Reaktion auf arthrotische Gelenkveränderungen gewertet.

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Herrn Prof. Dotzauer zum 60. Geburtstag.