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Rab GTPases, the largest group of small monomeric GTPases, have been shown to participate in membrane trafficking involving many cellular processes. However, their roles during osteoblastic differentiation remain to be elucidated. In this study, we investigated Rab GTPase involvement in osteoblastic differentiation. Protein levels of a series of Rabs (Rab4, Rab5, Rab7, Rab9a, Rab11a/b, and Rab27) were increased during osteoblastic differentiation of MC3T3-E1 cells, and the Rab11a/b levels were particularly pronounced in the presence of Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, an activator of osteoblastogenesis. We subsequently investigated the functional contribution of Rab11a and Rab11b during osteoblastic differentiation. The alkaline phosphatase (ALP) levels were reduced by Rab11b depletion but not by Rab11a depletion. Because our result suggested that Rab11a and Rab11b could be regulated downstream of Runx2 (Runt-related transcription factor 2), a key transcription factor for osteoblastic differentiation, we investigated the effects of the double knockdown of Runx2 and Rab11a or Rab11b on osteoblastic phenotypes. The double knockdown significantly reduced ALP activity as well as collagen deposition compared with single Runx2 knockdown. Furthermore, the Rab11a and Rab11b response to mechanical stress in vivo was investigated using a mouse orthodontic tooth movement model. Rab11a and Rab11b expression was enhanced in the periodontal ligament, where bone formation is activated by tensile stress. This study shows that Rab11a and Rab11b are regulated downstream of Runx2 in osteoblastic differentiation, and their expressions are also controlled by tensile stress.

DOI： 10.1016/j.bbrc.2022.07.061

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Periodontitis is one of the most common oral diseases resulting in gingival inflammation and tooth loss. Growing evidence indicates that it results from dysbiosis of the oral microbiome, which interferes with the host immune system, leading to bone destruction. Immune cells activate periodontal ligament cells to express the receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) and promote osteoclast activity. Osteocytes have active roles in periodontitis progression in the bone matrix. Local proteins are involved in bone regeneration through functional immunological plasticity. Here, we discuss the current knowledge of cellular and molecular mechanisms in periodontitis, the roles of local proteins, and promising synthetic compounds generating a periodontal regeneration effect. It is anticipated that this may lead to a better perception of periodontitis pathophysiology.

DOI： 10.3390/ijms23105540

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Hyperammonemia is known to cause various neurological dysfunctions such as seizures and cognitive impairment. Several studies have suggested that hyperammonemia may also be linked to the development of Alzheimer's disease (AD). However, the direct evidence for a role of ammonia in the pathophysiology of AD remains to be discovered. Herein, we report that hyperammonemia increases the amount of mature amyloid precursor protein (mAPP) in astrocytes, the largest and most prevalent type of glial cells in the central nervous system that are capable of metabolizing glutamate and ammonia, and promotes amyloid beta (Aβ) production. We demonstrate the accumulation of mAPP in astrocytes was primarily due to enhanced endocytosis of mAPP from the plasma membrane. A large proportion of internalized mAPP was targeted not to the lysosome, but to the endoplasmic reticulum, where processing enzymes β-secretase BACE1 (beta-site APP cleaving enzyme 1) and γ-secretase presenilin-1 are expressed, and mAPP is cleaved to produce Aβ. Finally, we show the ammonia-induced production of Aβ in astrocytic endoplasmic reticulum was specific to Aβ42, a principal component of senile plaques in AD patients. Our studies uncover a novel mechanism of Aβ42 production in astrocytes and also provide the first evidence that ammonia induces the pathogenesis of AD by regulating astrocyte function.

DOI： 10.1016/j.jbc.2022.101933

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Most oral squamous cell carcinomas (OSCCs) arise from a premalignant lesion, oral epithelial dysplasia; however, useful markers for the early detection of OSCC are lacking. The present study aimed to establish a novel experimental model to observe changes in the sequential expression patterns of mRNAs and proteins in a rat model of tongue cancer using liquid-based cytology techniques. Cytology specimens were collected at 2, 5, 8, 11, 14, 17 and 21 weeks from rats treated with 4-nitroquinoline 1-oxide to induce tongue cancer. The expression of candidate biomarkers was examined by performing immunocytochemistry and reverse transcription-quantitative PCR. The percentage of positively stained nuclei was calculated as the labeling index (LI). All rats developed OSCC of the tongue at 21 weeks. The mRNA expression levels of bromodomain protein 4 (Brd4), c-Myc and Tp53 were upregulated during the progression from negative for intraepithelial lesion or malignancy to squamous cell carcinoma (SCC). Brd4- and c-Myc-LI increased in low-grade squamous intraepithelial lesion, high-grade squamous intraepithelial lesion and SCC specimens. p53-LI was significantly increased in SCC specimens. This novel experimental model allowed the observation of sequential morphological changes and the expression patterns of mRNAs and proteins during carcinogenesis. Combining immunocytochemistry with cytology-based diagnoses may potentially improve the diagnostic accuracy of OSCC.

DOI： 10.3892/ol.2022.13196

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OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.

DOI： 10.1111/odi.14162

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The periodontal ligament (PDL) is a specialized connective tissue that provides structural support to the tooth and is crucial for oral functions. The mechanical properties of the PDL are mainly derived from the tissue-specific composition and structural characteristics of the extracellular matrix (ECM). The ECM also plays key roles in determining cell fate in the cellular microenvironment thus crucial in the PDL tissue homeostasis. In the present study, we determined the comprehensive ECM profile of mouse molar PDL using laser microdissection and mass spectrometry-based proteomic analysis with ECM-oriented data curation. Additionally, we evaluated changes in the ECM proteome under mechanical loading using a mouse orthodontic tooth movement (OTM) model and analyzed potential regulatory networks using a bioinformatics approach. Proteomic changes were evaluated in reference to the novel second harmonic generation (SHG)-based fiber characterization. Our ECM-oriented proteomics approach succeeded in illustrating the comprehensive ECM profile of the mouse molar PDL. We revealed the presence of type II collagen in PDL, possibly associated with the load-bearing function upon occlusal force. Mechanical loading induced unique architectural changes in collagen fibers along with dynamic compositional changes in the matrisome profile, particularly involving ECM glycoproteins and matrisome-associated proteins. We identified several unique matrisome proteins which responded to the different modes of mechanical loading in PDL. Notably, the proportion of type VI collagen significantly increased at the mesial side, contributing to collagen fibrogenesis. On the other hand, type XII collagen increased at the PDL-cementum boundary of the distal side. Furthermore, a multifaceted bioinformatics approach illustrated the potential molecular cues, including PDGF signaling, that maintain ECM homeostasis under mechanical loading. Our findings provide fundamental insights into the molecular network underlying ECM homeostasis in PDL, which is vital for clinical diagnosis and development of biomimetic tissue-regeneration strategies.

DOI： 10.3389/fphys.2022.899699

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Laminin, a basement membrane heterotrimeric glycoprotein composed of α/β/γ subunits, has important tissue-specific functions in the control of cellular behavior. Our recent study showed the colocalization of CD163+ M2-like macrophages with Schwann cells in human dental pulp, leading us to hypothesize that the laminin isoform of Schwann cells is associated with CD163 expression. The present study investigated the distribution of laminin isoforms in human dental pulp and the underlying mechanisms that affect macrophage phenotypes. Immunofluorescence analysis indicated that blood vessels were exclusively positive for laminin α4 and α5, whereas laminin α2 was associated with Schwann cells. Unexpectedly, laminin α3/laminin-332 (α3β3γ2) was detected on lymphatic vessels. In intact and carious teeth, CD163+ cells were associated with laminin α2, whereas CD206 single-positive cells were present inside, outside, and along blood vessels. In vitro incubation of THP-1 macrophages in plates coated with laminin-211/511 or its functionally analogous E8 fragments of α-chain (E8-α) indicated that cell shapes differed between macrophages grown on laminin-211/E8-α2 and macrophages grown on laminin-511/E8-α5. Laminin-211/E8-α2-coated plates upregulated CD163 expression, compared with laminin-511/E8-α5-coated plates. Integrin α3- and integrin α6-neutralizing Abs altered the shape of THP-1 macrophages and upregulated mRNA levels of CD206 and CD163 in macrophages grown on laminin-511; the neutralizing Abs did not affect macrophages grown on laminin-211. These findings suggest that laminin isoforms differentially regulate macrophage behavior via distinct integrin-laminin affinities. Of note, laminin-332 is expressed by pulpal lymphatic vessels, the existence of which has been debated; laminin-211 might have a role in maintaining CD163 expression on macrophages.

DOI： 10.4049/immunohorizons.2100110

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This study aimed to evaluate the anticariogenic biofilm activity of a novel zinc-containing glass ionomer cement, Caredyne Restore (CR), using a flow-cell system that reproduces Stephan responses. Streptococcus mutans biofilms were cultured on either CR or hydroxyapatite (HA) discs mounted on a modified Robbins device. The media were allowed to flow at a speed of 2 mL/min for 24 h while exposed to an acidic buffer twice for 30 min to mimic dietary uptake. Acid exposure enhanced biofilm inhibition in the CR group, which showed 2.6 log CFU/mm2 in viable cells and a 2 log copies/mL reduction in total cells compared to the untreated group after 24 h of incubation, suggesting enhanced anticariogenic activity due to the release of fluoride and zinc ions. However, there was no difference in the number of viable and total cells between the two experimental groups after 24 h of incubation in the absence of an acidic environment. The anticariogenic biofilm activity of CR occurs in acidic oral environments, for example in the transient pH drop following dietary uptake. CR restorations are recommended in patients at high risk of caries due to hyposalivation, difficulty brushing, and frequent sugar intake.

DOI： 10.3390/antibiotics10080977

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Hh signaling has been shown to be activated in intact and injured peripheral nerve. However, the role of Hh signaling in peripheral nerve is not fully understood. In the present study, we observed that Hh signaling responsive cells [Gli1(+) cells] in both the perineurium and endoneurium. In the endoneurium, Gli1(+) cells were classified as blood vessel associated or non-associated. After injury, Gli1(+) cells around blood vessels mainly proliferated to then accumulate into the injury site along with endothelial cells. Hh signaling activity was retained in Gli1(+) cells during nerve regeneration. To understand the role of Hedgehog signaling in Gli1(+) cells during nerve regeneration, we examined mice with Gli1(+) cells-specific inactivation of Hh signaling (Smo cKO). After injury, Smo cKO mice showed significantly reduced numbers of accumulated Gli1(+) cells along with disorganized vascularization at an early stage of nerve regeneration, which subsequently led to an abnormal extension of the axon. Thus, Hh signaling in Gli1(+) cells appears to be involved in nerve regeneration through controlling new blood vessel formation at an early stage.

DOI： 10.1016/j.neures.2021.06.003

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Wnt signaling plays a critical role in the development of many organs, including the major movable craniofacial organs tongue, lip, and eyelid. Four members of the R-spondin family (Rspo1-4) bind to Lgr4/5/6 to regulate the activation of Wnt signaling. However, it is not fully understood how Rspos/Lgrs regulate Wnt signaling during the development of movable craniofacial organs. To address this question, we examined the expression of Rspos, Lgrs, and Axin2 (major mediator of canonical Wnt signaling) during tongue, lip, and eyelid development. The expression of Axin2, Rspos and Lgrs was observed in many similar regions, suggesting that Rspos likely activate canonical Wnt signaling through the Lgr-dependent pathway in these regions. Lgr expression was not detected in regions where Axin2 and Rspos were expressed, suggesting that Rspos might activate canonical Wnt signaling through the Lgr-independent pathway in these regions. In addition, the expression of Rspos and Lgrs were observed in some other regions where Axin2 was not expressed, suggesting the possibility that Rspos and/or Lgrs are involved in non-canonical Wnt signaling or the Wnt-independent pathway. Thus, we identified a dynamic spatiotemporal expression pattern of Rspos and Lgrs during the development of the eyelid, tongue, and lip.

DOI： 10.1016/j.gep.2021.119195

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OBJECTIVES: Previous spinal nerve injury studies have reported brain-derived neurotrophic factor (BDNF) mRNA upregulation in either the ipsilateral dorsal root ganglion (DRG) neurons or both the contralateral and ipsilateral DRG neurons from early period after peripheral nerve injury. This BDNF elevation induces hyperalgesia in the injured and/or uninjured sites, but this detailed mechanism remains unknown. This study aimed to investigate the BDNF mRNA expression in bilateral DRG neurons caused by unilateral nerve injury and to explore the possible mechanisms by which nitric oxide (NO) mediates BDNF production in the DRG, resulting in contralateral hyperalgesia. METHODS: Early changes in BDNF mRNA expression in the bilateral trigeminal ganglia, within 1 day after mental nerve transection, were examined. Additionally, the effects on BDNF production of the NO synthase inhibitor N(ω)-nitro-l-arginine methyl ester (L-NAME) were investigated in the bilateral trigeminal ganglia. The relationship between injured neurons and BDNF production in the trigeminal ganglia was then assessed using immunohistochemical and retrograde tracing methods. RESULTS: Reverse transcription-PCR analysis demonstrated that unilateral transection of the mental nerve induced a rapid elevation of BDNF mRNA expression, which was inhibited by the intracerebroventricular administration of L-NAME prior to nerve transection. This effect was observed in both the ipsilateral and contralateral sides to the nerve transection. BDNF immunostaining combined with FluoroGold retrograde tracing revealed two types of BDNF-reactive neurons, FluoroGold-labelled and non-FluoroGold-labelled neurons, in the ipsilateral and contralateral sides of the trigeminal ganglia. BDNF-positive cells were also observed in the trigeminal ganglia of other trigeminal nerve branches. CONCLUSIONS: Unilateral nerve injury upregulates BDNF production in the bilateral trigeminal ganglia by NO-mediated and/or indirect activation of afferent neurons, resulting in contralateral hyperalgesia.

DOI： 10.1097/WNR.0000000000001635

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Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

Macrolides are used to treat various infectious diseases, including periodontitis. Furthermore, macrolides are known to have immunomodulatory effects; however, the underlying mechanism of their action remains unclear. DEL-1 has emerged as an important factor in homeostatic immunity and osteoclastogenesis. Specifically, DEL-1 is downregulated in periodontitis tissues. Therefore, in the present study, we investigated whether the osteoclastogenesis inhibitory effects of erythromycin (ERM) are mediated through upregulation of DEL-1 expression. We used a ligature-induced periodontitis model in C57BL/6Ncrl wild-type or DEL-1-deficient mice and in vitro cell-based mechanistic studies to investigate how ERM inhibits alveolar bone resorption. As a result of measuring alveolar bone resorption and gene expression in the tooth ligation model, ERM treatment reduced bone loss by increasing DEL-1 expression and decreasing the expression of osteoclast-related factors in wild-type mice. In DEL-1-deficient mice, ERM failed to suppress bone loss and gene expression of osteoclast-related factors. In addition, ERM treatment downregulated osteoclast differentiation and calcium resorption in in vitro experiments with mouse bone marrow-derived macrophages. In conclusion, ERM promotes the induction of DEL-1 in periodontal tissue, which may regulate osteoclastogenesis and decrease inflammatory bone resorption. These findings suggest that ERM may exert immunomodulatory effects in a DEL-1-dependent manner.

DOI： 10.3390/antibiotics10030312

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Mandibular anomalies are often seen in various congenital diseases, indicating that mandibular development is under strict molecular control. Therefore, it is crucial to understand the molecular mechanisms involved in mandibular development. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating the level of gene expression. We found that the mesenchymal conditional deletion of miRNAs arising from a lack of Dicer (an essential molecule for miRNA processing, Dicerfl/fl ;Wnt1Cre), led to an abnormal groove formation at the distal end of developing mandibles. At E10.5, when the region forms, inhibitors of Hh signaling, Ptch1 and Hhip1 showed increased expression at the region in Dicer mutant mandibles, while Gli1 (a major mediator of Hh signaling) was significantly downregulated in mutant mandibles. These suggest that Hh signaling was downregulated at the distal end of Dicer mutant mandibles by increased inhibitors. To understand whether the abnormal groove formation inDicer mutant mandibles was caused by the downregulation of Hh signaling, mice with a mesenchymal deletion of Hh signaling activity arising from a lack of Smo (an essential molecule for Hh signaling activation, Smofl/fl ;Wnt1Cre) were examined. Smofl/fl ;Wnt1Cre mice showed a similar phenotype in the distal region of their mandibles to those in Dicerfl/fl ;Wnt1Cre mice. We also found that approximately 400 miRNAs were expressed in wild-type mandibular mesenchymes at E10.5, and six microRNAs were identified as miRNAs with binding potential against both Ptch1 and Hhip1. Their expressions at the distal end of the mandible were confirmed by in situ hybridization. This indicates that microRNAs regulate the distal part of mandibular formation at an early stage of development by involving Hh signaling activity through controlling its inhibitor expression level.

DOI： 10.1111/joa.13328

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Language：Japanese   Publisher：新潟歯学会  

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A previous study has reported that a novel fluoro-zinc-silicate glass ionomer cement (Caredyne Restore) showed superior anti-biofilm effects by interfering with bacterial adhesion. However, the active ions may degrade with time. This study aimed to assess the valid anti-biofilm effects of Caredyne Restore after being aged by water immersion for 3 weeks. Streptococcus mutans biofilm was allowed to grow on the surface before and after water aging for 24 h using a modified Robbins device flow-cell system. The results showed water aging promoted biofilm formation. Insufficient amount of fluoride and zinc ions were released from Caredyne Restore after water aging under neutral pH condition. An acidic pH is needed to exert effective anti-biofilm properties. As the release of active ions from Caredyne Restore will gradually decrease after the restoration,  the restoration may not prevent biofilm formation after 3 weeks while neutral pH is maintained by the buffering capacity of saliva.

DOI： 10.1080/08927014.2020.1856371

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OBJECTIVE: Hypohidrotic ectodermal dysplasia (HED) is a hereditary disorder characterized by abnormal structures and functions of the ectoderm-derived organs, including teeth. HED patients exhibit a variety of dental symptoms, such as hypodontia. Although disruption of the EDA/EDAR/EDARADD/NF-κB pathway is known to be responsible for HED, it remains unclear whether this pathway is involved in the process of enamel formation. EXPERIMENTAL SUBJECTS AND METHODS: To address this question, we examined the mice overexpressing Ikkβ (an essential component required for the activation of NF-κB pathway) under the keratin 5 promoter (K5-Ikkβ). RESULTS: Upregulation of the NF-κB pathway was confirmed in the ameloblasts of K5-Ikkβ mice. Premature abrasion was observed in the molars of K5-Ikkβ mice, which was accompanied by less mineralized enamel. However, no significant changes were observed in the enamel thickness and the pattern of enamel rods in K5-Ikkβ mice. Klk4 expression was significantly upregulated in the ameloblasts of K5-Ikkβ mice at the maturation stage, and the expression of its substrate, amelogenin, was remarkably reduced. This suggests that abnormal enamel observed in K5-Ikkβ mice was likely due to the compromised degradation of enamel protein at the maturation stage. CONCLUSION: Therefore, we could conclude that the overactivation of the NF-κB pathway impairs the process of amelogenesis.

DOI： 10.1111/odi.13384

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Pluripotent stem cells are classified as naïve and primed cells, based on their in vitro growth characteristics and potential to differentiate into various types of cells. Human-induced pluripotent stem cells (iPSCs, also known as epiblast stem cells [EpiSCs]) have limited capacity to differentiate and are slightly more differentiated than naïve stem cells (NSCs). Although there are several in vitro protocols that allow iPSCs to differentiate into pancreatic lineage, data concerning generation of β-cells from these iPSCs are limited. Based on the pluripotentiality of NSCs, it was hypothesized that NSCs can differentiate into pancreatic β-cells when placed under an appropriate differentiation induction condition. We examined whether NSCs can be efficiently induced to form potentially pancreatic β cells after being subjected to an in vitro protocol. Several colonies resembling in vitro-produced β-cell foci, with β-cell-specific marker expression, were observed when NSC-derived embryoid bodies (EBs) were induced to differentiate into β-cell lineage. Conversely, EpiSC-derived EBs failed to form such foci in vitro. Intrapancreatic grafting of the in vitro-formed β-cell foci into nude mice (BALB/c-nu/nu) generated a cell mass containing insulin-producing cells (IPCs), without noticeable tumorigenesis. These NSCs can be used as a promising resource for curing type 1 diabetes.

DOI： 10.3390/jcm9092838

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Cleft palate is one of the most common congenital diseases. Therefore, elucidating the molecular mechanisms of palate development is crucial for basic science and the clinical field. Cleft palate in mouse with targeted gene mutation is an excellent experimental model for clarifying the mechanisms driving palate development. Cleft palate occurs in two forms: a cleft between the primary and secondary palates (primary cleft palate) and a cleft between the secondary palates (secondary cleft palate). It remains unclear whether primary palate development is under similar molecular control as secondary palate formation, since most of studies have focused on secondary palate development using mutant mice. CL/Fraser (CL/Fr) is a mouse strain that often exhibits spontaneous primary cleft palate; however, the molecular changes in primary cleft palate in CL/Fr mice are not fully understood. Several signaling pathways, including Shh, Fgf, Bmp, and Wnt signaling, have been shown to play key roles in secondary palate development. In the present study, we found that Shh and Wnt signaling pathways were downregulated in the primary cleft palate region in CL/Fr mice.

DOI： 10.1016/j.ajoms.2019.12.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Clinical Investigation  

Macrolide antibiotics exert antiinflammatory effects; however, little is known regarding their immunomodulatory mechanisms. In this study, using 2 distinct mouse models of mucosal inflammatory disease (LPS-induced acute lung injury and ligature-induced periodontitis), we demonstrated that the antiinflammatory action of erythromycin (ERM) is mediated through upregulation of the secreted homeostatic protein developmental endothelial locus-1 (DEL-1). Consistent with the anti-neutrophil recruitment action of endothelial cell-derived DEL-1, ERM inhibited neutrophil infiltration in the lungs and the periodontium in a DEL-1-dependent manner. Whereas ERM (but not other antibiotics, such as josamycin and penicillin) protected against lethal pulmonary inflammation and inflammatory periodontal bone loss, these protective effects of ERM were abolished in Del1-deficient mice. By interacting with the growth hormone secretagogue receptor and activating JAK2 in human lung microvascular endothelial cells, ERM induced DEL-1 transcription that was mediated by MAPK p38 and was CCAAT/enhancer binding protein-β dependent. Moreover, ERM reversed IL-17-induced inhibition of DEL-1 transcription, in a manner that was dependent not only on JAK2 but also on PI3K/AKT signaling. Because DEL-1 levels are severely reduced in inflammatory conditions and with aging, the ability of ERM to upregulate DEL-1 may lead to a novel approach for the treatment of inflammatory and aging-related diseases.

DOI： 10.1172/jci.insight.136706

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Hedgehog (Hh) signaling has been shown to be involved in regulating both intact and injured peripheral nerves. Therefore, it is critical to understand how Hh signaling is regulated in the peripheral nerve. One of the transcription factors of the Hh signaling pathway, Gli3, functions as both a repressor and an activator of Hh signaling activity. However, it remains unclear whether Gli3 is involved in controlling the intact and/or injured peripheral nerves. We found that Gli3 act as a repressor in the Schwann cells (SCs) of intact sciatic nerves. Although Dhh and Ptch1 expression were present, Hh signaling was not activated in these SCs. Moreover, heterozygous Gli3 mutation (Gli3-/+) induced ectopic Hh signaling activity in SCs. Hh signaling was thus suppressed by Gli3 in the SCs of intact sciatic nerves. Minor morphological changes were observed in the intact nerves from Gli3-/+ mice. Gli3 expression was significantly decreased following injury and ligand expression switched from Dhh to Shh, which activated Hh signaling in SCs from wild-type mice. Changes of these ligands was found to be important for nerve regeneration in which the downregulation of Gli3 was also involved. In fact, Gli3-/+ mice exhibited accelerated ligand switching and subsequent nerve regeneration. Both suppression of Hh signaling with Gli3 in the intact nerves and activation of Hh signaling without Gli3 in the injured nerve were observed in the SCs in an autocrine manner. Thus, Gli3 is a key factor in the control of intact peripheral nerve homeostasis and nerve regeneration.

DOI： 10.1016/j.neuroscience.2020.02.036

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BACKGROUND: Periodic patterning of iterative structures is diverse across the animal kingdom. Clarifying the molecular mechanisms involved in the formation of these structures helps to elucidate the genetic commonality of developmental processes, as organs with these structures are believed to share the same molecular mechanisms and fundamental processes. Palatal rugae are periodic corrugated structures on the hard palate and are conserved in all mammals. Although the numbers and patterns of the palatal rugae are species specific, they are consistent in each mammalian species, except humans. HIGHLIGHT: Palatal rugae development is thus under strict genetic control in most mammals and is an excellent model to investigate the genetic commonality of developmental processes to form periodic patterning. CONCLUSION: This review highlights the current understanding of the molecular mechanisms of palatal rugae development.

