Biomedical subjects

Yoshiro Takano

Publications and source records attributed to Yoshiro Takano.

At least 19 recordsLinked to original sources

PubMed2006

Nerve-epithelium association in the periodontal ligament of guinea pig teeth.

Several lines of evidence have suggested that periodontal nerves have other roles besides sensory function. Exploring the distribution pattern of nerves in relation to other structures within the periodontal ligament of various species should be important to understand their roles within the ligament. This study investigated whether any association exists between the nerves and the epithelial cells in the periodontal ligament of continuously erupting guinea pig molars, which show distinct enamel epithelium layers among the cementum pearls. Ten guinea pigs were fixed by vascular perfusion and jaw sections were processed for immunohistochemistry of protein gene product 9.5 (PGP 9.5), growth-associated protein-43 (GAP-43) and glia-specific S-100 protein, and for enzyme histocytochemistry of cholinesterase. Nerves that were immunopositive for the above neuronal markers were located predominantly in the alveolus-related part of the periodontal ligament. Some nerves, immunoreactive for PGP 9.5 and GAP-43, were also found in the tooth-related part (TRP) of the periodontal ligament close to the tooth surface. PGP 9.5-positive nerves in the TRP appeared very thin and terminated by making loops or plexus-like structures in close apposition to the epithelium layers, overlying the enamel surface in between cementum pearls. Such an intimate association between nerves and the enamel epithelium was not found in the labial periodontal tissue of incisors or the apical growing end of the molar, where periodontal fibre attachment was indistinct. The association between nerves and epithelium in the periodontal ligament of guinea pig molar is site specific and is only seen in the presence of cementum, suggesting that this association is related to the attachment function of the ligament.

Animals↗

PubMed2005

Identification of genes preferentially expressed in periodontal ligament: specific expression of a novel secreted protein, FDC-SP.

Gene expression in human periodontal ligament (PDL) was examined by suppression subtractive hybridization to identify genes that are preferentially expressed in tissue compared to cultured PDL fibroblasts. The most enriched genes in a subtracted cDNA library are primarily genes for extracellular matrix components, types I and III collagen, lumican, periostin, and asporin, among others, whose expression conveys unique mechanical properties to the PDL. Also within this group is the gene for follicular dendritic cell secreted protein (FDC-SP), a small protein like statherin in saliva, not previously found in PDL. FDC-SP's presence in PDL was confirmed by in situ hybridization in mouse which also showed that it was definitely present in the parotid gland, but, surprisingly, not in the other salivary glands: submandibular and sublingual. Since only normal tissue was examined, these findings suggest that FDC-SP plays an important but previously unsuspected role within oral connective tissue.

Cells, Cultured↗

PubMed2004

Site-specific localization of two distinct phosphatases along the osteoblast plasma membrane: tissue non-specific alkaline phosphatase and plasma membrane calcium ATPase.

In osteoblasts, alkaline phosphatase has been reported to be restricted to the basolateral domains. In recent studies, we have demonstrated phosphatase activities different from those of tissue non-specific alkaline phosphatase (TNSALP) along the osteoidal aspect of osteoblast membrane at alkaline and neutral pH on undecalcified freshly frozen sections of rat bones. In the present study, we sought to further characterize and define the nature of membrane-associated phosphatases along the osteoidal aspect of osteoblasts. Histochemical properties of the enzymes and their localization in vivo were examined in long bones of normal Wistar rats and TNSALP null mutant mice and their wild type littermates. Molecular profiles of the enzymes in the osteoblast extracts were also examined. The enzymatic activity of the phosphatase along the osteoidal surface of osteoblasts proved to be activated by both Mg2+ and Ca2+. Unlike TNSALP, the activity was inhibited by vanadate but resistant to levamisole, implicating a similarity between this enzyme and plasma membrane Ca2+ transport ATPase (PMCA). Immunohistochemistry showed that PMCA immunoreactions were restricted to the osteoidal domain of the plasma membrane. Native-PAGE analysis of osteoblast extracts suggested the presence of two phosphatases corresponding, respectively, to TNSALP and PMCA. Western blot analysis after SDS-PAGE of osteoblast extracts confirmed the existence of PMCA (140 kDa) and TNSALP (80 kDa). Gel-chemical analysis of the osteoblast extract from TNSALP null mutant mice depicted phosphatase activity, which was resistant to levamisole. These data suggest the presence of a phosphatase different from TNSALP, most plausibly PMCA, on the osteoidal surface of osteoblasts.

