Excess NF-κB induces ectopic odontogenesis in embryonic incisor epithelium.

Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKß, an essential component of the NF-κB pathway, under keratin 5 promoter (K5-Ikkß). K5-Ikkß mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkß mice. The supernumerary incisors in K5-Ikkß mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.

Zoledronic acid impairs re-epithelialization through down-regulation of integrin αvß6 and transforming growth factor beta signalling in a three-dimensional in vitro wound healing model.

This study examined the negative effects of zoledronic acid on the re-epithelialization of oral mucosa in a three-dimensional in vitro oral mucosa wound healing model. A living oral mucosa equivalent was constructed by seeding a mixture of primary human oral keratinocytes and fibroblasts, at a cell density of 1.5 × 10(5)cm(2) each, onto human cadaver dermis. This was cultured in a submerged condition in 1.2mM Ca(2+) EpiLife for 5 days, and then in an air-liquid interface for 14 days. The equivalent was wounded by excising a linear 2-mm-wide epithelial layer on day 8 and subsequently incubated with 10 µM zoledronic acid for an additional 11 days. Histological and immunohistochemical observations revealed zoledronic acid to significantly suppress the epithelial thickness and Ki-67-labelling index. Zoledronic acid also abolished integrin αvß6 expression, implying impaired keratinocyte migration. Zoledronic acid did not attenuate the total transforming growth factor beta 1 (TGF-ß1) production into the supernatant, but down-regulated TGF-ß receptor types I and II expression and Smad3 phosphorylation, as was also confirmed by immunofluorescence microscopy. This study therefore showed zoledronic acid to abrogate integrin αvß6 expression, cause the down-regulation of TGF-ß/Smad signalling in oral keratinocytes, and impair re-epithelialization, suggesting compromised oral mucosa homeostasis in patients receiving zoledronic acid.

Changes in jaw reflexes by stimulation of the hypothalamus in anesthetized rabbits.

Changes in the masseteric monosynaptic reflex (MMR) and jaw-opening reflex (JOR) responses resulting from conditioning stimulation in the hypothalamus were studied in anesthetized rabbits. Stimulation of the lateral hypothalamus evoked a facilitation of the MMR and an inhibitory or facilitatory effect on the JOR. The facilitatory effect on JOR was stronger than that on the MMR. The facilitatory effective site for the JOR was in the dorsal and lateral directions as compared to the inhibitory field. The results suggest two functionally distinct regions in the lateral hypothalamus that separately project to the jaw-opening muscles.

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.

Immunocytochemical detection of superoxide dismutases (SODs) in the periodontal Ruffini endings of the rat incisor.

The expression of immunoreactivities for superoxide dismutases (SODs), Mn-SOD and Cu/Zn-SOD, was immunohistochemically investigated in the lingual periodontal ligament and toe pads of adult rats. Immunocytochemistry for SODs revealed that the axon terminals of both the periodontal Ruffini endings and cutaneous Meissner's corpuscles showed mitochondrial Mn-SOD immunoreactivity, but not cytosolic Cu/Zn-SOD immunoreactivity, indicating Mn-SOD is a useful marker for identifying the mechanoreceptors. It is likely that Mn-SOD in the axon terminals of mechanoreceptors exerts protective action against nerve injury and neuronal death under severe conditions, serving to scavenge free radicals from the axon terminals.

Transient expression of heat shock protein (Hsp)25 in the dental pulp and enamel organ during odontogenesis in the rat incisor.

The expression of heat shock protein (Hsp) 25 during odontogenesis in the dental pulp and enamel organ of rat incisors was investigated by immunocytochemistry and confocal microscopy. In the process of dentin formation, immature odontoblasts first exhibited Hsp 25-immunoreactivity, and increased in immunointensity with the advance of their differentiation. In the dental pulp, in contrast, intense immunoreaction in the mesenchymal cells became weak or negative in parallel with the progress of cell differentiation. The immunoreaction for Hsp 25 in the enamel organ revealed a characteristic stage-related alteration during amelogenesis. In secretory ameloblasts, the immunoreaction for Hsp 25 was found throughout their cell bodies, intense reactivity being located near the proximal and distal terminal webs. At the maturation stage, ruffle-ended ameloblasts (RA) consistently showed Hsp 25-immunoreactivity throughout the cell bodies, whereas smooth-ended ameloblasts (SA) lacking a ruffled border were weak in immunoreaction at the distal cytoplasm. Other cellular elements of the enamel organ were negative. The subcellular localization of Hsp 25-immunoreactivity in this study appeared essentially identical to that of actin filaments as demonstrated by confocal microscopy using rhodamine-labeled phalloidin. These immunocytochemical data suggest that the Hsp 25 molecule is involved in reinforcement of the cell layer following cell movement during odontogenesis and in the formation and maintenance of the ruffled border of RA.

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.

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.

Immunohistochemical localization of S-100beta in the dental pulp of the rat molar.