DOI： 10.1016/j.job.2019.12.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

OBJECTIVES: This study is aimed at evaluating the effect of a new glass ionomer cement (GIC) containing fluoro-zinc-silicate fillers on biofilm formation and ion incorporation. MATERIALS AND METHODS: Streptococcus mutans biofilms were developed on two GIC materials: Caredyne Restore (CD) and Fuji VII (FJ); and hydroxyapatite (HA) for 24 h at 37 °C using a flow cell system. The morphological structure and bacterial viability were analyzed using a confocal laser scanning microscopy. Bacterial adhesion during the initial 2 h was also assessed by viable cell counting. To study the ion incorporation, restored cavities prepared on the root surfaces of human incisors were subjected to the elemental mapping of the zinc and fluoride ions in the GIC-dentin interface using a wavelength-dispersive X-ray spectroscopy electron probe microanalyzer. RESULTS: Morphological observations revealed that biofilm formation in the CD group was remarkably inhibited compared with the HA and FJ groups, exhibiting sparse, thinner biofilm clusters. The microorganisms adhering to the CD group were significantly inhibited, revealing 2.9 ± 0.4 for CD, 4.9 ± 0.2 for FJ, and 5.4 ± 0.4 log colony-forming units (CFU) for HA. The CD zinc ion incorporation depth was 72.2 ± 8.0 μm. The fluoride penetration of CD was three times deeper than that of FJ; this difference was statistically significant (p < 0.05). CONCLUSIONS: Enhanced by the incorporation of zinc and fluoride ions, the new GIC inhibited biofilm formation by interfering with bacterial adhesion. CLINICAL RELEVANCE: A novel GIC comprised of fluoro-zinc-silicate fillers may improve clinical outcomes, such as root caries and minimally invasive dentistry.

DOI： 10.1007/s00784-019-02991-0

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The mandible is a crucial organ in both clinical and biological fields due to the high frequency of congenital anomalies and the significant morphological changes during evolution. Primary cilia play a critical role in many biological processes, including the determination of left/right axis patterning, the regulation of signaling pathways, and the formation of bone and cartilage. Perturbations in the function of primary cilia are known to cause a wide spectrum of human diseases: the ciliopathies. Craniofacial dysmorphologies, including mandibular deformity, are often seen in patients with ciliopathies. Mandibular development is characterized by chondrogenesis and osteogenesis; however, the role of primary cilia in mandibular development is not fully understood. To address this question, we generated mice with mesenchymal deletions of the ciliary protein, Ift88 (Ift88fl/fl ;Wnt1Cre). Ift88fl/fl ;Wnt1Cre mice showed ectopic mandibular bone formation, whereas Ift88 mutant mandible was slightly shortened. Meckel's cartilage was modestly expanded in Ift88fl/fl ;Wnt1Cre mice. The downregulation of Hh signaling was found in most of the mesenchyme of Ift88 mutant mandible. However, mice with a mesenchymal deletion of an essential molecule for Hh signaling activity, Smo (Smofl/fl ;Wnt1Cre), showed only ectopic mandibular formation, whereas Smo mutant mandible was significantly shortened. Ift88 is thus involved in chondrogenesis and osteogenesis during mandibular development, partially through regulating Sonic hedgehog (Shh) signaling.

DOI： 10.1111/joa.13096

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Periodic patterning of iterative structures is a fundamental process during embryonic development, since these structures are diverse across the animal kingdom. Therefore, elucidating the molecular mechanisms in the formation of these structures promotes understanding of the process of organogenesis. Periodically patterned ridges, palatal rugae (situated on the hard palate of mammals), are an excellent experimental model to clarify the molecular mechanisms involved in the formation of periodic patterning of iterative structures. Primary cilia are involved in many biological events, including the regulation of signaling pathways such as Shh and non-canonical Wnt signaling. However, the role of primary cilia in the development of palatal rugae remains unclear. We found that primary cilia were localized to the oral cavity side of the interplacode epithelium of the palatal rugae, whereas restricted localization of primary cilia could not be detected in other regions. Next, we generated mice with a placodal conditional deletion of the primary cilia protein Ift88, using ShhCre mice (Ift88 fl/fl;ShhCre). Highly disorganized palatal rugae were observed in Ift88 fl/fl;ShhCre mice. Furthermore, by comparative in situ hybridization analysis, many Shh and non-canonical Wnt signaling-related molecules showed spatiotemporal expression patterns during palatal rugae development, including restricted expression in the epithelium (placodes and interplacodes) and mesenchyme. Some of these expression were found to be altered in Ift88 fl/fl;ShhCre mice. Primary cilia is thus involved in development of palatal rugae.

DOI： 10.1016/j.gep.2019.119062

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In this study, we aimed to clarify the role of ascorbic acid in collagen synthesis in periodontal ligaments using osteogenic disorder Shionogi (ODS)/ShiJcl-od/od rats lacking L-gulonolactone oxidase. These rats cannot synthesize ascorbic acid in vivo. Eight-week-old ODS/ShiJcl-od/od male rats were administered ascorbic acid solution at a concentration of 200 mg/dL (control group, n = 6) or ascorbic acid solution at concentration of 0.3 mg/dL (insufficient group, n = 12). Six rats of the insufficient group were then given with ascorbic acid solution at concentration of 200 mg/dL for additional 3 weeks (rescued group, n = 6), and then, their mandibles were histochemically examined. Consequently, the insufficient group specimens were seen to possess fewer collagen fibers, and silver impregnation revealed numerous fine, reticular fiber-like fibrils branching off from collagen in the periodontal ligaments. In control group, faint immunoreactivities for matrix metalloproteinase (MMP)2 and cathepsin H were seen in the periphery of blood vessels and throughout the ligament, respectively. In contrast, in the insufficient group, intense MMP2-immunoreactivity was observed to be associated with collagen fibrils in the periodontal ligaments, and cathepsin H-immunopositivity was seen in ligamentous cells. The rescued group showed abundant collagen fibers filling the periodontal ligament space. Under transmission electron microscopy, ligamentous fibroblasts incorporated collagen fibrils into tubular endosomes/lysosomes while simultaneously synthesizing collagen fibril bundles. Thus, ascorbic acid insufficiency affected the immunolocalization of cathepsin H and MMP2; however, ligamentous fibroblasts appear to possess the potential to synthesize collagen fibers when supplied with ascorbic acid.

DOI： 10.1093/jmicro/dfz021

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Tooth cusp is a crucial structure, since the shape of the molar tooth is determined by number, shape, and size of the cusp. Bone morphogenetic protein (Bmp) signaling is known to play a critical role in tooth development, including in initiation. However, it remains unclear whether Bmp signaling is also involved in cusp formation. To address this question, we examined cusp in two different transgenic mouse lines: mice with overexpression of Bmp4 (K14-Bmp4), and those with Bmp inhibitor, Noggin, (K14-Noggin) under keratin14 (K14) promoter. K14-Noggin mice demonstrated extra cusps, whereas reduced number of cusps was observed in K14-Bmp4 mice. To further understand how Bmps are expressed during cusp formation, we performed whole-mount in situ hybridisation analysis of three major Bmps (Bmp2, Bmp4, and Bmp7) in murine maxillary and mandibular molars from E14.5 to P3. The linear expressions of Bmp2 and Bmp4 were observed in both maxillary and mandibular molars at E14.5. The expression patterns of Bmp2 and Bmp4 became significantly different between the maxillary and mandibular molars at E16.5. At P3, all Bmps were expressed in all the cusp regions of the maxillary molar; however, the patterns differed. All Bmps thus exhibited dynamic temporo-spatial expression during the cusp formation. It could therefore be inferred that Bmp signaling is involved in regulating cusp formation.

DOI： 10.1016/j.gep.2019.04.002

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OBJECTIVE: The development of the maxillary bone is under strict molecular control because of its complicated structure. Primary cilia play a critical role in craniofacial development, since defects in primary cilia are known to cause congenital craniofacial dysmorphologies as a wide spectrum of human diseases: the ciliopathies. The primary cilia also are known to regulate bone formation. However, the role of the primary cilia in maxillary bone development is not fully understood. DESIGN: To address this question, we generated mice with a mesenchymal conditional deletion ofIft88 using the Wnt1Cre mice (Ift88fl/fl;Wnt1Cre). The gene Ift88 encodes a protein that is required for the function and formation of primary cilia. RESULTS: It has been shown thatIft88fl/fl;Wnt1Cre mice exhibit cleft palate. Here, we additionally observed excess bone formation in the Ift88 mutant maxillary process. We also found ectopic apoptosis in the Ift88 mutant maxillary process at an early stage of development. To investigate whether the ectopic apoptosis is related to the Ift88 mouse maxillary phenotypes, we generated Ift88fl/fl;Wnt1Cre;p53-/- mutants to reduce apoptosis. The Ift88fl/fl;Wnt1Cre;p53-/- mice showed no excess bone formation, suggesting that the cells evading apoptosis by the presence of Ift88 in wild-type mice limit bone formation in maxillary development. On the other hand, the palatal cleft was retained in the Ift88fl/fl;Wnt1Cre;p53-/- mice, indicating that the excess bone formation or abnormal apoptosis was independent of the cleft palate phenotype in Ift88 mutant mice. CONCLUSIONS: Ift88 limits bone formation in the maxillary process by suppressing apoptosis.

DOI： 10.1016/j.archoralbio.2019.02.017

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Neuroma formation at sites of injury can impair peripheral nerve regeneration. Although the involvement of semaphorin 3A has been suggested in neuroma formation, this detailed process after injury is not fully understood. This study was therefore undertaken to examine the effects of semaphorin 3A on peripheral nerve regeneration during the early stage after injury. Immunohistochemistry for semaphorin 3A and PGP9.5, a general neuronal marker, was carried out for clarify chronological changes in their expressions after transection of the mouse inferior alveolar nerve thorough postoperative days 1 to 7. At postoperative day 1, the proximal stump of the damaged IAN exhibited semaphorin 3A, while the distal stump lacked any immunoreactivity. From this day on, its expression lessened, ultimately disappearing completely in all regions of the transected inferior alveolar nerve. A local administration of an antibody to semaphorin 3A into the nerve transection site at postoperative day 3 inhibited axon sprouting at the injury site. This antibody injection increased the number of trigeminal ganglion neurons labeled with DiI (paired t-test, p < 0.05). Immunoreactivity of the semaphorin 3A receptor, neuropilin-1, was also detected at the proximal stump at postoperative day 1. These results suggest that nerve injury initiates semaphorin 3A production in ganglion neurons, which is then delivered through the nerve fibers to the proximal end, thereby contributes to the inhibition of axonal sprouting from the proximal region of injured nerves in the distal direction. To our knowledge, this is the first report to reveal the involvement of Sema3A in the nerve regeneration process at its early stage.

DOI： 10.1038/s41598-018-37819-6

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BACKGROUND: The timing, location, and level of gene expression are crucial for normal organ development, because morphogenesis requires strict genetic control. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating gene expression level. Although miRNAs are known to be involved in many biological events, the role of miRNAs in organogenesis is not fully understood. Mammalian eyelids fuse and separate during development and growth. In mice, failure of this process results in the eye-open at birth (EOB) phenotype. RESULTS: It has been shown that conditional deletion of mesenchymal Dicer (an essential protein for miRNA processing; Dicer fl/fl ;Wnt1Cre) leads to the EOB phenotype with full penetrance. Here, we identified that the up-regulation of Wnt signaling resulted in the EOB phenotype in Dicer mutants. Down-regulation of Fgf signaling observed in Dicer mutants was caused by an inverse relationship between Fgf and Wnt signaling. Shh and Bmp signaling were down-regulated as the secondary effects in Dicer fl/fl ;Wnt1Cre mice. Wnt, Shh, and Fgf signaling were also found to mediate the epithelial-mesenchymal interactions in eyelid development. CONCLUSIONS: miRNAs control eyelid development through Wnt. Developmental Dynamics 248:201-210, 2019. © 2019 Wiley Periodicals, Inc.

DOI： 10.1002/dvdy.10

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Several studies have demonstrated the remarkable properties of microbiota and their metabolites in the pathogenesis of several inflammatory diseases. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a bioactive metabolite generated by probiotic microorganisms during the process of fatty acid metabolism, has been studied for its protective effects against epithelial barrier impairment in the intestines. Herein, we examined the effect of HYA on gingival epithelial barrier function and its possible application for the prevention and treatment of periodontal disease. We found that GPR40, a fatty acid receptor, was expressed on gingival epithelial cells
activation of GPR40 by HYA significantly inhibited barrier impairment induced by Porphyromonas gingivalis, a representative periodontopathic bacterium. The degradation of E-cadherin and beta-catenin, basic components of the epithelial barrier, was prevented in a GPR40-dependent manner in vitro. Oral inoculation of HYA in a mouse experimental periodontitis model suppressed the bacteria-induced degradation of E-cadherin and subsequent inflammatory cytokine production in the gingival tissue. Collectively, these results suggest that HYA exerts a protective function, through GPR40 signaling, against periodontopathic bacteria-induced gingival epithelial barrier impairment and contributes to the suppression of inflammatory responses in periodontal diseases.

DOI： 10.1038/s41598-018-27408-y

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Activation of Shh signaling is known to be observed following injury of the peripheral nerves such as the sciatic nerve. However, the precise role of Shh signaling during peripheral nerve regeneration is not fully understood. The inferior alveolar nerve (IAN) is most commonly injured during oral surgery. Unlike the sciatic nerve, the IAN is isolated from other craniofacial tissues, as it resides in a long bony canal within the mandible. The IAN is thus an excellent experimental model for investigating peripheral nerve regeneration. In this study, the role of Shh signaling in peripheral nerve regeneration was investigated using the mouse IAN transection model. During regeneration, Shh signaling was activated within the entire distal region of the IAN and proximal stumps. Inhibition of Shh signaling by cyclopamine application at the transection site led to abnormal axon growth in random directions, a reduced number of macrophages, and an increase in myelin debris within the distal region. Shh signaling is thus involved in peripheral nerve regeneration via the regulation of myelin degradation.

DOI： 10.1016/j.neulet.2017.12.051

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The tongue is a critical organ, involved in functions such as speaking, swallowing, mastication, and degustation. Although Sox genes are known to play critical roles in many biological processes, including organogenesis, the expression of the Sox family members during tongue development remains unclear. We therefore performed a comparative in situ hybridization analysis of 17 Sox genes (Sox1-14, 17, 18, and 21) during murine tongue development. Sox2, 4, 6, 8, 9, 10, 11, 12, and 21 were found to be expressed in the tongue epithelium, whereas Sox2, 4-6, 8-11, 13, and 21 showed expression in the mesenchyme of the developing tongue. Expression of Sox1, 4, 6, 8-12, and 21 were observed in the developing tongue muscle. Sox5 and 13 showed expression only at E12, while Sox1 expression was observed only on E18. Sox6, 8, 9, and 12 showed expression at several stages. Although the expression of Sox2, 4, 10, 11, and 21 was detected during all the four stages of tongue development, their expression patterns differed among the stages. We thus identified a dynamic spatiotemporal expression pattern of the Sox genes during murine tongue development. To understand whether Sox genes are involved in the development of other craniofacial organs through similar roles to those in tongue development, we also examined the expression of Sox genes in eyelid primordia, which also contain epithelium, mesenchyme, and muscle. However, expression patterns and timing of Sox genes differed between tongue and eyelid development. Sox genes are thus related to organogenesis through different functions in each craniofacial organ.

DOI： 10.1155/2018/1601363

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Periodic patterning of iterative structures is diverse across the animal kingdom. Clarifying the molecular mechanisms involved in the formation of these structure helps to elucidate the process of organogenesis. Turing-type reaction-diffusion mechanisms have been shown to play a critical role in regulating periodic patterning in organogenesis. Palatal rugae are periodically patterned ridges situated on the hard palate of mammals. We have previously shown that the palatal rugae develop by a Turing-type reaction-diffusion mechanism, which is reliant upon Shh (as an inhibitor) and Fgf (as an activator) signaling for appropriate organization of these structures. The disturbance of Shh and Fgf signaling lead to disorganized palatal rugae. However, the mechanism itself is not fully understood. Here we found that Lrp4 (transmembrane protein) was expressed in a complementary pattern to Wise (a secreted BMP antagonist and Wnt modulator) expression in palatal rugae development, representing Lrp4 expression in developing rugae and Wise in the inter-rugal epithelium. Highly disorganized palatal rugae was observed in both Wise and Lrp4 mutant mice, and these mutants also showed the downregulation of Shh signaling, which was accompanied with upregulation of Fgf signaling. Wise and Lrp4 are thus likely to control palatal rugae development by regulating reaction-diffusion mechanisms through Shh and Fgf signaling. We also found that Bmp and Wnt signaling were partially involved in this mechanism.

DOI： 10.1371/journal.pone.0204126

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Peripheral nerve injury has been suggested to up-regulate mRNA for the vascular endothelial growth factor (VEGF) which enhances nerve regeneration. VEGF is known to regulate angiogenesis by binding with a specific receptor, the vascular endothelial growth factor receptor (VEGFR). However, little is known about the involvement of VEGF-VEGFR signaling in the nerve regeneration at early stages though previous studies contained a lengthy observation. The present study examined that relationship between angiogenesis and peripheral nerve regeneration at the early stage after nerve transection by focusing on the chronological changes in the expression patterns of VEGF-VEGFR signaling. This study used our previously reported experimental model for nerve regeneration following the transection of the inferior alveolar nerve (IAN) in mice. In a double staining of PGP9.5 and CD31, respective markers for the nerve fibers and endothelial cells, CD31 immunoreactions first appeared in the injury site on postoperative (PO) day 2 when the transected nerve fibers had not been re-connected. The most intense immunoreaction for CD31 was found around the regenerating nerve fibers extending from the proximal stump on PO day 3, but it gradually lessened to disappear by PO day 7. The expression patterns of VEGFR1 and VEGFR2 showed similar chronological changes through the observation periods, with most intense immunoreaction found on PO day 3. Western blotting of total protein extracted from the injury site demonstrated the clear bands for VEGF-A and VEGF-B on PO day 2, indicating a time lag for the expression of ligands and receptors. A local administration of antibody to VEGF-A inhibited the elongation of the nerve fibers from the proximal stump. Furthermore, this administration of VEGF-A antibody inhibited the expression of CD31 in the gap between proximal and distal stumps. These results indicated that a nerve injury initiates productions in VEGF-A and VEFG-B, followed with the expression of VEGFR1 and VEGFR2 at early stages after the nerve injury. Taken these findings together, it is reasonable to postulate that immediate response of VEGF-VEGFR signaling to nerve injury plays a crucial role in local angiogenesis, resulting in a trigger for the regeneration of the nerve fibers in mouse IAN.

DOI： 10.2220/biomedres.39.287

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The tongue is a critical organ, involved in functions such as speaking, swallowing, mastication, and degustation. Although Sox genes are known to play critical roles in many biological processes, including organogenesis, the expression of the Sox family members during tongue development remains unclear. We therefore performed a comparative in situ hybridization analysis of 17 Sox genes (Sox1-14, 17, 18, and 21) during murine tongue development. Sox2, 4, 6, 8, 9, 10, 11, 12, and 21 were found to be expressed in the tongue epithelium, whereas Sox2, 4-6, 8-11, 13, and 21 showed expression in the mesenchyme of the developing tongue. Expression of Sox1, 4, 6, 8-12, and 21 were observed in the developing tongue muscle. Sox5 and 13 showed expression only at E12, while Sox1 expression was observed only on E18. Sox6, 8, 9, and 12 showed expression at several stages. Although the expression of Sox2, 4, 10, 11, and 21 was detected during all the four stages of tongue development, their expression patterns differed among the stages. We thus identified a dynamic spatiotemporal expression pattern of the Sox genes during murine tongue development. To understand whether Sox genes are involved in the development of other craniofacial organs through similar roles to those in tongue development, we also examined the expression of Sox genes in eyelid primordia, which also contain epithelium, mesenchyme, and muscle. However, expression patterns and timing of Sox genes differed between tongue and eyelid development. Sox genes are thus related to organogenesis through different functions in each craniofacial organ.

DOI： 10.1155/2018/1601363

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Objective. Mandibular distraction surgery is a critical treatment for jaw deformity. However, abnormal mandibular condylar bone resorption is often seen as complication after surgery. Our previous study using a rat mandibular distraction model suggested that overloading leads to mandibular condylar resorption. Host factors are also believed to influence the resorption. To understand the relationship between host factors and resorption, we investigated the effect of changing bone mass and architecture on the mandibular condyle using FK506.
Study Design. FK506, an immunosuppressant, was used to compromise bone mass and architecture in this study. Animals were divided into 4 groups: distraction surgery (Dist), FK506 administration (FK), distraction surgery with FK506 administration (FK + Dist), and no surgery or FK506 administration (Cont).
Results. The FK group showed reduced bone mass and impaired bone architecture. The Dist group exhibited abnormal bone resorption on the surface of the condyles, which was slightly exacerbated in the FK + Dist group. Bone defect length decreased over time as a result of bone apposition in the Dist group. However, in the FK + Dist group, the bone defect length remained the same.
Conclusions. These results suggest that bone mass and architecture strongly affect the tolerance to the overloading and adaptation with bone apposition in condylar resorption site.

DOI： 10.1016/j.oooo.2017.05.472

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Language：Japanese   Publisher：(一社)日本歯科医学教育学会  

本邦では、社会の急速な高齢化に伴い、高度な治療が求められる患者や全身的な疾患を有する患者が増加している。また、患者の意識変化によって、より質の高い治療を求められることが多くなっている結果、臨床実習に協力していただける患者が減少している。このような背景から、学生は限られた患者を対象に臨床技能の習得をし、卒業時には相応の知識と臨床能力を確保しなければならない。この問題を解決するための手段として、新潟大学歯学部では1つの模型の中に様々な病態を具備した6種類のオリジナルの模型を開発し、治療計画立案を含めた統合的な模型実習を導入している。実習は臨床実習直前の5年前期に実施され、基礎模型実習と臨床実習をつなぐと共に、不足する臨床実習を補完する役割を果たす。模型には歯石除去必要部位、抜歯、充填、歯内治療、分割抜歯適応歯および歯冠修復、不良補綴歯、欠損補綴適応部位を含む。学生は診断・治療計画立案から歯科処置までをファントムに装着した模型上で実際の臨床をシミュレートする形で行う。現在、本実習の評価はルーブリックを用いて行っているが、評価方法に関しては今後も継続的に検討する必要がある。実習後の学生アンケートからは実習が有意義なものであったとの回答を得ており、知識や技術を使いこなす統合的な能力の育成に有効であることが示唆された。(著者抄録)

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Language：Japanese   Publisher：日本歯科医学教育学会  

近年、歯学部・歯科大学では技能教育の改善が求められ、診療参加型臨床実習の実践が推奨されている。本学では以前より、学生が直接歯科医療行為に参加する臨床実習を実施しているが、その学習成果、すなわち、現実的な状況で知識や技術を使いこなせる統合的な能力としての「臨床能力」を、いかにして直接評価すべきか対応に苦慮してきた。また、学生が実際の診療の中でどのようなことを感じ、学習し、どのような指導を受けたか、という学習過程そのものが、必ずしも整理された形で実体化されていないという問題点があった。そこで、臨床能力を評価するためのルーブリックを定め、学生の臨床能力を客観性のある到達レベルとして把握するとともに、日々の臨床実習での学習活動を記載するポートフォリオと組み合わせて運用することにより、臨床実習で行っている教育そのものを経時的に記録・評価し、実体化するという発想にいたった。このような背景から電子ポートフォリオの開発と運用を開始したが、学生・教員の受け入れはスムースであった。臨床実習が進むにつれ、ポートフォリオに記載された教員による学生の評価は向上しており、経験を積むことによる学生の臨床能力向上が反映されたものと推察された。以上のことから、臨床実習における電子ポートフォリオの導入は、学生自身の振り返りや教員の学生指導にとって有用であると考えられた。(著者抄録)

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS INC  

The Wingless/integrase-1 (Wnt) family of protein ligands and their functional antagonists, secreted frizzled-related proteins (sFRPs), regulate various biological processes ranging from embryonic development to immunity and inflammation. Wnt5a and sFRP5 comprise a typical ligand/antagonist pair, and the former molecule was recently detected at the messenger RNA (mRNA) level in human periodontitis. The main objective of this study was to investigate the interrelationship of expression of Wnt5a and sFRP5 in human periodontitis (as compared to health) and to determine their roles in inflammation and bone loss in an animal model. We detected both Wnt5a and sFRP5 mRNA in human gingiva, with Wnt5a dominating in diseased and sFRP5 in healthy tissue. Wnt5a and sFRP5 protein colocalized in the gingival epithelium, suggesting epithelial cell expression, which was confirmed in cultured human gingival epithelial cells (HGECs). The HGEC expression of Wnt5a and sFRP5 was differentially regulated by a proinflammatory stimulus (lipopolysaccharide [LPS] from Porphyromonas gingivalis) in a manner consistent with the clinical observations (i.e., LPS upregulated Wnt5a and downregulated sFRP5). In HGECs, exogenously added Wnt5a enhanced whereas sFRP5 inhibited LPS-induced inflammation, as monitored by interleukin 8 production. Consistent with this, local treatment with sFRP5 in mice subjected to ligature-induced periodontitis inhibited inflammation and bone loss, correlating with decreased numbers of osteoclasts in bone tissue sections. As in humans, mouse periodontitis was associated with high expression of Wnt5a and low expression of sFRP5, although this profile was reversed after treatment with sFRP5. In conclusion, we demonstrated a novel reciprocal relationship between sFRP5 and Wnt5a expression in periodontal health and disease, paving the way to clinical investigation of the possibility of using the Wnt5a/sFRP5 ratio as a periodontitis biomarker. Moreover, we showed that sFRP5 blocks experimental periodontal inflammation and bone loss, suggesting a promising platform for the development of a new host modulation therapy in periodontitis.