Alkaline Phosphatase↗

PubMed2004

Appearance of electron-dense segments: indication of possible conformational changes of pre-mineralizing collagen fibrils in the osteoid of rat bones.

To elucidate precise mechanisms of appositional mineralization of bone, structural features of mineralizing collagen fibrils of the osteoid in normal and hypocalcaemic rats were examined in detail by transmission electron microscopy. Ultrathin sections of the osteoid of various types of bones of the rats fed with regular or normal calcium diet often displayed electrondense segments in the specific regions of the collagen fibrils located immediately adjacent to the mineralization front or to the mineralization nodules. Such dense segments appeared only after Ur-Pb staining and were more distinct in undecalcified specimens. Dense segments were undetectable in ultrathin sections picked up on ethylene glycol instead of water in the trough, even after Ur-Pb staining. Collagen fibrils in the widened osteoid of hypocalcaemic rats fed with calcium-free diet failed to show electron-dense segments. A careful comparison between the hydrously or anhydrously processed adjacent sections of a normal rat bone indicated a drastic dissolution of electron-dense material from the bone matrix near the mineralization front in hydrously processed sections and, thus, implicated the presence of labile mineral-matrix complexes in the recently mineralized bone matrix. Such labile sediments were readily dissociated within the ultrathin sections while the sections were floating on water and immediately adsorbed onto the pre-mineralizing collagen fibrils, where some conformational changes might have occurred. These data indicate that highly electron-dense segments appearing in the osteoidal collagen fibrils are a type of process-induced product, which indirectly represent possible structural alterations in the segmental portions of pre-mineralizing collagen fibrils in the osteoid of rat bones.

Animals↗

PubMed2004

Eccentric localization of osteocytes expressing enzymatic activities, protein, and mRNA signals for type 5 tartrate-resistant acid phosphatase (TRAP).

Enzymatic activity of type 5 tartrate-resistant acid phosphatase (TRAP) has been regarded as one of the reliable markers for osteoclasts and their precursors. The presence of TRAP activity in osteocytes near the bone resorbing surface has also been pointed out in some reports. However, the significance of TRAP reactions in osteocytes remains controversial and, in fact, there is no agreement as to whether the histochemical enzyme reactions in osteocytes represent the TRAP enzyme generated by the respective osteocytes or is a mere diffusion artifact of the reaction products derived from the nearby osteoclasts. Current histochemical, immunohistochemical, and in situ hybridization studies of rat and canine bones confirmed TRAP enzyme activity, TRAP immunoreactivity, and the expression of Trap mRNA signals in osteocytes located close to the bone-resorbing surface. TRAP/Trap- positive osteocytes thus identified were confined to the areas no further than 200 microm from the bone-resorbing surface and showed apparent upregulation of TRAP/Trap expression toward the active osteoclasts. Spatial and temporal patterns of TRAP/Trap expression in the osteocytes should serve as a valuable parameter for further analyses of biological interactions between the osteocytes and the osteoclasts associated with bone remodeling.

Acid Phosphatase↗

PubMed2004

Age-related changes and the possible adaptability of rat jaw muscle spindles: immunohistochemical and fine structural studies.