The present study was undertaken to reveal whether S-100alpha or S-100beta or both are present in the nerve fibers in the rat molar tooth pulp. No immunoreactivity for S-100alpha was observed in the molar pulp. In the root pulp, thick smooth-surfaced structures accompanying the blood vessel showed S-100beta-like immunoreactivity (-LI), and occasionally a very few thin beaded elements exhibited S-100beta-LI. In the coronal pulp, S-100beta-like immunoreactive (-IR) structures arborized repeatedly and extensively; they had a predominantly thick, smooth-surfaced appearance, though parts appeared thin and beaded. Numerous thin varicose S-100beta-IR structures ran through the odontoblast cell layer, and further penetrated into the predentin alongside the dentinal tubules. They could be traced for approximately 10-20 micrometers into the predentin from the pulp-predentin border. Immunoelectron microscopy revealed that the Schwann cells in the root pulp showed S-100beta-LI, and that S-100beta-LI was present in the axoplasm as well as Schwann cells in the coronal pulp. The S-100beta-IR axons were rarely surrounded by S-100beta-IR Schwann cells. In the predentin, S-100beta-IR nerve fibers terminated in a position close to the odontoblast processes. The present findings indicate that S-100beta, not S-100alpha, is present in the axon in the dental pulp and predentin as well as in the Schwann cells.

Immunocytochemical demonstration of heat shock protein 25 in the rat temporomandibular joint.

The expression of heat shock protein 25 (Hsp 25) was investigated in the rat temporomandibular joint by immunocytochemistry combined with confocal and electron microscopy. Immunostaining with an antibody to Hsp25 was able to demonstrate various cellular elements in the synovial membrane of the joint. Intense immunoreaction for Hsp25 was recognized in certain cells comprising the synovial lining layer. Confocal microscopic observation revealed two characteristic profiles of the Hsp25-positive cells with cytoplasmic processes: one extended thick and long processes towards the articular cavity, and the other prejected horizontally slender processes which covered the synovial membrane. Under the electron microscope, the immunoreactive synovial lining cells were characterized by a well-developed rough endoplasmic reticulum and secretory granules, suggesting that they can be categorized as fibroblastic type B cells. The covering by the cytoplasmic extensions was confirmed by immuno-electron microscopic observations. This cytoplasmic covering presumably performs a barrier function and expedites the effective secretion/resorption of synovial fluids. Since it has been proposed that Hsp 25 is associated with an estrogen receptor, the immunopositive synovial lining cells were considered estrogen-target cells. Immunoreactivity for Hsp25 was also observed in the chondrocytes of the maturative and hypertrophic cell layers as well as in the cells of the articular disk. A suggestion was made that Hsp25 might be involved in the inhibition of apoptosis of those cells.

Immunocytochemical detection of S-100beta in the periodontal Ruffini endings in the rat incisor.

Subcellular localization of S-100 protein, a kind of calcium binding proteins, was examined immunohistochemically in the Ruffini ending, a primary mechanoreceptor, in the periodontal ligament of the rat incisor. The periodontal ligament of the rat incisor was found to contain many S-100beta-immunoreactive (-IR) structures but no S-100alpha-IR elements. The S-100beta-IR structures ramified extensively to form Ruffini endings and were frequently associated with round cells, the terminal Schwann cells, which also showed S-100beta-like immunoreactivity. In many periodontal Ruffini endings, S-100beta-IR products were recognized in the cytoplasm of Schwann cells, but not in the axoplasm. However, some axon terminals which had fewer or shorter axonal fingers, were filled with S-100beta-IR products. The present findings indicated the existence of S-100beta, not S-100alpha, in axon terminals of the periodontal mechanoreceptive endings which were identified as type II Ruffini endings.

Immunocytochemical demonstration of the synovial membrane in experimentally induced arthritis of the rat temporomandibular joint.

The present study is first to report an experimental model of adjuvant-induced arthritis in the rat temporomandibular joint (TMJ). Arthritis was induced by simultaneous intradermal administrations of Freund's complete adjuvant, one at the parietal scalp and the other at the base of the tail. In this model, we demonstrated responses of the synovial membrane by immunocytochemistry using antibodies to OX6 and ED1 which recognize Ia antigen in MHC class II antigen-expressing cells and the macrophage/monocyte lineage, respectively. Three weeks after administration, no remarkable signs of inflammation were macroscopically recognizable in the TMJ, but microscopically the synovial membrane in the TMJ revealed marked changes such as enhanced vascularization and hemostasis in the sublining layer and a thickening in the synovial lining cell layer. Intense OX6-immuno-reactivity was found in the synovial lining cells at lesions in the experimental group but not in the control group. Immunoelectron microscopy revealed that these OX6-immunopositive synovial lining cells developed dense cytoplasmic processes and numerous vacuoles and vesicles, resembling type A cells. Part of the type A cells also showed ED1-immunoreactivity. The expression of OX6 or ED1 immunoreactivity in the synovial lining cells might be involved in the initial immune responses in this arthritis model because the synovial membranes are exposed to the synovial fluids which have been believed to contain antigenic substances.