DOI： 10.1177/0022034516687248

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Language：Japanese   Publisher：日本歯科医学教育学会  

歯学教育を改善するために、各大学歯学部・歯科大学においてさまざまな取り組みが行われている。なかでも、診療参加型の臨床実習を充実させることは重視されており、それぞれの歯学部・歯科大学は信頼される歯科医師を輩出するという最も大きな社会的命題を果たすべく努力を続けている。新潟大学歯学部では、卒業生の質の保証を目的に、臨床実習においてACCEPT Projectを立ち上げ、「ACKPIS」と称する方法を用いて学生の臨床能力を評価してきた。ACKPISは診療を自験する学生に対し、どのような専門領域においても必要不可欠となる6つの基本項目を、それぞれの専門処置を評価課題として確認するものである。今回、ACKPISの効果を検証するために、平成28年度の受検生にアンケート調査を行った。ACKPISの基本項目は、ADEAやGDCが提唱する歯科医師に求められるコンピテンシーの中にも該当する記述がみられ、国内外で医師に対して行われている臨床能力評価法同様、診療現場でのフィードバックを含んでいることから、歯学生の臨床能力を評価するために適当であると考えられた。また、アンケート結果から、受検した学生もACKPISの必要性や重要性を認識し、受検方法や合否判定の妥当性を認めていることが明らかになった。以上のことから、ACKPISは臨床実習中の学生の臨床能力を評価するための有用な方法となり得ることが示唆された。(著者抄録)

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Background: Non-gustatory filiform papillae play critical roles in helping to grip food, drawing food to the esophagus, cleaning the mouth, and spreading saliva. The molecular mechanisms of filiform tongue papillae development however are not fully understood. Results: We found Ikk alpha and Irf6 expression in developing tongue epithelium, and describe here specific tongue abnormalities in mice with mutation of these genes, indicating a role for Ikk alpha and Irf6 in filiform papillae development. Ikk alpha and Irf6 mutant tongues showed ectopic vertical epithelium at the midline, while lateral sides of mutant tongues adhered to the oral mucosa. Both the ectopic median vertical epithelium and adhered epithelium exhibited the presence of filiform tongue papillae, whereas epithelium between the median vertical epithelium and adhered tongue showed a loss of filiform tongue papillae. Timing of filiform papillae development was found to be slightly different between the midline and lateral regions of the wild-type tongue. Conclusions: Filiform papillae thus develop through distinct molecular mechanisms between the regions of tongue dorsum in the medio-lateral axis, with some filiform papillae developing under the control of Ikk alpha and Irf6. Developmental Dynamics 245: 937-946, 2016. (C) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/DVDY.24427

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Background: Non-gustatory filiform papillae play critical roles in helping to grip food, drawing food to the esophagus, cleaning the mouth, and spreading saliva. The molecular mechanisms of filiform tongue papillae development however are not fully understood. Results: We found Ikk alpha and Irf6 expression in developing tongue epithelium, and describe here specific tongue abnormalities in mice with mutation of these genes, indicating a role for Ikk alpha and Irf6 in filiform papillae development. Ikk alpha and Irf6 mutant tongues showed ectopic vertical epithelium at the midline, while lateral sides of mutant tongues adhered to the oral mucosa. Both the ectopic median vertical epithelium and adhered epithelium exhibited the presence of filiform tongue papillae, whereas epithelium between the median vertical epithelium and adhered tongue showed a loss of filiform tongue papillae. Timing of filiform papillae development was found to be slightly different between the midline and lateral regions of the wild-type tongue. Conclusions: Filiform papillae thus develop through distinct molecular mechanisms between the regions of tongue dorsum in the medio-lateral axis, with some filiform papillae developing under the control of Ikk alpha and Irf6. Developmental Dynamics 245: 937-946, 2016. (C) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/DVDY.24427

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Members of the Sox gene family play critical roles in many biological processes including organogenesis. We carried out comparative in situ hybridisation analysis of seventeen Sox genes (Sox1-14, 17, 18 and 21) during murine palatogenesis from initiation to fusion of the palatal shelves above the dorsal side of the tongue. At palatal shelf initiation (E12.5), the localized expression of six Sox genes (Sox2, 5, 6, 9,12 and 13) was observed in the shelves, whereas Sox4 and Sox11 showed ubiquitious expression. During the down growth of palatal shelves (E13.5), Sox4, Sox5, and Sox9 exhibited restricted expression to the interior side of the palatal shelves facing the tongue. Following elevation of the palatal shelves (E14.5), Sox2, Sox11 and Sox21 expression was present in the midline epithelial seam. We thus identify dynamic spatio-temporal expression of Sox gene family during the process of palatogenesis. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gep.2016.05.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Identifying substandard tissue-engineered oral mucosa grafts with a poor epithelium before clinical use is critical to ensure quality assurance/control in regenerative medicine, leading to success of grafting. This study investigated the effects of one of the C-xylopyranoside derivatives, -D-xylopyranoside-n-propane-2-one (XPP), on oral epithelial regeneration. Using a three-dimensional oral mucosa model, we analyzed changes of the epithelial structure, glycosaminoglycan (GAG) synthesis, the expression levels of basement membrane zone markers, and substrates of Akt/mTOR signaling. Compared with the control, 2mM XPP treatment increased the mean and minimal epithelial thickness, and reduced the variation of epithelial thickness. It also stimulated expressions of decorin and syndecan-1 with change of GAG amount and/or composition, and enhanced the expressions of integrin 6, CD44, and Akt/mTOR signaling substrates. These findings suggest that XPP supplementation contributes to consistent epithelial regeneration. Moreover, upregulation of those markers may play a role in increasing the quality of the oral mucosal epithelium.

DOI： 10.1080/09168451.2016.1153957

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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 PDGFRb) but not for the other markers of myogenic cells (MyoD and myogenin) or a contractile apparatus (aSMA 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. Key words: angiogenesis; synovial lining cell; synovial membrane; temporomandibular joint; tomato lectin.

DOI： 10.1111/joa.12426

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Quintessence Publishing Co. Inc.  

Aims: To examine the effects of local brain-derived neurotrophic factor (BDNF) produced after nerve injury on the functional regeneration of the damaged nerve. Methods: The inferior alveolar nerve was transected in adult male rats and 1 μg or 10 μg of BDNF antibody was administered at the injury site
a third group of rats received saline and a fourth group underwent nerve ligation. BDNF mRNA was quantified in the transected tissue and trigeminal ganglion by using realtime polymerase chain reaction (PCR). Head withdrawal thresholds following mechanical (tactile) stimulation (with von Frey filaments) of the mental region were measured for 3 weeks postoperatively. Electromyographic activity of the jaw opening reflex (JOR) was recorded from the anterior belly of the digastric muscle. Results: Within 24 hours, transection induced significant elevation of BDNF mRNA expression in the injured tissue (unpaired t test, P &lt
.01). The head withdrawal threshold to mechanical stimulation increased at 1 day after transection and then decreased (two-way repeated measures analysis of variance [ANOVA], P &lt
.001). At 2 weeks after surgery, the head withdrawal threshold was higher than before surgery in the group that received a higher dose of BDNF antibody (ANOVA, P &lt
.001), but not in the group that received a smaller dose (ANOVA, P &gt
.05). No significant differences were observed in the latency or threshold of the JOR between saline- and antibody-treated rats (unpaired t test, P &gt
.05). Conclusion: These results suggest that locally administered BDNF antibody neutralizes nerve injury-induced BDNF at the injury site and thus influences sensorimotor recovery.

DOI： 10.11607/ofph.1651

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Denture-wearing affects the quality and quantity of epithelial cells in the underlying healthy oral mucosa. The physiologic mechanisms, however, are poorly understood. This study aimed to compare histologic changes and cellular responses of an epithelial cell layer to cyclic mechanical pressure-loading mimicking denture-wearing using an organotypic culture system to develop a three-dimensional in vitro oral mucosa model (3DOMM). Primary human oral keratinocytes and fibroblasts were serially grown in a monolayer culture, and cell viability was measured under continuous cyclic mechanical pressure (50kPa) for 7days (cycles of 60min on, 20s off to degas and inject air). Upon initiation of an air-liquid interface culture for epithelial stratification, the cyclic pressure, set to the mode above mentioned, was applied to the 3DOMMs for 7days. Paraffin-embedded 3DOMMs were examined histologically and immunohistochemically. In the monolayer culture, the pressure did not affect the viability of oral keratinocytes or fibroblasts. Few histologic changes were observed in the epithelial layer of the control and pressure-loaded 3DOMMs. Immunohistochemical examination, however, revealed a significant decrease in Ki-67 labelling and an increase in filaggrin and involucrin expression in the suprabasal layer of the pressure-loaded 3DOMMs. Pressure-loading attenuated integrin 1 expression and increased matrix metalloproteinase-9 activity. Incomplete deposition of laminin and type IV collagen beneath the basal cells was observed only in the pressure-loaded 3DOMM. Cyclic pressure-loading appeared to disrupt multiple functions of the basal cells in the 3DOMM, resulting in a predisposition towards terminal differentiation. Thus, denture-wearing could compromise oral epithelial homeostasis.

DOI： 10.1111/joor.12254

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS INC  

Nuclear factor kappa B (NF-kappa B) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-kappa B signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-kappa B signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKK beta, an essential component of the NF-kappa B pathway, under keratin 5 promoter (K5-Ikk beta). K5-Ikk beta 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-Ikk beta mice. The supernumerary incisors in K5-Ikk beta mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-kappa B activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.

DOI： 10.1177/0022034514556707

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：UNIV BASQUE COUNTRY UPV-EHU PRESS  

Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox 1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.

DOI： 10.1387/ijdb.150192ao

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Objective. This study investigated the bone resorption process of the rat mandibular condyle after mandibular distraction.
Study Design. Male Wistar rats at 10 weeks of age underwent unilateral mandibular distraction at 0.175 mm per 12 hours for 10 days. Histologic and histochemical analyses were performed at postoperative day 1 and weeks 1 and 3.
Results. High-resolution computed tomography (micro-CT) observations showed that deformation of the condyle occurred in the anterior region, where a discontinuity of the condylar cartilage layer was found in histologic sections. This destroyed area gathered many osteoclasts. In the central region, disorganization with a thin hypertrophic cell layer was recognizable by day 1 but later thickened. Morphologic recovery of the mandibular condyle could be attained by week 3 in this animal model.
Conclusions. These morphologic findings indicate that rapid deformation of the condyle, with destruction of the cartilage layer and bone resorption, was caused by artificial distraction.

DOI： 10.1016/j.0000.2014.05.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

This study aimed to examine the contributions of brain-derived neurotrophic factor (BDNF) at the injury site toward neuroma formation and nerve regeneration after inferior alveolar nerve transection. Histological analysis confirmed neuroma formation at 2 weeks after complete transection of the inferior alveolar nerve. A local administration of an antibody to BDNF inhibited connective tissue proliferation at the injury site and promoted nerve fiber integrity. Fluorogold labeling showed a significantly higher number of labeled cells in the trigeminal ganglion in the anti-BDNFtreated group compared with the vehicle control group. In-situ hybridization histochemistry showed intense signals for tropomyosin receptor kinase B mRNA in the area of the injury site containing fibrous or granular tissue in the anti-BDNF-treated group. In contrast, these signals were close to the detection limit in the area of the perineurium in intact nerve trunks, indicating that the signals were expressed by fibroblasts within the connective tissue. These findings suggest that antagonization of endogenous BDNF induced by nerve injury reduces neuroma formation, without inhibiting damaged axon regeneration. (C) 2014 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

DOI： 10.1097/WNR.0000000000000231

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Background: Tooth development is highly regulated in mammals and it is regulated by networks of signaling pathways (e. g. Tnf, Wnt, Shh, Fgf and Bmp) whose activities are controlled by the balance between ligands, activators, inhibitors and receptors. The members of the R-spondin family are known as activators of Wnt signaling, and Lgr4, Lgr5, and Lgr6 have been identified as receptors for R-spondins. The role of R-spondin/Lgr signaling in tooth development, however, remains unclear. Results: We first carried out comparative in situ hybridization analysis of R-spondins and Lgrs, and identified their dynamic spatio-temporal expression in murine odontogenesis. R-spondin2 expression was found both in tooth germs and the tooth-less region, the diastema. We further examined tooth development in R-spondin2 mutant mice, and although molars and incisors exhibited no significant abnormalities, supernumerary teeth were observed in the diastema. Conclusions: R-spondin/Lgr signaling is thus involved in tooth development. Developmental Dynamics 243:844-851, 2014. (c) 2014 Wiley Periodicals, Inc.

DOI： 10.1002/dvdy.24124

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHURCHILL LIVINGSTONE  

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 x 10(5) cm(2) each, onto human cadaver dermis. This was cultured in a submerged condition in 1.2 mM Ca2+ 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 mu M 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 alpha v beta 6 expression, implying impaired keratinocyte migration. Zoledronic acid did not attenuate the total transforming growth factor beta 1 (TGF-beta 1) production into the supernatant, but down-regulated TGF-beta 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 alpha v beta 6 expression, cause the down-regulation of TGF-beta/Smad signalling in oral keratinocytes, and impair re-epithelialization, suggesting compromised oral mucosa homeostasis in patients receiving zoledronic acid.

DOI： 10.1016/j.ijom.2013.06.016

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Language：Japanese   Publisher：新潟大学教育・学生支援機構  

新潟大学歯学部では,初年次教育として大学学習法を実施している.この授業は講義・演習での知識や技能をもとに,レポート作成とプレゼンテーションを行うもので,その学習活動を通して,間題解決能力,論理的思考力,表現力の開発をねらいとしている.今回,学習成果の直接評価に向けて,パフォーマンス評価を導入すべく,レポートからこれら能力を読み解くルーブリックづくりに取り組んだ.また,この経験からみえてきたパフォーマンス評価の難点とその対処について検討した.その結果,パフォーマンス評価により能力を把握できる可能性が示されたが,導入には教員の理解と協力,能力と関連した適切な評価観点の設定,評価の信頼性を高める工夫,評価と指導の一体化を考慮した授業デザインなど,いくつかの課題があることも明らかになった.The curriculum of Niigata University Faculty of Dentistry provides the &quot;Study Skills&quot; course as first year education. The ultimate goal of this course is to develop student abilities of problem solving, logical thinking and expression. For that purpose, each student is guided through the process of writing a report and presenting its content. To access the degree to which these abilities were achieved, we created a rubric and used it to evaluate the student reports. Based on the experience of this trial, we discussed problems for the introduction of performance assessment into the course. Although performance assessment has a high potential to grasp these abilities, there are some practical problems that may interfere with its introduction, such as &quot;how to involve the faculty in assessment&quot;, &quot;how to establish dimensions of the rubric that appropriately relate with the student abilities&quot;, &quot;how to increase the reliability of assessment&quot;, &quot;how to design the course to allow for a connection between assessment and instruction&quot;.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.

DOI： 10.1007/s00418-012-1064-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

We have histologically examined vascular invasion and calcification of the hypertrophic zone during endochondral ossification in matrix metalloproteinase (MMP)-9 deficient (MMP-9(-/-)) mice and in their littermates at 3 days, 3 weeks and 6 weeks after birth. Capillaries and osteoclasts at the chondro-osseous junction showed an intense MMP-9 immunopositivity, suggesting that they recognize chemical properties of cartilaginous matrices, and then release MMP-9 for cartilage degradation. CD31-positive capillaries and tartrate-resistant acid phosphatase-reactive osteoclasts could be found in the close proximity in the region of chondro-osseous junction in MMP-9(-/-) mice, while in wild-type mice, vascular invasion preceded osteoclastic migration into the epiphyseal cartilage. Although MNP-9(-/-) mice revealed larger hypertrophic zones, the index of calcified area was significantly smaller in MMP-9(-/-) mice. Interestingly, the lower layer of the MMP-9(-/-) hypertrophic zone showed intense MMP-13 staining, which could not be observed in wild-type mice. This indicates that MMP-13 may compensate for MMP-9 deficiency at that specific region, but not to a point at which the deficiency could be completely rescued. In conclusion, it seems that MMP-9 is the optimal enzyme for cartilage degradation during endochondral ossification by controlling vascular invasion and subsequent osteoclastic migration.

DOI： 10.2220/biomedres.34.119

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We surveyed medical and dental schools to promote the exchange of information about university efforts to increase the number of research-oriented doctors. Periods in which students rotate through laboratories to conduct research were reported by more than two thirds of universities. Many comments asserted that these efforts are effective. However, a small number of respondents reported low student motivation and insufficient time for laboratory experience. MD-PhD courses, in which students take a leave of absence in the middle of undergraduate training and follow a PhD curriculum, have been employed by more than 10 universities. However, relatively few students have chosen such programs. Modified MD-PhD courses have recently been introduced by several universities. In these courses, by taking part of the graduate school curriculum in advance, undergraduate students can shorten the time they spend in graduate school. Students who take such courses are increasing. There were many opinions that extra positions and financial support for research-oriented doctors are effective and should be enhanced. There were also many opinions that emphasize the importance of identifying research-oriented students, improving laboratory working environments, attending academic meetings and inter-university consortia to maintain students' motivation, and promoting collaboration with departments of clinical medicine.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：QUINTESSENCE PUBLISHING CO INC  

Purpose: Although the recent success rates of dental implants are quite high, it is still true that loading during the bone healing period may be more likely to discourage osseointegration and that occlusal overloading might result in implant failure, even after osseointegration is established. The purposes of this study were to establish an appropriate experimental animal model and to histologically assess degenerative changes in established osseointegration under early and excessive occlusal loading. Materials and Methods: Forty rats were divided into control and experimental groups. The maxillary first and second molars on both sides were extracted, and machined-surface titanium implants were placed. In the experimental group, 2 or 4 weeks after implant placement, abutments that were designed to overload the implants were attached. Control group implants did not receive abutments and remained in situ 2 or 4 weeks. Sections were prepared and observed histologically. Results: Attrition of occluding opposite teeth and shiny spots on the abutments indicated that this model was useful for histologic investigation of the remodeling and bone changes around implants. Specimens showed remarkable bone loss and deterioration of osseointegration when overloading began at 2 weeks. Overloading applied after 4 weeks of healing induced active bone resorption in remote areas of the implants after 15 days of occlusion, while bone resorption at the interface was limited. Conclusion: The authors successfully established an implant occlusion model using rats. This model revealed degenerative changes in osseointegration and/or in the bone around implants upon excessive occlusal loading. These results emphasize the risks associated with immediate loading and overloading. This is the first study to reveal the possibility of bone loss around overloaded implants in the absence of infection using a small animal model.

DOI： 10.11607/jomi.2388

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

This study was designed to (1) assess the in vitro biocompatibility of a chitosancollagen composite scaffold (C3) constructed by blending commercial chitosan and tilapia scale collagen with oral mucosa keratinocytes, (2) histologically and immunohistochemically characterize an ex vivo-produced oral mucosa equivalent constructed using the C3 (EVPOME-C), and (3) compare EVPOME-C with oral mucosa constructs utilizing AlloDerm (R) (EVPOME-A), BioMend (R) Extend (TM) (EVPOME-B), and native oral mucosa. C3 scaffold had a well-developed fibril network and a sufficiently small porosity to prevent keratinocytes from growing inside the scaffold after cell-seeding. The EVPOME oral mucosa constructs were fabricated in a chemically defined culture system. After culture at an air-liquid interface, EVPOME-C and EVPOME-B had multilayered epithelium with keratinization, while EVPOME-A had a more organized stratified epithelium. Ki-67 and p63 immunolabeled cells in the basal layer of all EVPOMEs suggested a regenerative ability. Compared with native oral mucosa, the keratin 15 and 10/13 expression patterns in all EVPOMEs showed a less-organized differentiation pattern. In contrast to the beta 1-integrin and laminin distribution in EVPOME-A and native oral mucosa, the subcellular deposition in EVPOME-C and EVPOME-B indicated that complete basement membrane formation failed. These findings demonstrated that C3 has a potential application for epithelial tissue engineering and provides a new potential therapeutic device for oral mucosa regenerative medicine. (C) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.

DOI： 10.1002/jbm.b.32746

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Increased expression of the transient receptor potential vanilloid 1 (TRPV1) channels, following nerve injury, may facilitate the entry of QX-314 into nociceptive neurons in order to achieve effective and selective pain relief. In this study we hypothesized that the level of QX-314/capsaicin (QX-CAP) - induced blockade of nocifensive behavior could be used as an indirect in-vivo measurement of functional expression of TRPV1 channels. We used the QX-CAP combination to monitor the functional expression of TRPV1 in regenerated neurons after inferior alveolar nerve (IAN) transection in rats. We evaluated the effect of this combination on pain threshold at different time points after IAN transection by analyzing the escape thresholds to mechanical stimulation of lateral mental skin. At 2 weeks after IAN transection, there was no QX-CAP mediated block of mechanical hyperalgesia, implying that there was no functional expression of TRPV1 channels. These results were confirmed immunohistochemically by staining of regenerated trigeminal ganglion (TG) neurons. This suggests that TRPV1 channel expression is an essential necessity for the QX-CAP mediated blockade. Furthermore, we show that 3 and 4 weeks after IAN transection, application of QX-CAP produced a gradual increase in escape threshold, which paralleled the increased levels of TRPV1 channels that were detected in regenerated TG neurons. Immunohistochemical analysis also revealed that non-myelinated neurons regenerated slowly compared to myelinated neurons following IAN transection. We also show that TRPV1 expression shifted towards myelinated neurons. Our findings suggest that nerve injury modulates the TRPV1 expression pattern in regenerated neurons and that the effectiveness of QX-CAP induced blockade depends on the availability of functional TRPV1 receptors in regenerated neurons. The results of this study also suggest that the QX-CAP based approach can be used as a new behavioral tool to detect dynamic changes in TRPV1 expression, in various pathological conditions.

DOI： 10.1371/journal.pone.0044023

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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This study examined the immunoexpression pattern of aquaporin-1 (AQP1), first identified as a water channel protein, in the periodontal ligament of rat molars during experimental tooth movement to clarify its role in periodontal responses in an overloaded model by the insertion of a piece of elastic band. In the control group without any treatment, the cementoblasts and osteogenic cells as well as the vascular endothelial cells showed AQP1 immunoreaction. In the experimental group, hyalinized tissue and intensely AQP1 positive amorphous structures which were identified as degenerated endothelial cells by immunoelectron microscopy, occurred at the compression side on Days 1 and 3. AQP1 immunoreaction came to be stronger in the intact endothelial cells around the hyalinized tissue. The hyalinized tissue had almost disappeared by Day 5 when many macrophages reactive to acid phosphatase activity appeared. The periodontal width on Day 7 became almost the same as that in the control group. These findings indicate that the hyalinized tissue and damaged AQP1 positive endothelial cells are phagocytized by macrophages which have temporally migrated, and suggest that the surviving endothelial cells with intense AQP1 reaction are involved in periodontal regeneration by capillary sprouting.