Afferent signals from jaw muscle spindles contribute to the feedback mechanism that regulates mastication. The integrity and adaptability of this proprioceptor to age-related changes of the surrounding structures are therefore essential to maintain an appropriate masticatory function throughout life. In this study, we examined muscle spindles obtained from temporal and masseter muscles of 10-week-, 12-, 18-, and 24-month-old Wistar rats, employing immunohistochemistry for protein gene product 9.5 (PGP 9.5) or growth-associated protein (GAP-43) in addition to transmission electron microscopy, in order to investigate their morphological changes in relation to the effect of aging on the adaptive potential of the receptors. Immunohistochemistry for PGP 9.5 showed virtually similar reactions at sensory nerve terminals in all age groups. On the other hand, immunoreactivity for GAP-43 in the sensory nerve ending of the muscle spindles was found 2 and 3 weeks after birth but became almost undetectable by 10 weeks. However GAP-43 immunoreactions occasionally reappeared in those of spindles in 12- and 18-month old animals, and vanished again by 24 months of age. Electron microscopic observations also revealed age-related morphological changes in the intrafusal muscle fibers of the rats in 12-month and older groups. The extent of degenerative and/or atrophic alterations of intrafusal fibers increased with age and involved the nerve elements of spindles by 24 months. These findings indicate that the adaptation potential of rat jaw muscle spindles is well preserved until middle age, but diminishes in elderly animals. Structural changes of muscle spindles in elderly animals probably contribute to the deterioration of the muscular function.

Age Factors↗

PubMed2004

The induction of enamel and dentin complexes by subcutaneous implantation of reconstructed human and murine tooth germ elements.

Tooth induction by xenogenic graft of reconstructed human tooth germ components has never been attempted. Here we report our first attempt at a transplantation of human tooth germ components, heterologously recombined with mouse dental epithelia, into immunocompromised animals. Human third molar tooth germs enucleated from young patients as prophylactic treatment for orthodontic reasons were collected. The whole or minced human dental papilla was reconstructed with human- or mouse molar enamel epithelium, and transplanted in the dorsal aspect of C.B-17/Icr-scid Jcl mice. The transplant of human dental papilla reconstructed with human enamel epithelium formed thin dentin and immature enamel layers by 3 to 4 weeks, but remained extremely small in quantity due to a shortage of epithelial components in the graft. The addition of E16 mouse molar enamel organs (n=10-12) to each graft augmented the formation of tooth germ-like structures, but the differentiation of mouse molar ameloblasts was suppressed. However, once a solid layer of mineralized dentin was established, mouse ameloblasts accelerated their differentiation, and completed the enamel matrix formation and maturation within the following 4 weeks, whereas human ameloblasts, which had interacted with human dental papilla, remained in the stage of matrix formation during the same period. These data imply that, in reconstructed transplants, the differentiation of mouse dental epithelia is restrained by putative suppressive factors derived from human dental papilla until they are separated by mineralized dentin layers that serve as a diffusion barrier. The mouse enamel organ nevertheless retains its own phenotypic characteristics and intrinsic timing of cell differentiation and function.

Amelogenin↗

PubMed2004

[Mineralization of dental hard tissues].

Tooth is a composite of the three different biological mineralized tissues (dentin, enamel, and cementum) that acquired supreme mechanical properties and function necessary for the mechanical digestion of the food throughout life. The underlining mechanisms of mineralization of each of these three components are unique and different from each other. Here the focus was placed on the mineralization process of dentin and enamel of the teeth of vertebrates.

Amelogenesis↗

PubMed2003

Pulpal regeneration after cavity preparation, with special reference to close spatio-relationships between odontoblasts and immunocompetent cells.

The regeneration process of the odontoblast cell layer incident to tooth injury, especially its relationship with immunocompetent cells in pulp healing, has not been fully understood. The purpose of the present study was to clarify this relationship between odontoblasts and immunocompetent cells in the process of pulp regeneration following cavity preparation in rat molars by immunocytochemistry for heat shock protein (Hsp) 25 as well as class II major histocompatibility complex (MHC) molecules. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin, whereas class II MHC-positive cells were predominantly located beneath the odontoblast cell layer. Cavity preparation caused the destruction of the odontoblast layer to form an edematous lesion and the shift of class II MHC-positive cells with the injured odontoblasts toward the pulp core at the affected site. Some damaged odontoblasts without apparent cytoplasmic processes, round in profile, retained the immunoreactivity for Hsp25, suggesting the survival of a part of the odontoblasts against artificial external stimuli. Twelve hours after cavity preparation, numerous class II MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules. By postoperative 72 hours, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border, but the class II MHC-positive cells moved from the pulp-dentin border to the subodontoblastic layer. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts. The functional roles of Hsp 25-positive odontoblasts and immunocompetent cells such as class II MHC-positive cells in the process of pulp regeneration after cavity preparation are discussed in conjunction with our previous experimental data.

Animals↗

PubMed2003

Development of a liquid culture system for megakaryocyte terminal differentiation: fibrinogen promotes megakaryocytopoiesis but not thrombopoiesis.

Megakaryocyte differentiation is composed of three distinct stages: formation of erythromegakaryocytic progenitor cells, maturation of megakaryocytes and production of platelets. We have developed a liquid culture system for megakaryocyte terminal differentiation from haematopoietic stem cells into proplatelets. In this system, CD34+ cells isolated from human cord blood, differentiated to CD41+ cells, were classified either as propidium iodide (PI)+ cells (large) or PI- cells (small) by fluorescence-activated cell sorting analysis on the late-stage CD41+ cells. Transmission electron microscopy showed that the cultured small cells were morphologically identical to platelets isolated from normal peripheral blood. Moreover, the number of differentiated cells that were CD42b-positive attained an approximately 60-fold expansion over that of the primary CD34+ cells in this culture system. Furthermore, gene expression of megakaryocytopoietic transcriptional factors, GATA-1 and NF-E2, and several megakaryocytic markers such as glycoprotein (GP)IIb and thromboxane synthase was observed in the individual differentiation stage. Treatment with fibrinogen, a ligand of GPIIb/IIIa, increased the number of CD41+/PI+ cells, but treatment in the late stage suppressed CD41+/PI- cell formation, suggesting that fibrinogen promotes megakaryocytopoiesis, but not thrombopoiesis. We conclude that this liquid culture system using human CD34+ cells may be used to mimic the physiological development from haematopoietic stem cells into megakaryocytes, as well as promote subsequent thrombopoiesis.

Antigens, CD34↗

PubMed2003

Possible role of dentin matrix in region-specific deposition of cellular and acellular extrinsic fibre cementum.

The mechanism whereby a region-specific deposition of the two types of cementum (cellular cementum and acellular extrinsic fibre cementum) is regulated on the growing root surface was tested using bisphosphonate-affected teeth of young rats and guinea pigs. The animals were injected subcutaneously with 8 or 10 mg P x kg body weight(-1) x day(-1) of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) for 1 or 2 weeks. In rat molars, HEBP prevented mineralization of newly formed root dentin matrix and totally inhibited de novo deposition of acellular extrinsic fibre cementum. Instead, thick cellular cementum was induced on the non-mineralized root dentin surface, irrespective of the position of the root. In both animals, cellular cementum was also induced on the non-mineralized surface of root analogue dentin in HEBP-affected incisors, where only acellular extrinsic fibre cementum is deposited under normal conditions. In normal rat molars, dentin sialoprotein (DSP) was concentrated along the dentin-cellular cementum border, but not that of dentin and acellular extrinsic fibre cementum. In HEBP-affected rat incisors, DSP was shown to penetrate through the non-mineralized dentin into the surrounding tissues, but not through the mineralized portions. These data suggest that, at the site of cellular cementum formation, putative inducing factors for cellular cementum might diffuse into the periodontal space through the newly deposited mantle dentin matrix before it is mineralized. At earlier stages of root formation, mantle dentin might mineralize more promptly not to allow such diffusion. The timing of mineralization of mantle dentin matrix might be the key determinant of the types of the cementum deposited on the growing root surface.

Animals↗

PubMed2003

[Not Available].

Explore the source record for details and available documents.

Journal Article↗

PubMed2002

Morphological influence of ascorbic acid deficiency on endochondral ossification in osteogenic disorder Shionogi rat.