DOI： 10.2220/biomedres.33.225

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Objective: This study aimed to clarify the effects of zoledronic acid (ZOL) on human primary oral mucosal keratinocytes growing in a monolayer culture and on a tissue-engineered oral mucosal construct.
Design: Changes in the viability and proliferation of oral keratinocytes incubated with ZOL were measured. Following treatment with 10 mu M ZOL, histological examinations and immunohistochemical analyses for Ki-67, Geminin, and phospho-histone (gamma)-H2A.X were performed on tissue-engineered oral mucosa. Cell cycle distribution and the degree of apoptosis were also measured by flow cytometry. Additionally, we measured the expression of cell cycle regulatory proteins as well as phospho-Chk1 and -Chk2.
Results: ZOL treatment suppressed cell viability and proliferation in a dose-dependent manner. Compared with untreated tissue-engineered oral mucosa, ZOL treatment resulted in a thinner epithelium in which the basal cells appeared less-organised. This is consistent with the observed significant reduction in the Ki-67 labelling index. In contrast, the Geminin labelling index was significantly higher than that in the untreated sample. In spite of the presence of a few apoptotic cells, ZOL induced an arrest in S-phase, which was confirmed by our observed alterations in the expression levels of cyclin A, B1, p27(KIP1), Rb and phospho-Rb. When the proteasome inhibitor MG132 was added to the ZOL-treated cells, we observed a partial restoration of the expression of cyclin A, cyclin B1, and p27(KIP1). Expression of phospho-Chk1 was detected, and a significant increase in the labelling index of gamma-H2A.X was also seen.
Conclusions: These results indicate that a 10-mu M ZOL treatment induces a DNA damage response in oral keratinocytes that activates the ubiguitin-mediated proteolysis of cell cycle regulators, resulting in cell cycle arrest and repressive effects on cell viability, proliferation, and epithelial turnover. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.archoralbio.2011.11.015

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The articular disc in the temporomandibular joint (TMJ) that serves in load relief and stabilizing in jaw movements is a dense collagenous tissue consisting of extracellular matrices and disc cells. The various morphological configurations of the disc cells have given us diverse names, such as fibroblasts, chondrocyte-like cells and fibrochondrocytes; however, the characteristics of these cells have remained to be elucidated in detail. The disc cells have been reported to exhibit heterogeneous immunoreaction patterns for intermediate filaments including glial fibrillary acidic protein (GFAP), nestin and vimentin in the adult rat TMJ. Because these intermediate filaments accumulate in the disc cells as tooth eruption proceeds during postnatal development, it might be surmised that the expression of these intermediate filaments in the disc cells closely relates to mechanical stress. The present study was therefore undertaken to examine the effect of a continuous compressive force on the immunoexpression of these intermediate filaments and an additional intermediate filament muscle-specific desmin in the disc cells of the TMJ disc using a rat experimental model. The rats wore an appliance that exerts a continuous compressive load on the TMJ. The experimental period with the appliance was 5 days as determined by previous studies, after which some experimental animals were allowed to survive another 5 days after removal of the appliance. Histological observations demonstrated that the compressive force provoked a remarkable acellular region and a decrease in the thickness of the condylar cartilage of the mandible, and a sparse collagen fiber distribution in the articular disc. The articular disc showed a significant increase in the number of desmin-positive cells as compared with the controls. In contrast, immunopositive cells for GFAP, nestin and vimentin remained unchanged in number as well as intensity. At 5 days after removal of the appliance, both the disc and cartilage exhibited immunohistological and histological features in a recovery process. These findings indicate that the mature articular cells are capable of producing desmin instead of the other intermediate filaments against mechanical stress. The desmin-positive disc cells lacked a-smooth muscle actin (a-SMA) in this study, even though desmin usually co-exists with a-SMA in the vascular smooth muscle cells or pericytes. Because the precursor of a pericyte has such an immunoexpression pattern during angiogenesis, there is a further possibility that the formation of new vessels commenced in response to the extraordinary compressive force.

DOI： 10.1111/j.1469-7580.2012.01501.x

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：(公社)日本顕微鏡学会  

歯の支持・固定装置である歯根膜は豊富な知覚神経支配を受けており、咀嚼システムの感覚入力系として機能している。歯根膜の感覚受容器は侵害受容性の自由神経終末と機械受容器にわけられる。歯に加わる刺激は歯根膜機械受容器を介して、さまざまな口腔反射を惹起し、円滑に咀嚼運動を制御している。歯根膜機械受容器として低閾値遅順応性II型の伸展受容器であるルフィニ神経終末が重要である。この神経終末は分枝を繰り返す太い軸索終末と終末シュワン細胞が付随するという特徴をもっている。軸索終末にはカルシウム結合タンパクなどさまざまなタンパクの発現がみられる。歯根膜ルフィニ神経終末の発育・成熟には歯の萠出力や咬合力のような機械刺激の付与が不可欠であり、また高い再生能力を有している。多種の神経栄養因子が時期依存的に作用することによって、歯根膜ルフィニ神経終末の発育・再生が制御されているようである。(著者抄録)

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歯の支持・固定装置である歯根膜は豊富な知覚神経支配を受けており、咀嚼システムの感覚入力系として機能している。歯根膜の感覚受容器は侵害受容性の自由神経終末と機械受容器にわけられる。歯に加わる刺激は歯根膜機械受容器を介して、さまざまな口腔反射を惹起し、円滑に咀嚼運動を制御している。歯根膜機械受容器として低閾値遅順応性II型の伸展受容器であるルフィニ神経終末が重要である。この神経終末は分枝を繰り返す太い軸索終末と終末シュワン細胞が付随するという特徴をもっている。軸索終末にはカルシウム結合タンパクなどさまざまなタンパクの発現がみられる。歯根膜ルフィニ神経終末の発育・成熟には歯の萠出力や咬合力のような機械刺激の付与が不可欠であり、また高い再生能力を有している。多種の神経栄養因子が時期依存的に作用することによって、歯根膜ルフィニ神経終末の発育・再生が制御されているようである。(著者抄録)

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CCN family proteins play diverse roles in many aspects of cellular processes such as proliferation, differentiation, adhesion, migration, angiogenesis and survival. In the bone tissue of vertebrate species, the expression of most CCN family members has been observed in osteoblasts. However, their spatial and temporal distributions, as well as their functions, are still only partially understood. In this study, we evaluated the localization of CCN family members in skeletal tissue in vivo and comparatively analyzed the gene expression patterns and functions of the members in murine osteoblasts in primary culture. Immunofluorescent analyses revealed that the CCN family members were differentially produced in osteoblasts and osteocytes. The presence of all Ccn transcripts was confirmed in those osteoblasts. Among the members, CCN1, CCN2, CCN4 and CCN5 were found in osteocytes. CCN4 and CCN5 were distributed in osteocytes located inside of bone matrix as well. Next, we investigated the expression pattern of Ccn family members during osteoblast differentiation. Along with differentiation, most of the members followed proper gene expression patterns: whereas, Ccn4 and Ccn5 showed quite similar patterns. Furthermore, we evaluated the effects of CCN family members on the osteoblastic activities by using recombinant CCN proteins and RNA interference method. Five members of this family displayed positive effects on osteoblast proliferation or differentiation. Of note, CCN3 drastically inhibited the osteoblast activities. Each Ccn specific siRNA could modulate osteoblast activities in a manner expected by the observed effect of respective recombinant CCN protein. In addition, we found that extracellular signal-regulated kinase1/2 and p38 mitogen-activated protein kinase pathways were critically involved in the CCN family member-mediated modification of osteoblast activities.
Collectively, all Ccn family members were found to be differentially expressed along with differentiation and therefore could participate in progression of the osteoblast lineage. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2011.06.033

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The articular disc is a dense collagenous tissue containing disc cells that are phenotypically described as chondrocyte-like cells or fibrochondrocytes. Despite the possible existence of these phenotypes in systemic joints, little is known about the detailed classification of the articular disc cells in the temporomandibular joint. In this immunocytochemical study we examined the localization and distribution patterns of nestin and glial fibrillary acidic protein (GFAP) in the articular disc of the rat temporomandibular joint at postnatal day 1, and weeks 1, 2, 4 and 8, based on the status of tooth eruption and occlusion. Nestin and GFAP are intermediate filament proteins whose expression patterns are closely related to cell differentiation and cell migration. Both types of immunopositive cell greatly increased postnatally to a stable level after postnatal week 4, but they showed different distribution patterns and cell morphologies. Nestin-reactive disc cells, which were characterized by a meagre cytoplasm and thin cytoplasmic processes, were scattered in the articular disc, whereas GFAP-positive cells, characterized by broader processes, existed exclusively in the deeper area. In mature discs, the major proportion of articular disc cells exhibited GFAP immunoreactivity. Furthermore, a double-immunostaining demonstrated that the nestin-negative cells, consisting of GFAP-positive and -negative cells, exhibited immunoreactions for heat shock protein 25. These findings indicate that the articular disc cells comprise at least three types in the rat temporomandibular joint and suggest that their expressions closely relate to mechanical loading forces within the joint, including occlusal force, as observed through postnatal development.

DOI： 10.1111/j.1469-7580.2011.01404.x

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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CiNii Article

CiNii Books

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CiNii Article

CiNii Books

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：日本歯科医学教育学会  

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The working group for the future planning of the Japanese Association of Anatomists (JAA) has been working to address the issues that were consulted from the president of JAA since October 2009. After making the interim report in March 2010, a public hearing for general members of the JAA was held and a final report was submitted to the President in January 2011. The report contains the analysis of the current situation, the directions in which we should proceed, and recommendations of concrete actions that JAA should take for each issue. The issues discussed were as follows: 1. Future prospects of anatomy and morphological sciences. How can we maintain the specialties of morphological and anatomical sciences in the rapidly advancing field of life sciences and develop collaborations with other fields? 2. Improvement of the JAA academic meetings. How can we increase JAA members and young participants in the academic meetings of the JAA? 3. Fostering the next generation of young researchers. How can we increase young researchers graduated from the schools of Medicine or Dentistry? 4. Future prospects of education of gross anatomy. Prospects of education in gross anatomy and the body donation registration system in relation with some new cadaver-related movements.

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Osseointegration is the most preferable interface of dental implants and newly formed bone. However, the cavity preparation for dental implants often gives rise to empty lacunae or pyknotic osteocytes in bone surrounding the dental implant. This study aimed to examine the chronological alternation of osteocytes in the bone surrounding the titanium implants using a rat model. The distribution of the osteocytic lacunar canalicular system (OLCS) in bone around the titanium implants was examined by silver impregnation according to Bodian&#039;s staining. We also performed double staining for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), as well as immunohistochemistry for fibroblast growth factor (FGF) 23--a regulator for the serum concentration of phosphorus--and sclerostin, which has been shown to inhibit osteoblastic activities. Newly formed bone and the injured bone at the early stage exhibited an irregularly distributed OLCS and a few osteocytes positive for sclerostin or FGF23, therefore indicating immature bone. Osteocytes in the surrounding bone from Day 20 to Month 2 came to reveal an intense immunoreactivity for sclerostin. Later on, the physiologica

DOI： 10.1002/ar.21391

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Osseointegration is the most preferable interface of dental implants and newly formed bone. However, the cavity preparation for dental implants often gives rise to empty lacunae or pyknotic osteocytes in bone surrounding the dental implant. This study aimed to examine the chronological alternation of osteocytes in the bone surrounding the titanium implants using a rat model. The distribution of the osteocytic lacunar canalicular system (OLCS) in bone around the titanium implants was examined by silver impregnation according to Bodian&apos;s staining. We also performed double staining for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), as well as immunohistochemistry for fibroblast growth factor (FGF) 23-a regulator for the serum concentration of phosphorus-and sclerostin, which has been shown to inhibit osteoblastic activities. Newly formed bone and the injured bone at the early stage exhibited an irregularly distributed OLCS and a few osteocytes positive for sclerostin or FGF23, therefore indicating immature bone. Osteocytes in the surrounding bone from Day 20 to Month 2 came to reveal an intense immunoreactivity for sclerostin. Later on, the physiological bone remodeling gradually replaced such immature bone into a compact profile bearing a regularly arranged OLCS. As the bone was remodeled, FGF23 immunoreactivity became more intense, but sclerostin became less so in the well-arranged OLCS. In summary, it seems likely that OLCS in the bone surrounding the dental implants is damaged by cavity formation, but later gradually recovers as bone remodeling takes place, ultimately inducing mature bone. Anat Rec, 294:1074-1082, 2011. (C) 2011 Wiley-Liss, Inc.

DOI： 10.1002/ar.21391

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Language：Japanese   Publisher：新潟歯学会  

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We tested a hypothesis that the spinal plasticity induced within a few hours after nerve injury may produce changes in cortical activities and an initial phase of neuropathic pain. Somatosensory cortical responses elicited by vibratory stimulation were visualized by transcranial flavoprotein fluorescence imaging in mice. These responses were reduced immediately after cutting the sensory nerves. However, the remaining cortical responses mediated by nearby nerves were potentiated within a few hours after nerve cutting. Nerve injury induces neuropathic pain. In the present study, mice exhibited tactile allodynia 1-2 weeks after nerve injury. Lesioning of the ipsilateral dorsal column, mediating tactile cortical responses, abolished somatic cortical responses to tactile stimuli. However, nontactile cortical responses appeared in response to the same tactile stimuli within a few hours after nerve injury, indicating that tactile allodynia was acutely initiated. Weinvestigated the trigger mechanisms underlying the cortical changes. Endogenous glial cell line-derived neurotrophic factor (GDNF), found in the Meissner corpuscles, induced basal firing_0.1Hzor less in itsA_ tactile afferents, and disruption of the basal firing triggered the potentiation of nontactile cortical responses. Application of 10nMLY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop- 1-yl]-3-(xanth-9-yl) propanoic acid], a specific antagonist of group II metabotropic glutamate receptors (mGluRs), on to the surface of the spinal cord also induced the potentiation of nontactile cortical responses. Together, it is suggested that low-frequency afferent firing produced by GDNF in touch-sensitive nerve fibers continuously activated spinal group II mGluRs and that failure of this activation triggered tactile allodynia. © 2011 the authors.

DOI： 10.1523/JNEUROSCI.6753-10.2011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

We tested a hypothesis that the spinal plasticity induced within a few hours after nerve injury may produce changes in cortical activities and an initial phase of neuropathic pain. Somatosensory cortical responses elicited by vibratory stimulation were visualized by transcranial flavoprotein fluorescence imaging in mice. These responses were reduced immediately after cutting the sensory nerves. However, the remaining cortical responses mediated by nearby nerves were potentiated within a few hours after nerve cutting. Nerve injury induces neuropathic pain. In the present study, mice exhibited tactile allodynia 1-2 weeks after nerve injury. Lesioning of the ipsilateral dorsal column, mediating tactile cortical responses, abolished somatic cortical responses to tactile stimuli. However, nontactile cortical responses appeared in response to the same tactile stimuli within a few hours after nerve injury, indicating that tactile allodynia was acutely initiated. We investigated the trigger mechanisms underlying the cortical changes. Endogenous glial cell line-derived neurotrophic factor ( GDNF), found in the Meissner corpuscles, induced basal firing similar to 0.1 Hz or less in its A beta tactile afferents, and disruption of the basal firing triggered the potentiation of nontactile cortical responses. Application of 10nM LY341495[(2S)-2-amino-2-[(1S, 2S)-2-carboxycyclopropl-yl]-3-(xanth-9-yl)propanoic acid], a specific antagonist of group II metabotropic glutamate receptors (mGluRs), on to the surface of the spinal cord also induced the potentiation of nontactile cortical responses. Together, it is suggested that low-frequency afferent firing produced by GDNF in touch-sensitive nerve fibers continuously activated spinal group II mGluRs and that failure of this activation triggered tactile allodynia.

DOI： 10.1523/JNEUROSCI.6753-10.2011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

The acid-sensing ion channel 3 (ASIC3), a member of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily, has been reported to participate in acid sensing, mechanosensation, and nociception. However, no information is available regarding the precise localization and function of this molecule in the periodontal ligament, which contains abundant sensory nerves originating from the trigeminal ganglion. The present study examined the expression of ASIC3 in the lingual periodontal ligament of mouse incisors by immunohistochemistry. Furthermore, the expression of ASIC3 in the trigeminal ganglion which innervates the periodontal ligament - was investigated at protein (immunohistochemistry and quantitative analysis) and mRNA levels (RT-PCR technique and in situ hybridization histochemistry). Immunohistochemistry for ASIC3 was able to demonstrate dendritic profiles of the periodontal Ruffini endings in the mouse incisors. No thin fibers terminating as nociceptive free nerve endings exhibited ASIC3 immunoreactivity. Double immunofluorescent staining revealed ASIC3 immunoreaction in the axoplasm but not in the ordinary Schwann cells - including the associated terminal Schwann cells. Observation of the trigeminal ganglia showed variously sized neurons expressing ASIC3 immunoreaction; the most intense immunopositivity was found in the small and medium-sized neurons, as confirmed by in situ hybridization histochemistry using a specific cRNA probe. Quantitative analysis on trigeminal ganglion neurons showed that 38.0% of ASIC3 neurons could be categorized as medium-sized neurons which mediate mechanotransduction. These findings suggest that ASIC3 functions as a molecule for mechanosensation in the periodontal Ruffini endings. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2010.11.023

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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The signaling axis comprising the parathyroid hormone (PTH)-related peptide (PTHrP), the PTH/PTHrP receptor and the fibroblast growth factor receptor 3 (FGFR3) plays a central role in chondrocyte proliferation. The Indian hedgehog (IHH) gene is normally expressed in early hypertrophic chondrocytes, and its negative feedback loop was shown to regulate PTH/PTHrP receptor signaling. In this study, we examined the regulation of PTH/PTHrP receptor gene expression in a FGFR3-transfected chondrocytic cell line, CFK2. Expression of IHH could not be verified on these cells, with consequent absence of hypertrophic differentiation. Also, expression of the PTH/PTHrP receptor (75% reduction of total mRNA) and the PTHrP (50% reduction) genes was reduced in CFK2 cells transfected with FGFR3 cDNA. Interestingly, we verified significant reduction in cell growth and increased apoptosis in the transfected cells. STAT1 was detected in the nuclei of the CFK2 cells transfected with FGFR3 cDNA, indicating predominance of the JAK/STAT signaling pathway. The reduction in PTH/PTHrP receptor gene in CFK2 cells overexpressing FGFR3 was partially blocked by treatment with an inhibitor of JAK3 (WHI-P131), but not with an inhibitor of MAPK (SB203580) or JAK2 (AG490). Altogether, these findings suggest that FGFR3 down-regulates PTH/PTHrP receptor gene expression via the JAK/STAT signaling in chondrocytic cells. The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved.

DOI： 10.1093/jmicro/dfq002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The signaling axis comprising the parathyroid hormone (PTH)-related peptide (PTHrP), the PTH/PTHrP receptor and the fibroblast growth factor receptor 3 (FGFR3) plays a central role in chondrocyte proliferation. The Indian hedgehog (IHH) gene is normally expressed in early hypertrophic chondrocytes, and its negative feedback loop was shown to regulate PTH/PTHrP receptor signaling. In this study, we examined the regulation of PTH/PTHrP receptor gene expression in a FGFR3-transfected chondrocytic cell line, CFK2. Expression of IHH could not be verified on these cells, with consequent absence of hypertrophic differentiation. Also, expression of the PTH/PTHrP receptor (75% reduction of total mRNA) and the PTHrP (50% reduction) genes was reduced in CFK2 cells transfected with FGFR3 cDNA. Interestingly, we verified significant reduction in cell growth and increased apoptosis in the transfected cells. STAT1 was detected in the nuclei of the CFK2 cells transfected with FGFR3 cDNA, indicating predominance of the JAK/STAT signaling pathway. The reduction in PTH/PTHrP receptor gene in CFK2 cells overexpressing FGFR3 was partially blocked by treatment with an inhibitor of JAK3 (WHI-P131), but not with an inhibitor of MAPK (SB203580) or JAK2 (AG490). Altogether, these findings suggest that FGFR3 down-regulates PTH/PTHrP receptor gene expression via the JAK/STAT signaling in chondrocytic cells.

DOI： 10.1093/jmicro/dfq002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Potentiation of inhibitory gamma-aminobutyric acid subtype A (GABA(A)) receptor function is involved in the mechanisms of anesthetic action. The present study examined the immobilizing action of the volatile anesthetic isoflurane in mice with double knockout (DKO) of phospholipase C-related inactive protein (PRIP)-1 and -2. Both of these proteins play important roles in the expression of GABA(A) receptors containing the gamma 2 subunit on the neuronal cell surface. Immunohistochemistry for GABA(A) receptor subunits demonstrated reduced expression of gamma 2 subunits in the spinal cord of the DKO mice. Immunohistochemistry also revealed up-regulation of the alpha 1 and beta 3 subunits even though there were no apparent differences in the immunoreactivities for the beta 2 subunits between wild-type and DKO mice. The tail-clamp method was used to evaluate the anesthetic/immobilizing effect of isoflurane and the minimum alveolar concentration (MAC) was significantly lower in DKO mice compared with wild-type controls (1.07 +/- 0.01% versus 1.36 +/- 0.04% atm), indicating an increased sensitivity to isoflurane in DKO mice. These immunohistochemical and pharmacological findings suggest that reduced expression of the GABA(A) receptor gamma 2 subunit affects the composition and function of spinal GABA(A) receptors and potentiates the immobilizing action of isoflurane. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2010.01.031

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Language：Japanese   Publisher：日本歯科医学会  

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DOI： 10.1016/j.neures.2010.07.2264

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DOI： 10.1016/j.neures.2010.07.371

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This study aimed to evaluate whether the immunolocalization of fibroblast growth factor (FGF) 23 and dentin matrix protein 1 (DMP1) is associated with the spatial regularity of the osteocyte lacunar canalicular system(s) (OLCS). Femora of 12-weeks-old male ICR mice were fixed with 4 paraformaldehyde, decalcified with a 10 EDTA solution and then embedded in paraffin. We have devised a triple staining procedure that combines silver impregnation, alkaline phosphatase (ALPase) immunohistochemistry and tartrate-resistant acid phosphatase (TRAPase) enzyme histochemistry on a single paraffin section. This procedure permitted the visualization of ALPase-positive plump osteoblasts and several TRAPase-positive osteoclasts on those bone matrices featuring irregularly arranged OLCS, and of ALPase-positive bone lining cells on the bone matrix displaying the well-arranged OLCS. As observations proceeded from the metaphysis toward the diaphysis, the endosteal cortical bone displayed narrower bands of calcein labeling, accompanied by increased regularity of the OLCS. This implies that the speed of bone deposition during bone remodeling would affect the regularity of the OLCS. While DMP1 was evenly localized in all regions of the cortical bones, FGF23 was more abundantly localized in osteocytes of cortical bones with regularly arranged OLCS. In cortical bones, the endosteal area featuring regular OLCS exhibited more intense FGF23 immunoreaction when compared to the periosteal region, which tended to display irregular OLCS. In summary, FGF23 appears to be synthesized principally by osteocytes in the regularly distributed OLCS that have been established after bone remodeling.

DOI： 10.1093/jmicro/dfp032

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

This study aimed to evaluate whether the immunolocalization of fibroblast growth factor (FGF) 23 and dentin matrix protein 1 (DMP1) is associated with the spatial regularity of the osteocyte lacunar canalicular system(s) (OLCS). Femora of 12-weeks-old male ICR mice were fixed with 4% paraformaldehyde, decalcified with a 10% EDTA solution and then embedded in paraffin. We have devised a triple staining procedure that combines silver impregnation, alkaline phosphatase (ALPase) immunohistochemistry and tartrate-resistant acid phosphatase (TRAPase) enzyme histochemistry on a single paraffin section. This procedure permitted the visualization of ALPase-positive plump osteoblasts and several TRAPase-positive osteoclasts on those bone matrices featuring irregularly arranged OLCS, and of ALPase-positive bone lining cells on the bone matrix displaying the well-arranged OLCS. As observations proceeded from the metaphysis toward the diaphysis, the endosteal cortical bone displayed narrower bands of calcein labeling, accompanied by increased regularity of the OLCS. This implies that the speed of bone deposition during bone remodeling would affect the regularity of the OLCS. While DMP1 was evenly localized in all regions of the cortical bones, FGF23 was more abundantly localized in osteocytes of cortical bones with regularly arranged OLCS. In cortical bones, the endosteal area featuring regular OLCS exhibited more intense FGF23 immunoreaction when compared to the periosteal region, which tended to display irregular OLCS. In summary, FGF23 appears to be synthesized principally by osteocytes in the regularly distributed OLCS that have been established after bone remodeling.

DOI： 10.1093/jmicro/dfp032

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH-driven bone anabolism. After administering PTH intermittently to wildtype and c-fos(-/-) mice, immunohistochemical, histomorphometrical, ultrastructural, and statistical examinations were performed. Structural and kinetic parameters related to bone formation were increased in PTH-treated wildtype mice, whereas in the osteoclast-deficient c-fos(-/-) mice, there were no significant differences between groups. In wildtype and knockout mice, PTH administration led to significant increases in the number of cells double-positive for alkaline phosphatase and BrdU, suggesting active pre-osteoblastic proliferation. Ultrastructural examinations showed two major pre-osteoblastic subtypes: one rich in endoplasmic reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted the most abundant preosteoblastic phenotype after PTH administration in the wildtype mice. In c-fos(-/-) mice, misER cells were present on the bone surfaces but did not seem to be actively producing bone matrix. Several misER cells were shown to be positive for EphB4 and were eventually seen rather close to osteoclasts in the PTH-administered wildtype mice. We concluded that the absence of osteoclasts in c-fos(-/-) mice might hinder PTH-driven bone anabolism and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration. J Bone Miner Res 2009;24:1586-1597. Published online on April 27, 2009; doi: 10.1359/JBMR.090413

DOI： 10.1359/JBMR.090413

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH-driven bone anabolism. After administering PTH intermittently to wildtype and c-fos(-/-) mice, immunohistochemical, histomorphometrical, ultrastructural, and statistical examinations were performed. Structural and kinetic parameters related to bone formation were increased in PTH-treated wildtype mice, whereas in the osteoclast-deficient c-fos(-/-) mice, there were no significant differences between groups. In wildtype and knockout mice, PTH administration led to significant increases in the number of cells double-positive for alkaline phosphatase and BrdU, suggesting active pre-osteoblastic proliferation. Ultrastructural examinations showed two major pre-osteoblastic subtypes: one rich in endoplasmic reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted the most abundant preosteoblastic phenotype after PTH administration in the wildtype mice. In c-fos(-/-) mice, misER cells were present on the bone surfaces but did not seem to be actively producing bone matrix. Several misER cells were shown to be positive for EphB4 and were eventually seen rather close to osteoclasts in the PTH-administered wildtype mice. We concluded that the absence of osteoclasts in c-fos(-/-) mice might hinder PTH-driven bone anabolism and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration. J Bone Miner Res 2009;24:1586-1597. Published online on April 27, 2009; doi: 10.1359/JBMR.090413

DOI： 10.1359/JBMR.090413

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

The terminal Schwann cells (TSCs) which play crucial roles in regeneration of the periodontal Ruffini endings (RE) exhibit immunoreaction for glial cell line-derived neurotrophic factor (GDNF). However, no information is available regarding the role of GDNF in the periodontal RE during nerve regeneration. This study was undertaken to examine the changes in GDNF expression in the rat periodontal RE following transection of the inferior alveolar nerve (IAN) using immunohistochemistry for GDNF and S-100 protein, a marker for the TSCs. We additionally investigated the changes in expression of GDNF in the trigeminal ganglion (TG) at protein and mRNA levels. A transection to IAN induced a disappearance of the TSCs from the alveolus-related part (ARP), followed by a migration of spindle-shaped cells with S-100 but without GDNF immunoreactions into the tooth-related part (TRP) by postoperative (PO) week 2. At PO week 2, GDNF immunoreacted cellular elements increased in number in the ARP although the spindle-shaped cells without GDNF reaction remained in the TRP After PO week 4, many GDNF-positive TSCs appeared in the ARP though the spindle-shaped cells vanished from the TRP. A real time RT-PCR analysis demonstrated the highest elevation of GDNT mRNA in the TG at PO week 2. These findings suggested the involvement of this molecule in the maturation and maintenance of the periodontal RE during regeneration. Taken together with our previous and current studies, it appears that the regeneration of the periodontal RE is controlled by multiple neurotrophins in a stage-specific manner. Anat Rec, 292:1182-1191, 2009. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/ar.20931

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Objectives
It is important to know the etiology of implant failure. It has been reported that heat stress during drilling was one of the causes for failure and the threshold was 47 degrees C. However, clinically, we encounter cases in which overheating does not seem to affect osseointegration eventually. The purpose of this study was to assess histologically the spatio-temporal effect of heat stress on bone formation after overheating the bone matrix.
Material and methods
Rat calvarial bone was heated to 37 degrees C, 43 degrees C, 45 degrees C and 48 degrees C for 15 min by a temperature stimulator. Paraffin sections were prepared 1, 3 and 5 weeks after heating and investigated histologically under light microscopy. Hematoxylin and eosin staining, alkaline phosphatase (ALP), osteopontin (OPN), heat shock protein 27 (Hsp27) and heat shock protein 70 (Hsp70) immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) enzyme histochemistry were carried out. The area of dead osteocytes was calculated and statistically analyzed. Apoptotic osteocytes were detected by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) method.
Results
Along with the temperature increase, the area of dead osteocytes increased and regeneration of the periosteal membrane was delayed. Hsps- and TUNEL-positive cells were only seen in the 48 degrees C group. Spatio-temporal changes of TRAP- and ALP-positive cell numbers were observed, while OPN expression was mostly absent. Even after 48 degrees C stimulation, bone formation on the calvarial surface was observed after 5 weeks.
Conclusions
Although there was a temperature-dependent delay in bone formation after heat stress, the 48 degrees C heat stress did not obstruct bone formation eventually. This delay was probably caused by slow periosteal membrane regeneration.
To cite this article:Yoshida K, Uoshima K, Oda K, Maeda T. Influence of heat stress to matrix on bone formation.Clin. Oral Impl. Res. 20, 2009; 782-790.doi: 10.1111/j.1600-0501.2008.01654.x.

DOI： 10.1111/j.1600-0501.2008.01654.x

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Objectives
It is important to know the etiology of implant failure. It has been reported that heat stress during drilling was one of the causes for failure and the threshold was 47 degrees C. However, clinically, we encounter cases in which overheating does not seem to affect osseointegration eventually. The purpose of this study was to assess histologically the spatio-temporal effect of heat stress on bone formation after overheating the bone matrix.
Material and methods
Rat calvarial bone was heated to 37 degrees C, 43 degrees C, 45 degrees C and 48 degrees C for 15 min by a temperature stimulator. Paraffin sections were prepared 1, 3 and 5 weeks after heating and investigated histologically under light microscopy. Hematoxylin and eosin staining, alkaline phosphatase (ALP), osteopontin (OPN), heat shock protein 27 (Hsp27) and heat shock protein 70 (Hsp70) immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) enzyme histochemistry were carried out. The area of dead osteocytes was calculated and statistically analyzed. Apoptotic osteocytes were detected by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) method.
Results
Along with the temperature increase, the area of dead osteocytes increased and regeneration of the periosteal membrane was delayed. Hsps- and TUNEL-positive cells were only seen in the 48 degrees C group. Spatio-temporal changes of TRAP- and ALP-positive cell numbers were observed, while OPN expression was mostly absent. Even after 48 degrees C stimulation, bone formation on the calvarial surface was observed after 5 weeks.
Conclusions
Although there was a temperature-dependent delay in bone formation after heat stress, the 48 degrees C heat stress did not obstruct bone formation eventually. This delay was probably caused by slow periosteal membrane regeneration.
To cite this article:Yoshida K, Uoshima K, Oda K, Maeda T. Influence of heat stress to matrix on bone formation.Clin. Oral Impl. Res. 20, 2009; 782-790.doi: 10.1111/j.1600-0501.2008.01654.x.

DOI： 10.1111/j.1600-0501.2008.01654.x

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OBJECTIVES: It is important to know the etiology of implant failure. It has been reported that heat stress during drilling was one of the causes for failure and the threshold was 47 degrees C. However, clinically, we encounter cases in which overheating does not seem to affect osseointegration eventually. The purpose of this study was to assess histologically the spatio-temporal effect of heat stress on bone formation after overheating the bone matrix. MATERIAL AND METHODS: Rat calvarial bone was heated to 37 degrees C, 43 degrees C, 45 degrees C and 48 degrees C for 15 min by a temperature stimulator. Paraffin sections were prepared 1, 3 and 5 weeks after heating and investigated histologically under light microscopy. Hematoxylin and eosin staining, alkaline phosphatase (ALP), osteopontin (OPN), heat shock protein 27 (Hsp27) and heat shock protein 70 (Hsp70) immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) enzyme histochemistry were carried out. The area of dead osteocytes was calculated and statistically analyzed. Apoptotic osteocytes were detected by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) method. RESULTS: Along with

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Dental treatments sometimes cause sensory impairment, especially in the region innervated by the third division of the trigeminal nerve. The most frequent symptoms are loss of sensation and abnormal sensation. Although most studies have addressed the neuropathic symptom "allodynia" using experimental animal models of the infraorbital nerve, there is little information regarding the sensory impairment that frequently occurs clinically. Therefore, different experimental models are required to clarify the mechanisms of the clinical effects, and previous experimental models have been limited to rats. Here, we report a sensory impairment model in mice whose mechanical touch threshold increased after tight ligation of the mental nerve. Habituation before surgery by mechanical touching of the face enabled us to observe the long-term chronological changes in sensation. The mechanical touch thresholds within the mental nerve region were measured for 70 postoperative (PC) days. Changes in the distribution of substance P (SP) were evaluated by immunohistochemistry to clarify the involvement of axonal flow in the sensory impairment and its recovery. The mechanical touch thresholds transiently increased by PO days 2-3, but decreased to the preoperative levels at around PO day 14. Apparent SP immunoreactivity was recognizable on the medial side to the ligation at PO days 2-3 and disappeared at PO day 7. These behavioural and immunohistochemical changes appeared to exhibit similar time courses, suggesting a possible relationship between them. Therefore, we suggest that our experimental mouse model could represent a new model for clarifying the mechanism of the sensory impairment caused by peripheral nerve injury. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jneumeth.2009.04.020

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Epithelial sodium channels (ENaCs) are a subfamily of ion channels within the degenerin/ENaC (DEG/ENaC) superfamily. Previous studies have shown the immunolocalization of ENaC in the neural elements of the Cutaneous mechanoreceptors as well as dorsal root and trigeminal ganglion neurons, indicating the involvement of this molecule in mechanotransduction. The present study examined the expression of ENaC beta, a major component of ENaC protein, in the mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisors by immunohistochemistry. The expression of ENaC beta in the trigeminal ganglion-which innervates the periodontal Ruffini endings-was also investigated at the mRNA and protein levels. Furthermore, double staining and a nerve injury experiment were applied to clarify its detailed localization in the periodontal Ruffini endings. ENaC beta immunoreaction in the trigeminal ganglion was recognizable in the comparatively large neurons which have been considered to mediate mechanotransduction. Immunohistochemistry for ENaC beta demonstrated dendritic ramifications of the Ruffini endings as well as the rounded cells in the periodontal ligament. Double staining with ENaC beta and either PGP9.5 or S-100 protein showed immunoreaction for ENaC beta in both the axonal and glial elements in the periodontal ligament. Some ENaC beta positive cells with rounded profiles were reactive to non-specific cholinesterase activity. Furthermore, a transection of the inferior alveolar nerve failed to eliminate the rounded cells with ENaC beta reaction, indicating that they were the terminal Schwann cells associated with the periodontal Ruffini endings. These findings suggest that ENaC beta is a key mechanotransducing channel in the periodontal Ruffini endings. Probably, the terminal Schwann cells together with the axon terminals regulate mechanotransduction in the periodontal endings.

DOI： 10.2220/biomedres.30.113

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Caveolae are involved in clathrin-independent endocytosis, transcytosis, signal transduction, and tumor suppression - all of which depend on their main constituent protein caveolin families. The periodontal Ruffini ending has been reported to develop a caveola-like structure on the cell membrane of both the axon terminals and Schwann sheaths, suggesting the existence of an axon-Schwann cell interaction in the periodontal Ruffini endings. However, little information is available concerning the functional significance of these caveolae. The present study was undertaken to examine the immunolocalization of caveolin-1, -3 (Cav-1, Cav-3) and Ca2+-ATPase in the periodontal Ruffini endings of the rat incisor. Decalcified sections of the upper jaws were processed for immunocytochemistry at the levels of light and electron microscopy. Some immunostained sections were treated with histochemistry for nonspecific cholinesterase (nChE) activity. Observations showed the periodontal Ruffini endings were immunopositive for Cav-1, but not Cav-3. Immunoreactive products for Cav-1 were confined to caveola-like structures in the cell membranes of the cytoplasmic extensions and cell bodies of the terminal Schwann cells associated with the periodontal Ruffini endings. However, the axonal membranes of the terminals did not express any Cav-1 immunoreaction. Double staining with Ca2+-ATPase and either protein gene product 9.5 (PGP 9.5) or S-100 protein disclosed the co-localization of immunoreactions in the axonal branches of the periodontal Ruffini endings, but not in the terminal Schwann cells. As Ca2+ plays an important role in mechanotransduction, these characteristic immunolocalizations show Cav-1/Ca2+-ATPase might be involved in the quick elimination of intracellular Ca2+ in mechanotransduction.

DOI： 10.1111/j.1469-7580.2008.01029.x

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Nestin is an intermediate filament which was first identified in neuroepithelial stem cells. This expression has also been reported in restricted locations in adults. Previous studies have suggested that the periodontal Ruffini endings remain immature in nature even in adulthood. The present study reports on a characteristic expression of immunoreaction for nestin in the periodontal Ruffini endings during postnatal development. RT-PCR analysis detected nestin mRNA in a reverse transcripted cDNA sample from both the rat trigeminal ganglion and periodontal ligament. The nestin immunoreaction existed in the periodontal ligament at postnatal day 3 (PO 3 days), when many spindle-shaped Schwann cells were positive for nestin immunoreaction. At PO 1 week, when periodontal nerve fibers displayed a dendritic fashion, the round cells came to show the nestin immunoreaction. These immunopositive cells were also reactive for S-100 protein and non-specific cholinesterase, indicating that these cells could be categorized as terminal Schwann cells associated with the periodontal Ruffini endings. Some ordinary Schwann cells also exhibited nestin immunoreaction. From PO 2 to 3 weeks, nestin positive terminal Schwarm cells increased in number in accordance with the postnatal development of the periodontal Ruffini endings, while this immuno-expression pattern remained unchanged. Nestin immunoreaction was also recognizable in the satellite cells - but never in the neurons - in the trigeminat ganglion throughout this observation period. This immuno-expression pattern suggests that nestin serves as an intermediate filament for mechanical stability in the periodontal Ruffini endings against external stimuli. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2008.11.001

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Osseointegration is regarded as the most appropriate implant-bone interface in dental implantation. However, damaged bone with empty osteocytic lacunae driven by implant cavity preparation remains even after the completion of osseointegration. Although previous studies have suggested the occurrence of bone remodeling around implants, information on its detailed process is meager. Our study aimed to examine the fate of bone around titanium implants after the establishment of osseointegration on an animal model using the rat maxilla. Titanium implants were inserted into prepared bone cavities of the rat maxilla. Bone formation and maturation processes were evaluated by double staining for alkaline phosphatase and tartrate-resistant acid phosphatase, immunohistochemistry for bone matrix proteins, vital staining with calcein, and elemental mapping with an electron probe microanalyzer. Bone with empty osteocytic lacunae or pyknosis remained between the intact preexisting and newly formed woven bones at post 1 month. It gradually decreased to disappear completely by active bone remodeling with a synchronized coordination of alkaline phosphatase-positive osteoblasts and tartrate-resistant acid phosphatase-reactive osteoclasts at post 3 months, thickening to be replaced by compact bone. Dynamic labeling showed two clear lines in the newly formed bone around the implant through this experimental period. Electron probe microanalyzer analysis demonstrated chronologically increased levels of Ca and P in the newly formed bone identical to those in the surrounding bone at post 2.5 months. These findings indicate that continuous bone remodeling after the achievement of osseointegration causes replacement of the damaged bone by compact bone as well as an improvement in bone quality. Anat Rec,292:38-47, 2009. (c) 2008 Wiley-Liss, Inc.

DOI： 10.1002/ar.20748

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：F HERNANDEZ  

It has been reported that the Mg-insufficient bone is fragile upon mechanical loading, despite its high bone mineral density, while vitamin K2 (MK-4: menatetrenone) improved the mechanical strength of Mg-insufficient bone. Therefore, we aimed to elucidate the ultrastructural properties of bone in rats with dietary Mg insufficiency with and without MK-4 supplementation. Morphological examinations including histochemistry, transmission electron microscopy, electron probe microanalysis (EPMA) and X-ray diffraction were conducted on the femora and tibiae of 4-week-old Wistar male rats fed with 1) a normal diet (control group, 0.09% Mg), 2) a Mg-insufficient diet (low Mg group, 0.006% Mg), or 3) a Mg-insufficient diet supplemented with MK-4 (MK-4 group, 0.006% Mg, 0.03% MK-4). MK-4 appeared to inhibit the osteoclastic bone resorption that is stimulated by Mg insufficiency. EPMA analysis, however, revealed an increased concentration of Ca paralleling Mg reduction in the low Mg group. Assessment by X-ray diffraction revealed an abundance of a particular synthetic form of hydroxyapatite in the low Mg group, while control bones featured a variety of mineralized crystals. In addition, Mg-deficient bones featured larger mineral crystals, i.e., crystal overgrowth. This crystalline aberration in Mg-insufficient bones induced collagen fibrils to mineralize easily, even in the absence of mineralized nodules, which therefore led to an early collapse of the fibrils. MK-4 prevented premature collagen mineralization by normalizing the association of collagen fibrils with mineralized nodules. Thus, MK-4 appears to rescue the impaired collagen mineralization caused by Mg insufficiency by promoting a re-association of the process of collagen mineralization with mineralized nodules.

DOI： 10.14670/HH-23.1353

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CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members. and its null mice display skeletal dysmorphisms. However, little is known concerning roles of the other CCN members in chondrocytes. Using both in vivo and in vitro approaches, We conducted a comparative analysis of CCN2-null and wildtype mice to study the roles of CCN2 and the other CCN proteins in cartilage development. Immunohistochemistry was used to evaluate the localization of CCN proteins and other chondrocyte-associated molecules in the two types of mice. Moreover, gene expression levels and the effects of exogenous CCN proteins oil chondrocyte proliferation, differentiation, and the expression of chondrocyte-associated genes in their primary chondrocytes were evaluated. Ccn3 was dramatically upregulated in CCN2-null cartilage and chondrocytes. This upregulation was associated with diminished cell proliferation and delayed differentiation. Consistent with the in vivo findings, CCN2 deletion entirely retarded chondrocyte terminal differentiation and decreased the expression of several chondrocyte-associated genes ill vitro. whereas CcO expression drastically increased. In contrast, the addition Of exogenous CCN2 promoted differentiation strongly and induced the expression of the associated genes. whereas decreasing, the CcO expression. These findings collectively indicate that CCN2 induces chondrocyte differentiation by regulating the expression of chondrocyte-associated genes but that these effects are counteracted by CCN3. The lack of CCN2 caused upregulation of CCN3 in CCN2-null mice, which resulted in the observed phenotypes, Such as the resultant delay of terminal differentiation. The involvement of the PTHrP-Ihh loop in the regulation of CCN3 expression is also suggested.

DOI： 10.1359/JBMR.080615

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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.

DOI： 10.1002/ar.20655

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The klotho gene-deficient mouse is known as an animal model for an accelerated gerontic state, mimicking osteoporosis, skin atrophy, ectopic calcification, and gonadal dysplasia. To elucidate the influence of klotho deficiency on bone mineralization, we examined the ultrastructures of osteoblasts and bone matrices in addition to performing the elemental mapping of calcium, phosphorus, and magnesium in the bone. Under anesthesia, 4- and 5-week-old klotho-deficient mice (klotho(-/-) mice) and their wild-type littermates were perfused with either 4% paraformaldehyde for light microscopic observation or 4% paraformaldehyde and 0.0125% glutaraldehyde for electron microscopic observation. The femurs and tibiae were processed for both observations. Paraffin sections were subject to alkaline phosphatase and tartrate resistant acid phosphatase histochemistry. Semithin and ultrathin sections obtained from epoxy resin-embedded specimens were used for detecting mineralization - according to von Kossa's staining method - and for elemental mapping by electron probe micro-analyzer, respectively. Alkaline phosphatase-positive plump osteoblasts adjacent to the growth plate normally developed cell organelles in the klotho(-/-) metaphyses. This, however, contrasted with the flattened osteoblasts covering the metaphyseal trabeculae and accompanied by small tartrate resistant acid phosphatase-positive osteoclasts. The wild-type mice displayed the mineralized matrix at the zone of hypertrophic chondrocyte of the growth plate and well-mineralized metaphyseal trabeculae parallel to the longitudinal axis of the bone. Alternatively, the klotho(-/-) mice demonstrated a thick mineralized matrix from the proliferative zone of the growth plate as well as the large non-mineralized area in the metaphyseal trabeculae. Consistently, electron probe micro-analysis verified sporadic distributions of higher or lower concentrations of calcium and phosphorus in each trabecule of the klotho(-/-) mice. The distribution of magnesium, however, was almost uniform. Under transmission electron microscopy, osteoblasts on the metaphyseal trabeculae displayed less-developed cell organelles in the klotho(-/-) mice. Thus, the klotho deficiency appears not only to reduce osteoblastic population, but also to disturb bone mineralization.

DOI： 10.1111/j.1469-7580.2008.00859.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and Ccn2 null mutant mice display skeletal dysmorphisms. However, little is known concerning the roles of CCN2 during bone formation. We herein present a comparative analysis of wild-type and Ccn2 null mice to investigate the roles of CCN2 in bone development. Multiple histochemical methods were employed to analyze the effects of CCN2 deletion in vivo, and effects of CCN2 on the osteogenic response were evaluated with the isolated and cultured osteoblasts. As a result, we found a drastic reduction of the osteoblastic phenotype in Ccn2 null mutants. Importantly, addition of exogenous CCN2 promoted every step of osteoblast differentiation and rescued the attenuated activities of the Ccn2 null osteoblasts. These results suggest that CCN2 is required not only for the regulation of cartilage and subsequent events, but also for the normal intramembranous bone development. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.11.155

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The calcitonin-gene related peptide (CGRP) is a primary afferent neurotransmitter in the trigeminal system. Although a neonatal administration of capsaicin eliminates substance P (SP)-mediated nociceptive responses to induce a permanent functional reduction in C-fibers, little information is available regarding changes in CGRP-immunoreaction in mice undergoing neonatal capsaicin treatment (CP mice). This study examined postnatal changes in the distribution of CGRP-immunoreaction in the trigeminal subnucleus caudalis and trigeminal ganglion of CP mice by immunohistochemical technique and a quantitative analysis. Immunohistochemistry for CGRP in the subnucleus caudalis (Vc) demonstrated two dense distributions of neurons in the CP mice as well as naive mice: in the marginal layer and the region 400-600 mu m deep. The quantitative analysis revealed no significant difference in the density of CGRP immunoreaction between naive and CP mice 1-8 weeks of age. In the trigeminal ganglion of both groups, the size distribution of CGRP-positive neurons displayed a distribution pattern with one peak in 200-300 mu m(2) at week 1 and with two peaks in 200-300 mu m(2) and 600-700 mu m(2) at week 8 but no significant difference in neural density existed between these regions. When double staining in the naive mice with CGRP or SP and VR1, a capsaicin receptor, was done, many trigeminal ganglion neurons co-expressed SP- and VR1-immunoreactions, but rarely exhibited CGRP/VR1-co-localization. Taken together with previous data, these current observations suggest that CGRP containing afferent neurons possibly performs differing roles in nociceptive afferent input transmission within the Vc from SP-containing neurons in mice.

DOI： 10.2220/biomedres.29.33

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

This study aimed to evaluate the histological characteristics of the new bone formed at dental implant placement sites concomitantly grafted with a self-setting tricalcium phosphate cement (BIOPEX-R (R)). Standardized defects were created adjacent to the implants in maxillae of 4-week-old male Wistar rats, and were concomitantly filled with BIOPEX-R (R). Osteogenesis was examined in two sites of extreme clinical relevance: (1) the BIOPEX-R (R)-grafted surface corresponding to the previous alveolar ridge (alveolar ridge area), and (2) the interface between the grafting material and implants (interface area). At the alveolar ridge area, many tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts had accumulated on the BIOPEX-R (R) surface and were shown to migrate toward the implant. After that, alkaline phosphatase (ALPase)-positive osteoblasts deposited new bone matrix, demonstrating their coupling with osteoclasts. On the other hand, the interface area showed several osteoclasts initially invading the narrow gap between the implant and graft material. Again, ALPase-positive osteoblasts were shown to couple with osteoclasts, having deposited new bone matrix after bone resorption. Transmission electron microscopic observations revealed direct contact between the implant and the new bone at the interface area, although few thin cells could still be identified. At both the alveolar ridge and the interface areas, newly formed bone resembled compact bone histologically. Also, concentrations of Ca, P, and Mg were much alike with those of the preexistent cortical bone. In summary, when dental implant placement and grafting with BIOPEX-R (R) are done concomitantly, the result is a new bone that resembles compact bone, an ideal achievement in reconstructive procedures for dental implantology.