The influences of chronic deficiency of L-ascorbic acid (AsA) on the differentiation of osteo-chondrogenic cells and the process of endochondral ossification were examined in the mandibular condyle and the tibial epiphysis and metaphysis by using Osteogenic Disorder Shionogi (ODS) rats that bear an inborn deficiency of L-gulonolactone oxidase. Weanling male rats were kept on an AsA-free diet for up to 4 weeks, until the symptoms of scurvy became evident. The tibiae and condylar processes of scorbutic rats displayed undersized and distorted profiles with thin cortical and scanty cancellous bones. In these scorbutic bones, the osteoblasts showed characteristic expanded round profiles of rough endoplasmic reticulum, and lay on the bone surface where the osteoid layer was missing. Trabeculae formation was deadlocked, although calcification of the cartilage matrix proceeded in both types of bone. Scorbutic condylar cartilage showed severe disorganization of cell zones, such as unusual thickening of the calcification zone, whereas the tibial cartilage showed no particular alterations (except for a moderately decreased population of chondrocytes). In condylar cartilage, hypertrophic chondrocytes were encased in a thickened calcification zone, and groups of nonhypertrophic chondrocytes occasionally formed cell nests surrounded by a metachromatic matrix in the hypertrophic cell zone. These results indicate that during endochondral ossification, chronic AsA deficiency depresses osteoblast function and disturbs the differentiation pathway of chondrocytes. The influence of scurvy on mandibular condyle cartilage is different from that on articular and epiphyseal cartilage of the tibia, suggesting that AsA plays different roles in endochondral ossification in the mandibular condyle and long bones.

Animals↗

PubMed2002

Growth-associated protein-43 immunohistochemical and ultrastructural changes in jaw muscle spindles of the rat following loss of occlusion.

The effects of complete loss of occlusion on the structural and functional status of these muscle spindles were investigated by immunohistochemistry either for protein gene product 9.5 (PGP 9.5) or growth-associated protein-43 (GAP-43) by light and electron microscopy. All the upper molars of 4-week-old Wistar rats were extracted and the erupted portions of the upper and lower incisors of the same animals were cut-off at the level of the interdental papilla every other day. In a control group, immunoreactivity for GAP-43 was positive in the developing annulospiral endings of 2-week-old rats, but was not detected in any of the muscle spindles after 3 weeks of age. At 4 weeks of age, the PGP 9.5 immunostained spindles had well-differentiated annulospiral endings. Ultrastructurally, these afferent endings showed lenticular or circular profiles in cross-sections, and were differentially indented into the intrafusal-fibres. The inner surfaces of the terminals formed rather smooth myoneural junctions, while the outer surfaces were covered only by basal lamina continuous with that of the underlying intrafusal muscle fibres. After the experimental elimination of occlusal contact, GAP-43 immunoreactivity reappeared in some nerve endings of muscle spindles by 3 days, and persisted for at least 28 days. During this period, the afferent-terminals exhibited various fine structural abnormalities such as irregular outlines and invaginated neuromuscular interfaces. Some sensory-terminal (ST) profiles were completely engulfed by intrafusal-fibres. However, GAP-43 expression and ultrastructural alterations became undetectable within a week of the end of incisal cutting and the recovery of incisal-contact. These data indicate that remodelling of nerve terminals in muscle spindles, as assessed by GAP-43 expression and ultrastructural changes, occurs soon after a loss of occlusion, and ceases if incisal-contact is restored. It is concluded that possible changes in jaw muscle function, as well as a sudden loss of proprioceptive sensory input from the periodontal mechanoreceptors of molars and incisors, induce the structural reorganisation of nerve terminations in jaw muscle spindles that is associated with the appearance and disappearance of GAP-43 immunoreactivity.

Animals↗

PubMed2002

The influence of parathyroid hormone-related protein (PTHrP) on tooth-germ development and osteoclastogenesis in alveolar bone of PTHrP-knock out and wild-type mice in vitro.

In a previous study, it was shown that tooth germs of neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are penetrated or compressed by the surrounding alveolar bone, suggesting an important role for PTHrP in the formation and activation of osteoclasts around growing tooth germs. In order to elucidate the role of PTHrP during the development of the tooth germ and related structures, mandibular explants containing cap stage tooth germs of embryonic day 14, homozygous mice were here cultured with or without surrounding alveolar bone. There was no difference in the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells around the first molars of homozygous and wild-type mice. After 10 days of culture, osteoclastic cells were rarely present in explants from homozygous mice and penetration of alveolar bone into the dental papilla was observed. The decline in osteoclast number was partly restored by the addition of PTHrP to the culture. Tooth germs of both wild-type and homozygous mice cultured without alveolar bone developed well, with no apparent structural abnormality; dentine formation was evident after 10 days. These data suggest that PTHrP is not required for the development of the tooth germ proper but is indispensable in promoting the osteoclast formation required to accommodate that development.