DOI： 10.1002/jemt.20532

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Klotho-deficient mice exhibit multiple pathological conditions resembling human aging. Our previous study showed alterations in the distribution of osteocytes and in the bone matrix synthesis in klotho-deficient mice. Although the bone and tooth share morphological features such as mineralization processes and components of the extracellular matrix, little information is available on how klotho deletion influences tooth formation. The present study aimed to elucidate the altered histology of incisors of klotho-deficient mice-comparing the findings with those from their wild-type litter-mates, by using immunohistochemistry for alkaline phosphatase (ALP), osteopontin, and dentin matrix protein-1 (DMP-1), terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) detection for apoptosis, and electron probe microanalyzer (EPMA) analysis on calcium (Ca), phosphate (P), and magnesium (Mg). Klotho-deficient incisors exhibited disturbed layers of odontoblasts, predentin, and dentin, resulting in an obscure dentin-predentinal border at the labial region. Several odontoblast-like cells without ALP activity were embedded in the labial dentin matrix, and immunopositivity for DMP-1 and osteopontin was discernible in the matrix surrounding these embedded odontoblast-like cells. TUNEL detection demonstrated an apoptotic reaction in the embedded odontoblast-like cells and pulpal cells in the klotho-deficient mice. EPMA revealed lower concentrations of Ca, P, and Mg in the klotho-deficient dentin, except for the dentin around abnormal odontoblast-like cells. These findings suggest the involvement of the klotho gene in dentinogenesis and its mineralization.

DOI： 10.1002/ar.20630

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

In this study, we present a novel guided bone regeneration (GBR) concept that consists of combining Boneject, a bone substitute containing atelocollagen and bovine hydroxyapatite particles, with thermoplastic, bioresorbable plates (DeltaSystem) known to resist mechanical loading. In rat calvariae, standardized bone defects were filled with Boneject and covered with a convex DeltaSystem plate. Tissue from rats at 1, 2, 4, 8, and 12 weeks postoperation were fixed with an aldehyde solution, and the new bone formed at the defects was histologically assessed. At 1 week, alkaline phosphatase (ALP)-negative cells deriving from the bottom region of the defect could be found up to half the height of the cavity. Boneject particle surfaces in the bottom region revealed an intense osteopontin immunopositivity whereas those in the upper region did not. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts accumulated on the surfaces of osteopontin-coated particles. A newly formed, woven-like bone featuring ALP-positive osteoblasts extended from the native bone. At the second week, the newly formed woven bone had surrounded the Boneject particles. Cement lines, which indicate active bone remodeling, could be observed in the new bone despite its immaturity. Four weeks after surgery, the new bone had reached the height of the DeltaSystem plate, and just beneath it a periostin-positive fibrous layer covered the mix of new bone and Boneject particles. By then, despite having acceptable histological features, electron probe microanalyzer (EPMA) and transmission electron microscope (TEM) analyses revealed that the new bone could not be regarded as compact bone. At 8 and 12 weeks, the new bone showed compact bone-like features according to TEM and EPMA assessments. Summarizing, the combination of a bone substitute such as Boneject and a rigid, bioresorbable plate appears to be osteoconductive and to promote bone remodeling, leading to the genesis of a tissue similar to the one that is regarded as the "gold standard" for bone regeneration: the compact bone.

DOI： 10.1007/s00774-007-0763-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHURCHILL LIVINGSTONE  

Angiogenesis is anticipated during wound healing. Vascular endothelial growth factor (VEGF) is a potent direct angiogenic factor that stimulates endothelial cell migration and proliferation in vitro and angiogenesis in vivo. Ex vivo produced oral mucosa equivalent (EVPOME) grafts have been reported to promote revascularization in the underlying tissue after grafting. The aim of this study was to evaluating the following: VEGF mRNA and its protein expression in EVPOME grafts, the protein levels in conditioned media produced by EVPOMEs, and the ability of VEGF to stimulate the growth of microvascular endothelial cells. VEGF mRNA expression and immunoreaction were found in basal and suprabasal layers. VEGF secreted by EVPOME was detected throughout the period of manufacture of the grafts, and became elevated for the first week at an air-liquid interface. Both EVPOME-conditioned media and a medium containing 10 ng/mL recombinant human VEGF induced endothelial cell proliferation, while neutralization of VEGF by an antibody blocked cell growth. These results suggest that VEGF secreted by EVPOME grafts might assist initial vascularization after grafting, which is critical to subsequent graft survival.

DOI： 10.1016/j.ijom.2007.06.013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

We have examined the morphological changes in chondrocytes after exposure to experimental hypergravity. Tibial epiphyseal cartilages of 17-days-old mouse fetuses were exposed to centrifugation at 3G for 16 It mimicking hypergravitational environment (experimental group), or subjected to stationary cultures (control group). Centrifugation did not affect the sizes of epiphyseal cartilage, chondrocyte proliferation, type X collagen-positive hypertrophic zone, and the mRNA expressions of parathyroid hormone-related peptide and fibroblast growth factor receptor Ill. However, centrifuged chondrocytes showed abnormal morphology and aberrant spatial arrangements, resulting in disrupted chondrocytic columns. Through histochemical assessments, actin filaments were shown to distribute evenly along cell membranes of control proliferative chondrocytes, while chondrocytes subjected to centrifugal force developed a thicker layer of actin filaments. Transmission electron microscopic observations revealed spotty electron-dense materials underlying control chondrocytes' cell membranes, while experimental chondrocytes showed their thick layer. In the intracolumnar regions of the control cartilage, longitudinal electron-dense fibrils were associated with short cytoplasmic processes of normal chondrocytes, indicating assumed cell-to-matrix interactions. These extracellular fibrils were disrupted in the centrifuged samples. Summarizing, altered actin filaments associated with cell membranes, irregular cell shape and disappearance of intracolumnar extracellular fibrils suggest that hypergravity disturbs cell-to-matrix interactions in our cartilage model.

DOI： 10.2220/biomedres.28.191

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

We aimed to histologically elucidate whether bioresorbable plates (DeltaSystem (R)) can induce cortical bone formation, which is essential for long-lasting bone augmentation. Standardized bone defects in rat calvariae were covered with a convexly-shaped DeltaSystem (R) plate, and then processed for histological observations. At 1 week, alkaline phosphatase-positive osteoblasts were seen in the newly-formed bone extending from the cavity's bottom, indicating accelerated osteogenesis. A thick layer of soft connective tissue positive for periostin, a hallmark of periosteum, covered this new bone. At 2 weeks, a spongy bone had filled the cavity up to half its height. The inner layer of the soft tissue facing the spongy bone revealed abundant periostin and osteopontin, and had many tartrate-resistant acid phosphatase-positive osteoclasts. At 4 weeks, this layer had given rise to thin new bony matrices without relation to the spongy bone arising from the cavity. These bone matrices had been thickened by 8 weeks, and turned into a thick cortical bone outlining the regenerated bone at 12 weeks. Thus, our study has provided histological evidences of cortical osteogenesis when DeltaSystem (R) plates are used for bone augmentation procedures.

DOI： 10.2220/biomedres.28.219

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Previous ultrastructural studies have suggested an axon-Schwarm cell interaction in the periodontal Ruffini ending, a primary mechanoreceptor. However, no information is available on the transport mechanism between them. The present study examined the immunolocalization of aquaporin-1 (AQP1) and -4 (AQP4), a member of the water-selective channel, in the periodontal Ruffini endings of the rat incisors and trigeminal ganglion. In addition, the expression of mRNA for AQP1 and 4 was detected in the trigeminal ganglion by a RT-PCR technique. A single PCR product of the sizes anticipated for AQP1 and 4 was detectable in a reverse transcripted cDNA sample from the trigeminal ganglion, whose neurons innervate the periodontal Ruffini endings. An AQP1 immunoreaction was recognizable in the axon terminals of the periodontal Ruffini endings as well as their associated terminal Schwann cells, as confirmed with a double staining with AQP1 and either PGP9.5 or S-100 protein. However, no immunoreaction for AQP4 was found in periodontal Ruffini endings. Although the AQP4 immunoreaction was localized in some satellite cells - but never in neurons - of the trigeminal ganglion, 16.1% trigeminal neurons showed the AQP1 immunoreaction. Furthermore, the AQP1 immunoreaction was found in certain satellite cells which surrounded AQP1-positive or -negative neurons. An analysis of a cross-sectional area of these positive neurons demonstrated that approximately 66.9% of the positive neurons were 400-1000 mu m(2) (671.4 +/- 172.4 mu m(2)), indicating that they could be categorized as medium-sized neurons which mediate mechanotransduction. These findings suggest that AQP1 controls water transport in the periodontal Ruffini endings. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.brainres.2007.04.033

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Language：English   Publisher：(一社)日本骨代謝学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Little is known about the role of neurotrophin-4/5 (NT-4/5) in the regeneration of mechano-receptors. Therefore, the present study examined the regeneration process of Ruffini endings in the periodontal ligament in nt-4/5-deficient and wildtype mice following transection of the inferior alveolar nerve by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker, and by computer-assisted quantitative image analysis. Furthermore, rescue experiments by a continuous administration of recombinant NT-4/5 were performed and analyzed quantitatively. At postoperative day 3 (PO 3d), almost all PGP 9.5-positive neural elements had disappeared; they began to appear in both types of animals at PO 7d. At PO 10d, almost all nerve fibers showed a beaded appearance, with fewer ramifications in both types of mice. Although the regeneration proceeded in the wildtype, a major population of the periodontal Ruffini endings continued to display smooth outlines at PO 28d in the nt-4/5 homozygous mice. The reduction ratio of neural density reached a maximum at PO 3d, decreased at PO 10d, and later showed a plateau. In a rescue experiment, an administration of NT-4/5 showed an acceleration of nerve regeneration in the homozygous mice. These findings indicate that the nt-4/5depletion causes a delay in the regeneration of the periodontal Ruffini endings, but the delay is shortened by an exogenous administration of NT-4/5. Combined with our previous findings of bdnf-deficient mice (Harada et al. [2003] Arch Histol Cytol 66:183-194), these morphological and numerical data suggest that multiple neurotrophins such as NT-4/5 and brain-derived neurotrophic factor (BDNF) play roles in their regeneration in a stage-specific manner.

DOI： 10.1002/cne.21256

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The present study revealed that the fibroblast-like type B synoviocytes (covering the surface of the synovial membrane in the rat temporomandibular joint) had muscle-specific caveolin-3 protein in their caveolae. The existence of two kinds of type B synoviocytes (with and without caveolin-3-immunoreactions even in the synovial lining layer) might reflect the functional difference between them.

DOI： 10.1002/ar.20506

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The present study revealed that the fibroblast-like type B synoviocytes (covering the surface of the synovial membrane in the rat temporomandibular joint) had muscle-specific caveolin-3 protein in their caveolae. The existence of two kinds of type B synoviocytes (with and without caveolin-3-immunoreactions even in the synovial lining layer) might reflect the functional difference between them. © 2007 Wiley-Liss, Inc.

DOI： 10.1002/ar.20506

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Our recent study revealed an intense immunoreaction for GDNF and its receptors in the Ruffini endings, primary mechanoreceptors in the periodontal ligament, of young rats. However, no information is available for the expression of GDNF and its receptors during their development. The present study aimed to reveal postnatal changes in the immuno-expression of GDNF, GFR alpha l and RET in the periodontal Ruffini endings of the rat incisors by double immunofluorescent staining. At postnatal day 3 (PO 3d), no structure with GDNF-, GFR alpha l-, or RET-immunoreaction existed in the periodontal ligament. The PGP 9.5-positive nerve fibers without GDNF- and RET-immunoreaction displayed a dendritic fashion at PO lw, with a GFR alpha l-reaction found around these nerves. At PO 2w, GDNF-positive terminal Schwann cells occurred near the thick and dendritic axons, a part of which showed a RET-reaction, with no reactive cells near the thin nerves. The terminal Schwann cells became positive for GFR alpha l, but lacked RET-immunoreaction. At PO 3w, when the formation of the periodontal Ruffini endings had proceeded, GDNF-positive terminal Schwann cells began to increase in number. This stage-specific immuno-expression pattern suggests that GDNF is a key molecule for the maturation and maintenance of the periodontal Ruffini endings. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2006.11.012

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

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.

DOI： 10.1002/ar.a.20331

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The submandibular gland (SMG) has been regarded as an age-stable organ in spite of reports on its structural changes with aging. Although the klotho gene is involved in aging, little information is available regarding its effects on morphological changes of SMGs. The present study examined the histological and immunohistochemical features of SMGs in klotho-deficient mice - which are well-established aging models - by immunohistochemical and histochemical techniques. Five kinds of cellular markers - against NGF, EGF, Mn- and Cu/Zn-SOD, and RITC-conjugated phalloidin were used for the identification of cell types. In klotho-deficient mice, the SMGs lost their granular ducts and each lobe diminished. The granular duct showed strong immunoreactivities for NGF and EGF in the wild-type mice, but the NGF- and EGF-immunopositive ducts decreased in number remarkably in klotho-deficient mice. Interestingly, instead of a loss of the granular duct, the striated duct located on the distal portion in the homozygous mice came to show NGF- and EGF-immunoreactions. Neither Mn- and Cu/Zn-SOD immunoreactivities in the duct system nor the phalloidin-reaction in the myoepithelial cells differed between the wild-type and klotho-deficient mice. Our findings suggest that the klotho gene inhibited the differentiation of the granular duct from the striated duct due to the repression and/or down-regulation of sexual and growth hormones.

DOI： 10.1679/aohc.69.119

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Our previous studies have revealed the involvement of signaling pathways of BDNF and NT-4/5 via TrkB in the development, regeneration, survival and maintenance of the Ruffini endings, primary mechanoreceptors in the periodontal ligament. However, the involvement of other neurotrophins remains unclear. The present study examined the expression of GDNF, GFR alpha 1, and RET in the incisor periodontal ligament and trigeminal ganglion of young rats by RT-PCR and immunocytochemistry. All these mRNAs were detected in both tissues by RT-PCR. These immunoreactions were found in the terminal Schwann cells associated with the periodontal Ruffini endings, as confirmed by histochemistry for non-specific cholinesterase activity. Their axonal branches showed GF alpha 1- and RET-immunoreactions but lacked GDNF-immunoreactivity. In the trigeminal ganglion. about 30% of the neurons were immunoreactive to GFR alpha 1 and RET. Averages of cross-sectional areas of their positive neurons demonstrated that they could mainly be categorized as medium-sized neurons. GDNF-immunoreaction was restricted to the satellite cells and not in trigeminal ganglion neurons. These findings indicate that GDNF mediates trophic effects on the survival and target innervation of the periodontal Ruffini endings via GFR alpha 1 and RET. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2006.02.015

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Language：Japanese   Publisher：(一社)日本顎関節学会  

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The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices.

DOI： 10.1002/jemt.20272

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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. © 2005 Wiley-Liss, Inc.

DOI： 10.1002/ar.a.20275

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

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. (C) 2005 Wiley-Liss, Inc.

DOI： 10.1002/ar.a.20275

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

Neurotrophin-4/5 (NT-4/5)-a member of the neurotrophic factors-is a ligand for TrkB, which has been reported to be expressed in the mechanoreceptive Ruffini endings of the periodontal ligament. The present study examined developmental changes in the terminal morphology and neural density in homozygous mice with a targeted disruption of the nt-4/5 gene and wild-type mice by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker, and by quantitative analysis using an image analyzer. Postnatal development of terminal Schwann cells was also investigated by enzymatic histochemistry for non-specific cholinesterase activity (ChE). Furthermore, the immuno-expression of TrkB and low affinity nerve growth factor receptor (p75-NGFR) was surveyed in the periodontal Ruffini endings as well as trigeminal ganglion. At postnatal 1 week, the lingual periodontal ligament of both types of mice contained PGP 9.5-positive nerve fibers showing a tree-like ramification with axonal swellings in their course. In both types of mice at 2 weeks of age, comparatively thick nerve fibers with a smooth outline increased in number, and frequently ramified to form nerve terminals with dendritic profiles. However, no typical Ruffini endings with irregular outlines observed in the adult wild-type mice were found in the periodontal ligament at this stage. At postnatal 3 weeks, typical Ruffini endings with irregular outlines were discernable in the periodontal ligament of the wild-type mice while the dendritic endings showing smooth outlines were restricted to the homozygous mice. After postnatal 8 weeks, both types of mice showed an increase in the number of Ruffini endings, but the morphology differed between the wildtype and NT-4/5 homozygous mice. In the wild-type mice, a major population of the Ruffini endings expanded their axonal branches and developed many microprojections, resulting in a reduction of endings with smooth outlines. In contrast, we failed to find such typical Ruffini endings in the periodontal ligament of the homozygous mice: A majority of the periodontal Ruffini endings continued to show smooth outlines at postnatal 12 weeks. Quantitative analysis on neural density demonstrated a reduction in the homozygous mice with a significant difference by postnatal 8 weeks. Enzymatic histochemistry for non-specific ChE did not exhibit a distinct difference in the distribution and density of terminal Schwann cells between wild-type and homozygous mice. Furthermore, TrkB and p75-NGFR mRNA and proteins did not differ in the trigeminal ganglion between the two types. The periodontal Ruffini endings also displayed immunoreactivities for TrkB and p75-NGFR in both phenotypes. These findings suggest that the nt-4/5 gene depletion caused a delay in the formation and maturation of the periodontal Ruffini endings in the mice by inhibiting the growth of the periodontal nerves at an early stage, and indicate that multiple neurotrophins such as NT-4/5 and BDNF might play roles in the development and/or maturation of the periodontal Ruffini endings.

DOI： 10.1679/aohc.68.267

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.

DOI： 10.1679/aohc.68.371

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The trigeminal subnucleus caudalis (Vc) is a critical relay site for processing nociceptive afferent input from the orofacial area in addition to its modulation by neuroplastic change. Although an administration of capsaicin in neonates induces a selective destruction of substance P (SP)-immunoreactive nerve fibers, little information is available regarding its detailed effects on the Vc, particularly during postnatal development. The present study examined postnatal changes in the distribution of SP in the Vc and trigeminal ganglion (TG) by immunohistochemical techniques in naive (NV) and neonatally capsaicin-treated (CP) mice, combined with a quantitative analysis. The neonatal mice received a single subcutaneous injection of capsaicin (50 mg/kg) at 48 hours after birth. The neural density of the SP-immunoreaction decreased to approximately a quarter of that in 1-week-old NV mice but increased to three-quarters of that in the NV in the superficial area after postnatal week 2. A double staining with SP and myelin basic protein confirmed the absence of any SP-immunoreaction in the myelinated nerve fibers in both NV and CP mice. The SP-immunoreaction never overlapped with non-peptidergic IB4-labeled neurons in the Vc and TG of either group. Neither the size distribution of SP-positive neurons nor their relative ratio in the TG differed between NV and CP mice at the ages of postnatal weeks 1 and 8. These findings indicate two putative origins for the emergent SP-immunoreaction in the superficial layer of the Vc of the CP mice: the surviving trigeminal neurons with SP against capsaicin treatment and/or intrinsic neurons/interneurons in the Vc without SP under normal conditions.

DOI： 10.1679/aohc.68.311

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DOI： 10.1016/j.devbrainres.2004.12.005

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This study was designed to evaluate the histological changes during ossification and cellular events including osteogenic differentiation responding to collagenous bioresorbable membranes utilized for GBR. Standardized artificial bony defects were prepared at rat maxillae, and covered with a collagenous bioresorbable membrane. These animals were sacrificed at 1, 2, 3 and 4 weeks after the GBR-operation. The paraffin sections were subject to tartrate resistant acid phosphatase (TRAP) enzyme histochemistry and immunohistochemistry for alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC). In the first week of the experimental group, woven bone with ALP-positive osteoblasts occupied the lower half of the cavity. The collagenous membrane included numerous ALP-negative cells and OP-immunoreactive extracellular matrices. At 2 weeks, the ALP-, OP- and OC-immunoreactivity came to be recognizable in the region of collagenous membrane. Since ALP-negative soft tissue separated the collagenous membrane and the new bone originating from the cavity bottom, the collagenous membrane appeared to induce osteogenesis in situ. At 3 weeks, numerous collagen fibers of the membrane were embedded in the adjacent bone matrix. At 4 weeks, the membrane-associated and the cavity-derived bones had completely integrated, showing the same height of the periosteal ridge as the surrounding alveolar bones. The collagen fibers of a GBR-membrane appear to participate in osteogenic differentiation. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biomaterials.2005.03.023

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Previous developmental studies on the temporomandibular joint (TMJ) have proposed several hypotheses on the formation of its articular cavity. However, detailed information is meager. The present study examined the formation process of the articular cavity in the rat TMJ by immunocytochemistry for CD31, RECA-1, and ED1, which are useful cellular markers for endothelial cells and monocyte/macrophage lineages, respectively. The upper articular cavity formation had begun by embryonic day 21 (E21) and was completed at postnatal day 1 (P1) in advance of the lower cavitation
the latter took place from P1 to P3. The occurrence and distribution pattern of the CD31-, RECA-1-, and ED1-positive cells differed between the upper and lower articular cavity-forming areas: the ED1-positive cells exclusively occurred in the area of the prospective upper articular cavity prior to its formation, while no ED1-positive cell appeared in the lower cavity-forming area. In contrast, the CD31- and RECA-1-positive endothelial cells were restricted to the lower cavity-forming area (never the prospective upper cavity) at E19 and diminished thereafter. Throughout the cavity formation, we failed to find any apoptotic cells in the cavity formation area, indicating no involvement of apoptosis in the cavity formation in TMJ. The present findings on the behaviors of endothelial cells and ED1-positive cells show a possibility of different mechanism in the cavity formation between the upper and lower articular cavities in the rat TMJ. The appearance of ED1-reactive cells and temporal vascularization may play crucial roles in the upper and lower articular cavity formation, respectively. © 2005 Wiley-Liss, Inc.

DOI： 10.1002/ar.a.20228

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Previous developmental studies on the temporomandibular joint (TMJ) have proposed several hypotheses on the formation of its articular cavity. However, detailed information is meager. The present study examined the formation process of the articular cavity in the rat TMJ by immunocytochemistry for CD31, RECA-1, and ED1, which are useful cellular markers for endothelial cells and monocyte/macrophage lineages, respectively. The upper articular cavity formation had begun by embryonic day 21 (E21) and was completed at postnatal day 1 (P1) in advance of the lower cavitation; the latter took place from P1 to P3. The occurrence and distribution pattern of the CD31-, RECA-1-, and ED1-positive cells differed between the upper and lower articular cavity-forming areas: the ED1-positive cells exclusively occurred in the area of the prospective upper articular cavity prior to its formation, while no ED1-positive cell appeared in the lower cavity-forming area. In contrast, the CD31- and RECA-1-positive endothelial cells were restricted to the lower cavity-forming area (never the prospective upper cavity) at E19 and diminished thereafter. Throughout the cavity formation, we failed to find any apoptotic cells in the cavity formation area, indicating no involvement of apoptosis in the cavity formation in TMJ. The present findings on the behaviors of endothelial cells and ED1-positive cells show a possibility of different mechanism in the cavity formation between the upper and lower articular cavities in the rat TMJ. The appearance of ED1-reactive cells and temporal vascularization may play crucial roles in the upper and lower articular cavity formation, respectively. (C) 2005 Wiley-Liss, Inc.

DOI： 10.1002/ar.a.20228

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：(一社)歯科基礎医学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The hypertrophic chondrocytes lack the ability to proliferate, thus permitting matrix mineralization as well as vascular invasion from the bone in both the mandibular condyle and the epiphyseal cartilage. This study attempted to verify whether the histological appearance of the hypertrophic chondrocytes is in a steady state during postnatal development of the mouse mandibular condyle. Type X collagen immunohistochemistry apparently distinguished the fibrous layer described previously as the "articular zone," "articular layer," and "resting zone" from the hypertrophic zone. Interestingly, the ratio of the type X collagen-positive hypertrophic zone in the entire condyle seemed higher in the early stages but decreased in the later stages. Some apparently compacted cells in the hypertrophic zone showed proliferating cell nuclear antigen (PCNA) immunoreaction, indicating the potential for cell proliferation at the early stages. As the mice matured, in contrast, they further enlarged and assumed typical features of hypertrophic chondrocytes. Apoptotic cells were also discernible in the hypertrophic zone at the early but not later stages. Consistent with morphological configurations of hypertrophic chondrocytes, immunoreactions for alkaline phosphatase, osteopontin, and type I collagen were prominent at the later stage, but not the early stage. Cartilaginous matrices demonstrated scattered patches of mineralization at the early stage, but increased in their volume and connectivity at the later stage. Thus, the spatial and temporal occurrence of these immunoreactions as well as apoptosis likely reflect the prematurity of hypertrophying cells at the early stage, and imply a physiological relevance during the early development of the mandibular condyles.

DOI： 10.1002/jemt.20211

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Magnesium (Mg) is most likely restored in bone matrix, implicating a pivotal role in bone mineralization. Mg-insufficient bone reveals fragility to mechanical loading despite normal or higher levels of bone mineral content, permitting stimulated osteoclastic bone resorption. In contrast, vitamin K(2) (MK-4:menatetrenone) inhibited osteoclastic bone resorption stimulated by the Mg-insufficiency, thereby normalizing bone remodeling. The Mg-insufficiency caused an increased concentration of calcium, which resulted in an extremely-high purity of hydroxyapatite (HA) crystal [Ca(10)(PO(4))(6)(OH)(2)] and accelerated mineralization in bone. In contrast, MK-4 did not affect the calcium-concentration nor HA-purity, but repressed mineralization accelerated by Mg-insufficiency. Thus, MK-4 appears to recover the "bone quality" lessened by the Mg-insufficiency by two mechanisms:controlling bone turnover and mineralization.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

The postnatal development of nociceptive afferent activity expansion and its modulation features were examined in mice using an optical imaging technique. Developing mice (1-2 weeks old (N 1-2w), 3-4 weeks old (N3-4w), 5-6 weeks old (N5-6w) and 7-8 weeks old (N7-8w)) and neonatally capsaicin-treated mice were used. The propagation of neuronal excitation was measured by changes in fluorescent intensity in horizontal brain stem slices evoked by electrical stimulation to the trigeminal spinal tract. A single-pulse stimulation evoked excitation propagation in the trigeminal caudalis (Vc). The propagation area was larger in N1-2w than in N7-8w, and no differences were observed between capsaicin-treated and naive mice in the same age groups. Repetitive stimulation (100 Hz, 30 pulses) elicited long-lasting and widespread excitation propagation. The excitation propagation area was significantly larger in N7-8w than in N1-2w, N3-4w and N5-6w. This propagation was suppressed by 5 mu M L-703.606, an NK1-receptor antagonist, suggesting that the repetitive stimulation-elicited excitation may require substance-P releases. Morphological observations demonstrated that the neural network in the Vc had grown by postnatal week 5. These results suggest that nociceptive afferent activity co-operatively matures with development of the network structure in the Vc, and that a mechanism for prolonged increase in central excitability is established during a later postnatal period. (c) 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2005.03.005

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

骨基質の石灰化ミネラルにはさまざまな元素が存在するが,特に体内のマグネシウムは半分以上が骨基質に存在しており,カルシウム,リンとともに重要な骨基質ミネラルを構成している.低マグネシウム環境では,破骨細胞の吸収活性が上昇するとともに骨芽細胞も活性化され,骨代謝回転が亢進する.一方,ビタミンK2(MK-4:menatetrenone)は破骨細胞の活性を抑制するために骨代謝回転の過剰な亢進を是正する.一方で,低マグネシウム環境では,骨基質内のカルシウム濃度が上昇するために,純度の高いハイドロキシアパタイト結晶を形成し,石灰化の過剰成長までも誘導してしまう.ビタミンK2はハイドロキシアパタイト結晶の純度には影響を与えないが,石灰化の過剰成長を抑制することが明らかとなった(著者抄録)

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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 B-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. © 2005 Wiley-Liss, Inc.

DOI： 10.1002/ar.a.20191

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Estrogen has a diverse function, including cell proliferation and differentiation via estrogen receptors (ER), which have been reported to be the case in various tissues in addition to female reproductive organs. A recent immunocytochemical study has reported the expression of ER&alpha;, a subtype of ER, in rat odontoblasts, suggesting an involvement of estrogen in the differentiation of tooth-forming cells. However, there is no information on the ER&alpha; immunoexpression in ameloblasts. The present study was therefore undertaken to examine the localization of ERa immunoreaction in rat ameloblasts during amelogenesis. A computer-assisted quantitative analysis under a confocal laser scanning microscope was employed to demonstrate the stage-specific localization pattern of ERa immunoreaction. Immunohistochemistry of the rat enamel organ revealed ERa expression as nuclear localization in ameloblasts, stratum intermedium, stellate reticulum, and papillary layer, in addition to mature and immature odontoblasts. The ratio of immunopositive nuclei to total nuclei (immunopositive ratio) in ameloblasts was high at the apical loop region and gradually declined at the presecretory stage to zero at the secretory stage with statistically significant difference. The ER&alpha; immunolabeling pattern exhibited a periodic change at the maturation stage proper with constant higher labeling in ruffle-ended ameloblasts than in smooth-ended ameloblasts. The positive ratio was then followed by a statistically significant increase in immunolabeling thereafter. This stage-specific immunolabeling pattern during amelogenesis suggests a possible role of ER&alpha; in ameloblast proliferation and differentiation. &COPY; 2005 Wiley-Liss, Inc.

DOI： 10.1002/ar.a.20190

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Language：English   Publishing type：Research paper (scientific journal)  

Estrogen has a diverse function, including cell proliferation and differentiation via estrogen receptors (ER), which have been reported to be the case in various tissues in addition to female reproductive organs. A recent immunocytochemical study has reported the expression of ERα, a subtype of ER, in rat odontoblasts, suggesting an involvement of estrogen in the differentiation of tooth-forming cells. However, there is no information on the ERα immunoexpression in ameloblasts. The present study was therefore undertaken to examine the localization of ERα immunoreaction in rat ameloblasts during amelogenesis. A computer-assisted quantitative analysis under a confocal laser scanning microscope was employed to demonstrate the stage-specific localization pattern of ERα immunoreaction. Immunohistochemistry of the rat enamel organ revealed ERα expression as nuclear localization in ameloblasts, stratum intermedium, stellate reticulum, and papillary layer, in addition to mature and immature odontoblasts. The ratio of immunopositive nuclei to total nuclei (immunopositive ratio) in ameloblasts was high at the apical loop region and gradually declined at the presecretory stage to zero at the secretory stage with statistically significant difference. The ERα immunolabeling pattern exhibited a periodic change at the maturation stage proper with constant higher labeling in ruffle-ended ameloblasts than in smooth-ended ameloblasts. The positive ratio was then followed by a statistically significant increase in immunolabeling thereafter. This stage-specific immunolabeling pattern during amelogenesis suggests a possible role of ERα in ameloblast proliferation and differentiation. © 2005 Wiley-Liss, Inc.

DOI： 10.1002/ar.a.20190

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

One series of our research has shown an intense expression of immuno-reaction 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. &COPY; 2005 Wiley-Liss, Inc.

DOI： 10.1002/ar.a.20191

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：日本骨形態計測学会  

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Language：Japanese   Publisher：日本骨形態計測学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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Language：Japanese   Publisher：(公社)日本補綴歯科学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Nociceptive afferent signals from the orofacial area are transmitted to the trigeminal subnucleus caudalis (Vc) through the release of glutamate and/or substance P (SP). Although nociceptive transmission and/or modulating mechanisms are known to develop during the postnatal period, the specific developmental changes in nociception and/or modulation remain unclear. The present study examined postnatal changes in the spatial relationship between SP and its receptor, the NK1 receptor (NK1R), in the mouse Vc by immunohistochemistry and quantitative analysis. The medulla was removed from C57BL/6N mice (1, 2, 4, and 8 weeks of age) after perfusion and fixation, and cut horizontally at a thickness of 40 mu m. The relative densities of SP- and NK1R-immunoreactive areas and their changes with age were assessed statistically. One- and 2-week-old mice showed relatively high densities of SP-positive structures in the marginal layer (Mar) and the deep part of the magnocellular layer (Mag). The SP distribution in the superficial Vc remained unchanged, but the density in the deep Mag gradually decreased with age, resulting in a complete loss after postnatal week 4. The NK1R-irnmunoreactivity exhibited a similar distribution pattern to that of SP, but the pattern remained unchanged during the postnatal period.
Double-iinrnunofluorescence staining for SP and NK1 R demonstrated only moderate direct contact of SP-positive structures with NK1R in the superficial area. These separate distributions and the postnatal changes in SP and NK1R suggest the possibility of another nociceptive afferent transmission mechanists, that is, volume transmission, in the Vc other than synapse-mediated transmission. (c) 2005 Elsevier B.V All rights reserved.

DOI： 10.1016/j.devbrainres.2004.12.005

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Language：Japanese   Publisher：(一社)日本解剖学会  

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Language：Japanese   Publisher：(一社)日本解剖学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

DOI： 10.1007/BF03026322

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Language：English   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Osteopetrotic (op/op) mice fail to exhibit bone remodeling because of a defective osteoclast formation due to a lack of macrophage colony-stimulating factor. In this study, we investigated the femora of op/op mice to clarify whether the osteoblastic population and bone mineralization are involved in osteoclasts or their bone resorption. The op/op mice extended the meshwork of trabecular bones from the chondro-osseous junction to the diaphyseal region. In the femoral metaphyses of op/op mice, intense alkaline phosphatase (ALPase)-positive osteoblasts were observed on the metaphyseal bone in close proximity to the erosion zone of the growth plates. Von Kossa's staining revealed scattered mineralized nodules and a fine meshwork of mineralized bone matrices while the wild-type littermates developed well-mineralized trabeculae parallel to the longitudinal axis. In contrast to the metaphysis, some op/op diaphyses showed flattened osteoblasts with weak ALPase-positivity, and the other diaphyses displayed bone surfaces without a covering by osteoblasts. It is likely, therefore, that the osteoblastic population and activity were lessened in the op/op diaphyses. Despite the osteopetrotic model, von Kossa's staining demonstrated patchy unmineralized areas in the op/op diaphyses, indicating that a lower population and/or the activity of osteoblasts resulted in defective mineralization in the bone. Transmission electron microscopy disclosed few osteoblasts on the diaphyseal bones, and instead, bone marrow cells and vascular endothelial cells were often attached to the unmineralized bone. Osteocytes were embedded in the unmineralized bone matrix. Thus, osteoclasts appear to be involved in the osteoblastic population and activity as well as subsequent bone mineralization. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.micron.2005.06.008

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Language：English   Publisher：Japanese Association for Oral Biology  

Human enamel tufts appeared as corrugated ribbon-like structures located on the dentin parallel to the tooth axis when observed under the binocular microscope and scanning electron microscope (SEM). SEM observation disclosed enamel tufts as bundles of well-extended tubular structures attributable to hypomineralized enamel sheaths. Plate-like structures, previously referred to as "tuft-root" ran in the center of the enamel tufts, connecting the dentin surface. When observing under the transmission electron microscope, the plates of tufts extended from the superficial layer of the dentin, penetrating the hypermineralized zone adjacent to the dentin-enamel (D-E) junction, and then, reaching the tuft region. In the tuft region, the plates of tufts ran mainly along the enamel sheaths and partially across the enamel prisms. The immuno-gold technique verified an intense immunoreactivity for amelogenin in the superficial layer of the dentin as well as the enamel prisms in the tufts, although no reaction was found over the "plates of tufts". The immunoreactivity for 13-17 kd sheath proteins, also denoted as sheathlin, ameloblastin or amelin, was detected over the filamentous structures closely associated with the enamel sheaths in the enamel tuft. Thus, our study demonstrated that enamel tufts consist of both well extended hypomineralized enamel prisms and "plates of tufts". The major organic substance of the enamel tufts is suggested to be 13-17 kd sheath proteins rather than amelogenin.

DOI： 10.2330/joralbiosci.47.33

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Language：Japanese  

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Previous reports have shown expression of immunoreactivity for periostin, originally identified as osteoblast-specific factor-2, in the periosteum and periodontal ligament. However, the developmental changes in its expression and the detailed immunolocalization have remained veiled. The present study was undertaken to examine the spatiotemporal expression of this protein in teeth and their associated tissues of mice during development at light and electron microscopic levels. In tooth germs at cap stage, periostin immunoreactivity was recognizable in the interface between inner enamel epithelium and preodontoblasts as well as in the mesenchymal tissues around cervical loop. Dental follicles around tooth germs at bell stage localized periostin immunopositivity in addition to the immunopositive areas observed in cap-staged tooth germs, although the functional significance of periostin has remained unclear in tooth development. Furthermore, periostin immunoreactivity was also found in the alveolar bone surface. In the incisors of both 7- and 21-day-old mice, immunoreaction for periostin was discernible in the lingual periodontal ligament and labial fibrous tissue adjacent to the papillary layer. After postnatal day 7, immunoreaction for periostin came to be restricted to the fibrous bundles in the periodontal ligament in accordance with the organization of the periodontal fibers, indicating its localization matched the morphogenesis of the periodontal ligament. Immunoelectron microscopic observation of the mature periodontal ligament verified the localization of periostin between the cytoplasmic processes of periodontal fibroblasts and cementoblasts and the adjacent collagen fibrils. Our findings suggest that periostin is involved at the sites of the cell-to-matrix interaction, serving as adhesive equipment for bearing mechanical forces, including occlusal force and tooth eruption.

DOI： 10.1002/ar.a.20080

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Sensory mechanisms in teeth are not well understood and may involve pulpal-neural interactions. Tooth cells that proliferate in vitro have polyclonal immunoreactivity (IR) for glial fibrillary acidic protein, (GFAP), growth-associated protein (GAP-43), And vimentin, plus glial-like ion channels. Here, we analyzed GFAP-IR patterns in dental and trigeminal tissues of rats, for comparison With green fluorescent protein (GFP) associated with GFAP transcription in transgenic mice, in order to better characterize glial-like cells in dental tissues. Astrocytes, ganglion satellite cells, And epineurial Schwann cells were demonstrated by anti-GFAP antibodies and GFP-GFAP, as expected. Odontoblasts did not, stain by any of these methods and cannot be the glial-like cells. Fibroblasts and undifferentiated mesenchymal cells in pulp had polyclonal GFAP-IR and vimentin-IR, while nerve fibers reacted only with polyclonal antibody. Some Schwann cell subtypes in trigeminal nerve and Oral mucosa were positive for GFP and for polyclonal anti-GFAP, but not for monoclonal antibody. In pulp. almost all Schwann cells were unstained, but many Schwann cells in periodontal ligament had polyclonal GFAP-IR. These results show greater heterogeneity for Schwann cells than expected, and suggest that the glial-like pulp cells are fibroblasts and/or undifferentiated mesenchymal cells or stem cells. We also found that polyclonal GFAP revealed intermediate filaments in preterminal sensory nerve fibers, thereby providing a useful marker for that neural subregion. GFP transcription by some Schwann cell subtypes in oral mucosae and trigeminal nerve, but not trigeminal root Was a novel finding that reveals more complexity in peripheral glia than previously recognized. (C) 2005 Wiley-Liss, Inc.

DOI： 10.1002/jemt.20130

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Language：Japanese   Publishing type：Research paper (bulletin of university, research institution)  

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Other Link： http://hdl.handle.net/10191/23317

Language：English   Publisher：(一社)歯科基礎医学会  

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Language：Japanese   Publisher：(一社)日本骨代謝学会  

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DOI： 10.1002/ar.a.20043

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

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. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/ar.a.20043

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DOI： 10.1016/j.brainres.2004.04.061

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

This study was designed to investigate the responses of bone cells to a deproteinized bovine bone material, Bio-Oss(R) (Geistlich-Pharma, Wolhunsen, Switzerland), which was grafted in artificial bone defects of rat femurs. Standardized bone defects in the cortical bone of the right femurs were grafted with Bio-Oss(R) particles. Narrow penetrations were prepared on the bottom of the cavity, enabling osteogenic cells to migrate from the bone marrow. A defect in the left femur without Bio-Oss(R) was used as a control. The treated femurs were histochemically examined at 1, 3, 5, 7, and 14 days after the operation. At day 1, no osteogenic migration into the cavities occurred in either the control or experimental groups. At day 3, alkaline phosphatase (ALPase) immunohistochemistry showed a migration of the positive cells at the bottom of the cavities of the experimental groups, but not in the control ones. At day 5, new bone formation was recognized at the bottom of the cavity of both groups. In the experimental group, ALPase-positive cells were localized on Bio-Oss(R) and/or on the thin bone matrix that covered this material. The superficial layer of Bio-Oss(R) underlying the newly formed bone exhibited osteocalcin immunoreactivity. Transmission electron microscopy revealed osteoblasts depositing bone matrices - including collagen fibers - on the surface of Bio-Oss(R). At days 7 and 14, woven bone occupied the previous cavities of both control and experimental groups, accompanied by osteoclasts. Thus, Bio-Oss(R) appears to serve as a scaffold for osteogenic cells as well as to promote osteoblastic differentiation and matrix synthesis.

DOI： 10.1111/j.1600-0501.2004.01012.x

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Language：English   Publisher：Japanese Association for Oral Biology  

Mice with a targeted deletion of parathyroid hormone (PTH)-related peptide (PTHrP) develop a form of dyschondroplasia resulting from diminished proliferation and precocious maturation of chondrocytes. The hypertrophic zone of the mutant epiphyseal cartilage revealed aberrant heterogeneous populations of chondrocytes, i.e., non-hypertrophic cells at different stages of differentiation. Therefore, PTHrP appears to play a central role in modulating cell proliferation and differentiation. The biological function of PTHrP is mediated by signaling pathway linked to the PTH/PTHrP receptor. However, the amino acids at 87-107 positions of PTHrP show a putative nucleolar targeting signal, and thought to involve in the resistance to apoptosis. Chondrocytic cell line, CFK2, transfected with truncated forms of PTHrP cDNA showed this peptide in the nucleoli mediated by translation initiating from AUG-codon and alternatively initiating from CUG codons. Thereby, PTHrP appears to act as a bipartite modulator of chondrocyte proliferation/differentiation, both through signal transduction linked to the PTH/PTHrP receptor and by its direct action in the nucleolus. Meanwhile, PTH/PTHrP receptor expression is controlled by two promoters in mouse, and the downstream promoter acts predominantly in bone and cartilage. We found 1,25-dihydroxyvitamin D3 [1,25 (OH)₂D₃] downregulated the activity of the downstream promoter in osteoblasts, but not in chondrocytes. This indicates that the interplay between PTH and 1,25 (OH)₂D₃ is specific to their overlapping roles of serum calcium regulation in bone, and not to their complementary effects on proliferation/differentiation of chondrocytes. Thus, we will review our recent examinations on cartilage development in conjunction with the biological role in PTHrP, PTH/PTHrP receptor and 1,25 (OH)₂D₃.

DOI： 10.2330/joralbiosci.46.79

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DOI： 10.1007/s00441-003-0840-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

This study aimed to elucidate the biological effects of a self-setting tricalcium phosphate bone substitute (BIOPEX(R)) applied in rat femoral cortical bone cavities. Narrow penetrations through the cavity and bone marrow were prepared to obtain cellular sources. In the experimental group at day 1, a thin cell layer intruded into a narrow space between the grafted BIOPEX(R) and the bottom of the cavity. From days 5 to 10, a range of tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts accumulated on the surface of the BIOPEX(R) facing the bottom of the cavity, whilst many alkaline phosphatase (ALPase)-positive osteoblasts were localized on the bone surface opposing the BIOPEX(R). However, at day 20, osteoblasts were localized neighboring the osteoclasts on the BIOPEX(R), and deposited bone matrices onto this material, implying a coupling between osteoclasts and osteoblasts. At days 30 and 40 post-operation, small remnants of BIOPEX(R) particles were present in the new bone with a profile of compact bone. Thus, BIOPEX(R) is resorbed by osteoclasts, and succeeded by osteoblastic bone apposition with a coupling of osteoclasts and osteoblasts at the later stage. In conclusion, the use of BIOPEX(R) provides adequate bone regeneration with the profile of compact bone. (C) 2003 Elsevier Ltd. All rights reserved.

DOI： 10.1016/S0142-9612(03)00550-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

Previous studies have confirmed the presence of corticosteroid receptors including mineralocorticoid (MCR) and glucocorticoid receptors (GCR) in the glia cells of both central and peripheral nervous systems. However, no report has been offered as yet on the detailed localization of these receptors in the specialized Schwann cells associated with mechanoreceptors. Thus, the present study examined the immunolocalization of MCR and GCR in the specialized Schwann cells (terminal and lamellar Schwann cells) associated with the mechanoreceptors in rat periodontal ligament and palatal mucosa by the use of immunohistochemical techniques and confocal laser scanning microscopy. It further attempted to detect the localization of 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD II), a key enzyme for signal transduction via MCR. Immunostaining with antisera against GCR and MCR demonstrated intense immunoreactions in the terminal or lamellar Schwann cells but not nerve fibers associated with the periodontal and palatal mechanoreceptors. A double staining with propidium iodide and either GCR- or MCR-antiserum revealed the intranuclear and cytoplasmic localization of these receptors in the terminal Schwann cells. 11 beta-HSD II-immunoreactivity was found in the cytoplasm and on the nuclear envelope of the terminal Schwann cells, indicating the co-localization of MCR and 11 beta-HSD II in them. These findings suggest that the terminal or lamellar Schwann cells are target tissues for corticosteroid hormones. Taken together with previous reports on the functional significance of the corticosteroids, the results indicate that glucocorticoids and mineralocorticoids might be respectively involved in the proliferation and/or differentiation and Na+/K+-homeostasis in the terminal or lamellar Schwann cells associated with the mechanoreceptors.

DOI： 10.2220/biomedres.25.35

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Bone development is regulated by conserved signalling pathways that are linked to multifunctional growth factors and their high affinity receptors. Parathyroid hormone-related peptide (PTHrP) and fibroblast growth factor receptor 3 (FGFR3) have been shown to play pivotal, and sometimes complementary, roles in the replication, maturation and death of chondrocytes during endochondral bone formation. To gain further insight into how these pathways coordinate cartilage and bone development, we generated mice lacking expression of both PTHrP and FGFR3. The phenotype of compound mutant mice resembled that of their PTHrP-deficient littermates with respect to neonatal lethality, facial dysmorphism and foreshortening of the limbs. The absence of PTHrP in the developing epiphyseal cartilage of PTHrP(- / -) and PTHrP(- / -) /FGFR3(- / -) mice resulted in a dominant hypo-proliferative phenotype. However, abnormalities such as the presence of nonhypertrophic cells among hypertrophic chondrocytes and excessive apoptosis seen in the hypertrophic zone of PTHrP(-) (/) (-) mice were absent in the PTHrP(- / -) /FGFR3(-) (/) (-) mice. Furthermore, the absence of FGFR3 in single and compound mutant mice led to decreased expression of vascular endothelial growth factor (VEGF) and an increase in depth of hypertrophic chondrocytes. These observations indicate that FGFR3 deficiency can rescue some of the defects seen in PTHrP-deficient mice and that it plays an important role in the regulation of chondrocyte differentiation and hypertrophy. These studies support a dominant role for PTHrP in regulating the pool of proliferating cells during limb development and suggest that signalling by FGFR3 plays a more prominent role in cartilage maturation and vascular invasion at the chondro-osseous junction. (C) 2003 Elsevier Inc. All rights reserved.

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

We present a unique case of malignant fibrous histiocytoma (MFH) developing around a masseter muscle close to the surgical margin from which a benign fibrous histiocytoma (BFH) of the mandible was excised 3 years and 10 months earlier. In the interval between the surgical removal of the original BFH and the MFH, several surgical procedures were performed in the related region. Clinical and histopathological evidence showed that this case represented two separate lesions rather than a single disease process. The clinical history suggests that the chronic repair process might have contributed to the tumorigenic factor of a MFH. © 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ooe.2003.12.001

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Language：English   Publisher：Japanese Association for Oral Biology  

The synovial lining layer in the temporomandibular joint (TMJ) consists of macrophage-like type A cells and fibroblast-like type B cells. Their identification, however, has been difficult because of the lack of a specific cell marker. This review summarizes the characteristic configuration and development of the type B cell in the murine TMJ. Immunocytochemistry for 25kDa-heat shock protein (Hsp25) revealed two profiles of the fibroblast-like type B cells with cytoplasmic processes in the adult rat TMJ : one projected horizontally slender processes which covered the synovial membrane, and the other extended a thick, long process towards the articular cavity. The former appeared earlier than the latter during the development of synovial membrane, in close relation with the formation of the articular cavity and the commencement of active jaw movement. Since immature type B cells with Hsp25-immunoreactivity were found in the mesenchymal tissue which corresponded to the future articular cavity, type B cells may differentiate directly from mesenchymal cells.