Acid Phosphatase↗

PubMed2002

On the origin of intrinsic matrix of acellular extrinsic fiber cementum: studies on growing cementum pearls of normal and bisphosphonate-affected guinea pig molars.

Cementum pearls (CPs) belong to a type of acellular extrinsic fiber cementum (AEFC) that form on the maturing enamel of guinea pig molars. This study aimed to elucidate the forming process of intrinsic matrix of AEFC using the CPs of normal and bisphosphonate-affected guinea pig molars as experimental models. A group of guinea pigs were subjected to continuous administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) for 2 wk to inhibit mineralization of growing CPs. Fenestration of the enamel organ and migration of periodontal cells on to the exposed surface of maturing enamel appeared to be unaffected by HEBP, whereas de novo formation as well as growth of pre-existing CPs did not proceed under the same conditions. Immunoreactions for osteopontin were located exclusively on the mineralized matrix of preformed CPs, implying the absence of additional deposition or accumulation of putative intrinsic cementum matrix on the affected CPs, where the propagation of mineral phase had been arrested. In both normal and HEBP-treated groups, distinct enzymatic reactions for alkaline phosphatase appeared on the cells of the periodontal ligament associated closely with the sites of CP formation, and along the mineralization front of CPs. These observations suggest that the mineralization process per se plays a central role in the deposition of AEFC matrix and that alkaline phosphatase of periodontal cells penetrating through the enamel organ to the maturing enamel surface plays a key role in the mineralization process of CPs.

Alkaline Phosphatase↗

PubMed2002

Expression of alternatively spliced RNA transcripts of amelogenin gene exons 8 and 9 and its end products in the rat incisor.

In addition to seven known exons of the amelogenin gene, recent studies have identified two exons downstream of amelogenin exon 7 in genomic DNA of mouse and rat. Here the spatial and temporal expression of mRNAs and of the translated proteins derived from alternative splicing of the amelogenin gene ending with exon 8 and exon 9 were examined by in situ hybridization (ISH) and immunohistochemistry (IHC). RNA signals for exons 8 and 9 were expressed in the ameloblast layer extending from early presecretory to postsecretory transitional stages of amelogenesis. IHC of amelogenin proteins that include sequences encoded by these exons demonstrated identical localization of these proteins in the ameloblast layer corresponding to RNA signals identified by ISH. There was intense immunostaining of the enamel matrix secreted by these cells. Western blotting analysis of rat enamel proteins revealed three distinct protein bands with sequences encoded by the new exons. These data confirmed the existence of the transcripts of alternatively spliced mRNAs coding for exons 8 and 9 of the amelogenin gene in rat tooth germs and suggest that the translated proteins contribute to the heterogeneity of amelogenins and have some significant roles in enamel formation and mineralization.

Alternative Splicing↗

PubMed2002

A unique localization of mechanoreceptors in the periodontal tissue of guinea pig teeth.

This study describes the unique distribution of Ruffini endings (RE) in the periodontal tissues of the guinea pig teeth with special references to their presence in the enamel-related aspects of the continuously growing incisors and molars. In guinea pig incisors, immunohistochemistry for PGP 9.5 and glia specific S-100 protein revealed a condensed distribution of well-developed RE in the bone-related part of the lingual periodontal ligament as has been reported in many other rodents. In most cases, some RE-like nerve elements characterized by dendritic ramification and rounded terminal Schwann cells were found to be located in the labial, enamel-related regions, where no periodontal ligament-like fiber arrangement was established. In the molar periodontal ligament, well-developed RE-like nerve elements were also distributed in the enamel-related part, but in intimate relation to thick periodontal fiber bundles inserted in the cementum pearls grown on the enamel surface. In some cases, few RE were located in the apical region of the alveolar socket, where no periodontal fiber bundles could be identified. Our data provide the first morphological evidence of the presence of RE-like nerve elements in the enamel-related, fibrous connective tissue of continuously erupting rodent incisors. These data indicate that RE in guinea pig periodontal tissues have variable spatial correlation to the surrounding fibers, implicating their diverse mechanoreceptive properties depending on the anatomical location.

Animals↗