DOI： 10.2330/joralbiosci.46.519

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

Tissue responses to titanium implantation with two different surface conditions in our established implantation model in rat maxillae were investigated by light and transmission electron microscopy and by histochemistry for tartrate-resistant acid phosphatase (TRAPase) activity. Here we used two types of implants with different surface qualities: titanium implants sandblasted with Al2O3 (SA-group) and implants coated with hydroxyapatite (HA-group). In both groups, bone formation had begun by 5 days postimplantation when the inflammatory reaction had almost disappeared in the prepared bone cavity. In the SA-group, however, the bone formation process in the bone cavity was almost identical to that shown in our previous report using smooth surfaced implants (Futami et al. 2000): new bone formation, which occurred from the pre-existing bone toward the implant, was preceded by active bone resorption in the lateral area with a narrow gap, but not so in the base area with a wide gap. In the HA-group, direct bone formation from the implant toward the pre-existing bone was recognizable in both lateral and base areas. Many TRAPase-reactive cells were found near the implant surface. On the pre-existing bone, new bone formation occurred with bone resorption by typical osteoclasts. Osseointegration around the implants was achieved by postoperative day 28 in both SA- and HA-groups except for the lateral area, where the implant had been installed close to the cavity margin. These findings indicate that ossification around the titanium implants progresses in different patterns, probably dependent on surface properties and quality.

DOI： 10.1046/j.0905-7161.2003.00960.x

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Language：English   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

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 IT 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 fibro-blast-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.

DOI： 10.1679/aohc.66.289

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

The distribution and type of immunocompetent cells were investigated in rat oral mucosae using immunocytochemistry and enzyme histochemistry, focusing on histological structures. We used two antibodies, OX6 and ED1, which recognize the rat la-antigen and macrophage/monocyte lineage, respectively. Enzymatic histochemistry for acid phosphatase (ACPase) activity and adenosine triphosphatase (ATPase) activity was also employed to identify macrophages and Langerhans cells, respectively. Many OX6-immunopositive cells, dendritic or irregular in shape, were recognizable in the lamina propria of oral mucosae: some cells extended their dendritic processes into the epithelial layer of the buccal and sublingual mucosae. Dendritic cells within the epithelium showed intense ATPase reaction, indicating they could be categorized as Langerhans cells. A small number of ED1-positive cells existed in the lamina propria, but none were present in the epithelial cell layer. Double staining either with OX6 and ED1 or OX6 and ACPase made it possible to divide the immunocompetent cells in the lamina propria into three types: the OX6-positive cells without ED1 or ACPase-reaction, the OX6-negative cells with ED1 and ACPase-reactions, and the OX6-ED1/ACPase-co-expressing cells, each of which possessed characteristic ultrastructural features demonstrated by immunoelectron microscopy. Taking these findings together with previous reports, these three types of cells were regarded as a dendritic-like cell, a macrophage without antigen-presentation ability, and a macrophage with antigen-presentation ability, respectively. There were regional differences in the distribution and density of these immunocompetent cells; they were densely distributed in order of the buccal and sublingual mucosae, the palatal mucosa, and the dorsal surface of tongue. The region-specific distribution and density of the immunocompetent cells might be due to the histological. structure of each oral mucosa, suggesting the presence of different immune-defense systems among each portion of the oral mucosae.

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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.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

The periodontal Ruffini ending has been reported to show immunoreactivity for tyrosine kinase B (trkB), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the periodontal ligament of the rat incisor. Furthermore, adult heterozygous BDNF-mutant mice showed malformation and reduction of the periodontal Ruffini endings. To investigate further roles of BDNF in these structures, the development, distribution, and terminal morphology of Ruffini endings were examined in the incisor periodontal ligament of heterozygous and homozygous BDNF mutant mice, as well as in the wild-type littermate by immunohistochemistry for protein gene product (PGP) 9.5, a general neuronal marker. A similar distribution and terminal formation of PGP 9.5-immunoreactive nerve fibers was recognized in the periodontal ligament of all phenotypes at postnatal week (PW) 1. At this stage, the nerve fibers had a beaded appearance, but did not form the periodontal Ruffini endings. At PW2, the heterozygous and wild-type mice started to show ramified nerve fibers resembling the mature shape of periodontal Ruffini endings. At PW3, the Ruffini endings occurred in the periodontal ligament of the wild-type and heterozygous mice. While the Ruffini endings of the wild-type mice appeared either ruffled or smooth, as reported previously, most of these structures showed a smooth outline in the heterozygous mice. The homozygous mice lacked the typical Ruffini endings at PW3. In the quantitative analysis, homozygous mice had the smallest percentages of PGP 9.5-immunoreactive areas at the same postnatal periods, but there were no significant differences between wild-type and heterozygous mice during PW1-3. These findings suggest a possible involvement of BDNF during the postnatal development and, in particular, the maturation of periodontal Ruffini endings. Furthermore, other neurotrophins may play a role in the development and/or early maturation of the periodontal nerve fibers, as indicated by the presence of nerve fibers in the BDNF-homozygous mice. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/ar.a.10094

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The present study examined the immunohistochemical localization of heat shock protein 25 (Hsp25) during the regeneration of nerve fibers and Schwann cells in the periodontal ligament of the rat lower incisor following transection of the inferior alveolar nerve. In the untreated control group, the periodontal ligament of rat incisor did not contain any Hsp25-immunoreaction. On postoperative day 3 (PO 3d), a small number of Schwann cells with slender cytoplasmic processes exhibited Hsp25-immunoreactivity. From PO 5d to PO 21d, Hsp25-positive nerve fibers and Schwann cells drastically increased in number in the alveolar half of the ligament. Although the axons of some regenerating Ruffini-like endings also showed Hsp25-immunoreactions, the migrated Schwann cells were devoid of Hsp25-immunoreaction. Thereafter, Hsp25-positive structures decreased in number gradually to disappear from the periodontal ligament by PO 56d. This temporal expression of Hsp25 in the periodontal ligament well-reflected the regeneration process of the nerve fibers. Hsp25 in the regenerating nerve fibers and denervated Schwann cells most likely serves in modulating actin dynamics and as a cellular inhibitor of apoptosis, respectively. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/S0006-8993(03)02889-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

A high-speed optical imaging technique was employed for visualizing neuronal excitation propagation elicited by afferent stimulation in the mouse trigeminal caudalis (Vc) to clarify the central nociceptive modulation mechanism. Membrane depolarization evoked by a single-pulse stimulation to the spinal trigeminal tract (Tr) was propagated rostrally to the Vc, which was suppressed by CNQX This is consistent with our morphological observation that axons expand from the Tr into the Vc. A trained-pulse (tetanus) stimulation to the Tr evoked a broad, persistent excitation in the Vc, while MK-801 suppressed it. Neonatally capsaicin-treated mice maintained a single-pulse response but a lacked tetanus-evoked one. These indicated that prolonged depolarization elicited by repetitive stimulation is a prerequisite to C-fiber excitation for activating the NMDA receptors.

DOI： 10.1097/01.wnr.0000078544.07662.cc

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Language：Japanese   Publisher：(株)日本メディカルセンター  

副甲状腺ホルモン関連ペプチド(PTHrP)シグナルが軟骨形成に重要な役割を演ずることが明らかにされ,特にPTHrPとIndian hedgehogのnegative feedbackによって軟骨分化が調節されることが明らかとなっている.又,PTH/PTHrP受容体のpoint mutationでJansen型及びBlomstrand型の軟骨異形成症が発症すること,更に内軟骨腫が発症することも明らかにされている.近年の報告では,PTH/PTHrP受容体にNa+/H+交換体調節因子が結合することにより,PTH/PTHrP受容体からのホスフォリパーゼCのシグナル伝達が活性化することが発見された.これら軟骨細胞の分化制御機構について解説した

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The present study employed immunohistochemistry for protein gene product 9.5 (PGP 9.5) to examine the regeneration process of Ruffini endings, the primary mechanoreceptor in the periodontal ligament, in heterozygous mice with targeted disruption of the brain-derived neurotrophic factor (BDNF) gene and their littermates, following transection of the inferior alveolar nerve. When immunostained for PGP 9.5, periodontal Ruffini endings appeared densely distributed in the periodontal ligament of the heterozygous mice, but the density of the positively stained nerve fibers in the ligament was 20% lower than that in the control littermates. At 3 days after surgery, the PGP 9.5-positive neural elements had disappeared; they began to appear in the periodontal ligament of both animals at 7 days. However, the recovery pattern of the PGP 9.5-positive nerves differed between heterozygous and wild type mice, typical periodontal Ruffini endings morphologically identical to those in the control group appeared in the wild-type mice at 7 days, whereas such Ruffini endings were detectable in the heterozygous mice at 28 days, though much smaller in number. On day 28, when PGP 9.5-positive nerves were largely regenerated in wild type mice, their distribution was much less dense in the ligament of the heterozygous mice than in the non-treated heterozygous mice. The density of PGP 9.5-positive nerve fibers was significantly lower in the heterozygous mice than in wild type mice at any stage examined. These data showing that a reduced expression of BDNF causes delayed regeneration of the periodontal Ruffini endings suggest the involvement of BDNF in the regeneration process of these mechanoreceptors.

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DOI： 10.1002/jemt.10286

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

The response of nerve fibres in the peri-implant epithelium to titanium implantation was investigated with an experimental model using rat maxilla and immunohistochemical techniques. The latter employed antibodies to protein gene product 9.5 (PGP9.5), and to calcitonin gene-related peptide (CGRP). In control rats without an implantation, a dense innervation of PGP9.5- and CGRP-positive nerve fibres was recognized throughout the junctional epithelium, as has been previously reported. A titanium-implantation induced a remarkable inflammatory reaction, as well as the destruction of covering epithelial cells. By 3-5 days post-implantation, inflammatory reaction showed a tendency to disappear, and the peri-implant epithelium showed proliferation and down-growth along the implant. At this stage, no nerve fibres were found around the peri-implant epithelium. At 10 days, a few nerve fibres reached the basal cell layers of the peri-implant epithelium, and entered it 15 days after implantation when the peri-implant epithelial cells showed morphological features roughly resembling those of normal junctional epithelial cells. At the complete osseointegration stage (days 20-30), the PGP9.5- and CGRP-positive nerve fibres, thin and beaded in appearance, were found distributed in the peri-implant epithelium. After 20 days, the numerical density of the intraepithelial nerves in the peri-implant epithelium appeared the same as, or less than, that in the normal junctional epithelium. These findings indicate that the peri-implant epithelium shows the same innervation as that in normal junctional epithelium, and that the intraepithelial nerve fibres in the peri-implant epithelium might have diverse functions, which have been suggested in the literature.

DOI： 10.1034/j.1600-0501.2003.140216.x

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Language：Japanese   Publishing type：Research paper (scientific journal)  

The epiphyseal cartilage is composed of the distinct zones of resting, proliferative and hypertrophic chondrocytes. The intercolumnar cartilage matrix of the hypertrophic zone is subjected to mineralization whereas the transverse partitions of the cartilage column are poorly mineralized. Therefore, mineralized cartilage matrices are formed parallel to the longitudinal axis of the epiphyseal cartilage. Vascular endothelial cells invade the cartilage by penetrating the poorly mineralized transverse partition at the chondro-osseous junction, resulting in the exposure of mineralized intercolumnar matrix to bony tissue. The exposed mineralized cartilage matrices appear to serve as scaffolds for osteoblastic attachment. These osteoblasts deposit bone matrices onto the cartilage cores, forming primary trabecular bones. Vascular endothelial growth factor, VEGF, is a strong angiogenic factor, and play a pivotal role in vascular invasion into cartilage. The invading endothelial cells possess the receptor for VEGF, and secret matrix metallo protatenase to digest the cartilage matrices of the unmineralized transverse partition of the column.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

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.

DOI： 10.1002/jemt.10289

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Language：English   Publisher：WILEY-LISS  

This review covers current information about the ability of dental nerves to regenerate and the role of tooth pulp in recruitment of regenerating nerve fibers. In addition, the participation of dental nerves in pulpal injury responses and healing is discussed, especially concerning pulp regeneration and reinnervation after tooth replantation. The complex innervation of teeth is highly asymmetric and guided towards specific microenvironments along blood vessels or in the crown pulp and dentin. Pulpal products such as nerve growth factor are distributed in the same asymmetric gradients as the dentinal sensory innervation, suggesting regulation and recruitment of those nerve fibers by those specific factors. The nerve fibers have important effects on pulpal blood flow and inflammation, while their sprouting and cytochemical changes after tooth injury are in response to altered pulpal cytochemistry. Thus, their pattern and neuropeptide intensity are indicators of pulp status, while their local actions continually affect that status. When denervated teeth are injured, either by pulp exposure on the occlusal surface or by replantation, they have more pulpal necrosis than occurs for innervated teeth. However, small pulp exposures on the side of denervated crowns or larger lesions in germ-free animals can heal well, showing the value of postoperative protection from occlusal trauma or from infection. Current ideas about dental neuroplasticity, neuro-pulpal interactions, and nerve regeneration are related to the overall topics of tooth biomimetics and pulp/dentin regeneration.

DOI： 10.1002/jemt.10291

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Language：Japanese   Publisher：(一社)日本解剖学会  

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Language：Japanese   Publisher：(一社)日本解剖学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

Innervation and terminal morphology in the lingual periodontal ligament of the incisor were investigated in brain derived neurotrophic factor (BDNF) heterozygous mice and littermate wild-type mice (aged two months) using immunohistochemistry for protein gene product 9.5 (PGP-9.5), a general neuronal marker. In addition, computerassisted quantitative analysis was performed for a comparison of neuronal density in the periodontal ligament between heterozygous and wild-type mice. In wild-type mice, the periodontal ligament was found to be richly innervated by the mechanoreceptive Ruffini endings and nociceptive free nerve endings in the alveolus-related part of the periodontal ligament. The periodontal Ruffini endings in the wild-type mice incisor ligament were classified into two types: type I with ruffled outlines, and type II with a smooth outline. BDNF heterozygous mice showed malformations of the type I Ruffini endings which included fewer nerve fibers and fewer ramifications than those in wild-type mice as well as smooth outlines of the axon terminals. Quantitative analysis under a confocal microscope showed a roughly 18% reduction in neuronal density in the periodontal ligament of the heterozygous mice. These findings suggest that the development and maturation of the periodontal Ruffini endings require BDNF.

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

骨端軟骨は大きく,静止層,増殖層,肥大化層に分けられる.肥大化層のカラム間基質は石灰化を受けるが,カラム内の横隔壁は石灰化をあまり受けない.そのために,石灰化軟骨基質は軟骨の長軸方向に形成される.血管内皮細胞は軟骨・骨境界部であまり石灰化を受けていない横隔壁を貫通することで,軟骨に侵入してゆき,その結果,石灰化したカラム間基質が骨組織へと露出する.この露出した軟骨基質は骨芽細胞が接着する足がかりとして役立つと考えられる.又,これらの骨芽細胞は骨基質を軟骨基質上へ分泌することにより,一次骨梁を形成する

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

The distribution of immunocompetent cells in the dentogingival junction of rat molars during root formation was investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6-antibody) and monocyte/macrophage lineage cells (ED1-antibody) as well as by histochemical reaction for periodic acid-Schiff (PAS). Two portions (the functional epithelium in the mesial gingiva of the first molar, and the interdental gingiva between the first and second molars) were selected for observations. At the eruption stage of the first molar (16-18 days after birth), OX6-positive cells, dendritic or oval in shape, were abundantly distributed in the connective tissue between the oral epithelium and tooth germ. Positive cells with slender cell processes were also found beneath the ameloblast layer. At the commencement stage of the first molar occlusion (24-28 days after birth), numerous OX6-positive cells displaying a dendritic fashion existed preferentially in the mesial gingiva, but were fewer in the interdental gingiva. In contrast, the interdental gingiva showed a denser distribution of ED1-positive cells and PAS-reactive polymorphonuclear leukocytes (PMLs) than the mesial gingiva. At the completion stage of root formation (100-120 days after birth), the OX6-immunopositive cells invaded the deeper position of the mesial gingiva with the downgrowth of the epithelium; they had a considerably higher cell density compared with those in the interdental gingiva where PAS-reactive PMLs persisted. These findings indicated that the immunocompetent cells showed a region-specific distribution and cell density by their roles in immune response.

DOI： 10.1034/j.1600-0765.2003.01622.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMEDICAL RESEARCH PRESS LTD  

The immunolocalization of periostin, previously termed osteoblast-specific factor 2, was investigated in developing long bones of 17-day-old fetal mice and of 1-, 2-, 3- and 8-week-old mice at the light and electron microscopic levels. Fetal femurs showed immunoreactions for periostin in the periosteum, perichondrium, articular surface of the epiphyseal cartilage, joint ligaments, and fascias of surrounding muscles. In particular, intense immunoreactivity for periostin was found in the fibrous layer of the periosteum and perichondrium. At postnatal 1-and 2-weeks, in contrast, the immunoreactivity was restricted to the periosteum and thick fascias of surrounding muscles when compared with the fetal bones. Immunoelectron microscopic observation of the periosteum demonstrated immunoreaction products for periostin at the junction of periosteal fibroblasts and collagen bundles, suggesting its competence in the cell-to-matrix interaction. Mice at 3 and 8 weeks, unlike 2-week-old mice, showed a periostin-immunoreaction dominantly in the osteoblastic layer but not in the fibroblastic layer of the periosteum. Furthermore, the perichondrium and fascias of surrounding muscles were devoid of immunoreaction. Thus, periostin was confirmed to be widely distributed in bone and concomitant tissues at the fetal stage. As mice grew, however, its immunoreactivity gradually came to be restricted to the osteoblastic layer of the periosteum. Our findings suggest that periostin acts at the site of the cell-to-matrix interaction in periosteum, fascias, and joint ligament during morphogenesis of these tissues.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Previous studies have reported enhanced osteoclastogenesis, increased bone resorption and osteoporosis in osteoprotegerin (OPG)-deficient mice. In the present study, we show that the tibial epiphyses contain abundant, thin trabeculae lined with numerous osteoclasts and cuboidal osteoblasts. The increase in osteoblasts and osteoclasts was associated with a dramatic increase in calcein labelling of the mineralization fronts and replacement of much of the intertrabecular marrow with numerous alkaline phosphatase-positive preosteoblasts. Furthermore, the discrete, linear cement lines seen in wild-type mice were replaced by a randomly oriented meshwork of cement lines that were stained intensely for tartrate-resistant acid phosphatase and osteopontin in the OPG(-/-) mice. These indices of accelerated bone remodelling in mutant bone were associated with irregular trabecular surfaces, a disorganized collagen matrix interspersed with amorphous ground substance and numerous fissures between old and new bone. In total, these observations indicate that enhanced osteoclastic activity in OPG(-/-) epiphyses led to a coupled increase in osteoblast differentiation and activity and an increase in bone remodelling. The high bone turnover, disorganized matrix and impaired attachment of new to old bone in the cement lines in OPG(-/-) mice appear to cause bone fragility.

DOI： 10.1093/jmicro/52.6.503

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Parathyroid hormone (PTH)-related peptide (PTHrP) has been believed to act by binding the common receptor to PTH (PTH/PTHrP receptor). However, PTHrP is localized not only in the secretory pathway, but also in nucleoli by virtue of its nucleolar targeting signal (NTS). This review demonstrates the bipartite action of PTHrP on chondrocytes, the receptor-mediated and -independent signaling pathway. Mice with deletion of the PTHrP gene were characterized by a chondrodysplasia due to markedly reduced proliferation of epiphyseal chondrocytes. The PTH/PTHrP receptor was localized mainly in proliferative chondrocytes in the epiphyseal cartilage, indicating that PTHrP modulates normal proliferation via the receptor. In contrast to the receptor-mediated action, the mid-region of the amino acid sequence of PTHrP contains an NTS. The PTHrP-translation was found to initiate from both methionine-coding AUG and downstream leucine-coding CUGs in its signal sequence. When translated from CUGs, PTHrP accumulated in the nucleoli, and the translation from AUG localized PTHrP in both the Golgi apparatus and nucleoli. Therefore, nucleolar PTHrP appears to be derived from the translation initiating from both AUG and CUGs. A chondrocytic cell line expressing a full-length PTHrP, but not PTHrP lacking NTS, were resistant to apoptosis caused by serum depletion, suggesting that the nucleolar PTHrP in chondrocytes serves as a survival factor against apoptosis. Thus, PTHrP regulates chondrocyte proliferation, differentiation and apoptosis by mediating its receptor or acting directly on the nucleolus.

DOI： 10.1046/j.0022-7722.2002.00032.x

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publisher：新潟歯学会  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI IRELAND LTD  

The expression of galanin receptor-1 (GALR1) was investigated in the rat trigeminal ganglion by using immunocytochemistry and in situ hybridization. In addition, the regional distribution of GALR1-immuno reactive pulpal nerves and their ultrastructure were examined in the molar teeth. In the trigeminal ganglion. the immunoreactivity for GALR1 was recognizable in about 30%. of the total number of neurons. Most of the cell bodies were small to medium in size. Analysis of serially cut sections alternately stained with GALR1 and galanin antisera demonstrated that some GALR1-positive cells displayed immunoreactivity for galanin. In situ hybridization analysis, expression of GALR1 mRNA was detected in trigeminal ganglion cells. The cell size distribution was similar to that of GALR1-immunoreactive cells, In the dental pulp. a small number of nerve fibers displayed immunoreactivity for GALR1. The labeled fibers formed terminal arbors in the coronal pulp around and within the odontoblast cell layer, but never penetrated into the predentin and dentin. Ultrastructurally, GALR1 immunoreactivity in the dental pulp was confined to the axoplasm of unmyelinated nerve fibers. The present study provided new evidence that unmyelinated primary afferents innervating dental pulp possessed galanin receptor. and suggests the existence of nociceptive primary afferents functioning as autocrine cells. (C) 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/S0168-0102(01)00323-6

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Endothelial cells play an important role in endochondral bone formation. In the chondro-osseous junction, endothelial cells appear to invade into cartilage by the cellular mechanism of angiogenesis evidenced by cell duplication, disappearance of basement membranes and activated migration. The endothelial cells penetrate the unmineralized transverse partition of the cartilage columns.

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

軟骨内骨化を担う細胞群の主役の1つとして血管内皮細胞があげられるが,血管内皮細胞の軟骨侵入に至る迄の軟骨細胞の整然とした細胞増殖・分化は重要なプロセスである.石灰化軟骨基質は軟骨カラム間に形成されており,血管内皮細胞はカラム中の石灰化されていない薄い横隔壁を貫通することで軟骨に侵入すると考えられる.軟骨内骨化における軟骨細胞と血管内皮細胞に焦点をあてて解説した

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Munksgaard  

The cellular heterogeneity of Malassez epithelium (ME) residing in the periodontal ligament has recently been reported, and the presence and coexistence of the neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP) in single cells in ME has been shown (1). However, the identity of these neuroendocrine cells has so far not been verified. This study was undertaken in order to elucidate the identity of the neuroendocrine cells in ME by means of transmission electron microscopy, confocal scanning microscopy and immunohistochemistry using antibodies to protein gene product (PGP) 9.5 and cytokeratin 20 (CK). Gingival tissue was included in the study as a positive control for identification of Merkel-like cells in oral epithelium. CK 20 immunopositive cells were present in both Malassez epithelium and in basal cell layers of gingival epithelium showing a distribution consistent with PGP 9.5 labelled cells in both epithelia. The results from PGP 9.5 immuno electron microscopy clearly evidenced the presence of single, intensely labelled cells and some nerve fibres invested between the Malassez epithelial cells. The conformity of the immunopositive cells in Malassez and gingival epithelium verified by double immunolabelling with PGP 9.5 and CK 20. indicates that the labelled neuroendocrine cells arc identical in ME and in gingival epithelium. This demonstrates that Malassez epithelium not only exhibits neuroendocrine cells, but additionally that the neuroendocrine cells represent Merkel-like cells. © Blackwell Munksgaard, 2002.

DOI： 10.1034/j.1600-0765.2002.01374.x

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The present immunocytochemical study reports on the expression of heat-shock protein (Hsp) 25 during odontogenesis in rat molars from postnatal 1 to 100 days. Hsp 25 immunoreactivity (IR) appeared in the immature dental mesenchymal cells and the differentiating and differentiated odontoblasts. At 30 days, the coronal odontoblasts retained intense Hsp25-IR, whereas the odontoblasts in the root and floor pulp were initially weak or negative but increased in IR in the later stages, indicating that the expression of Hsp 25 reflects the differentiation status of odontoblasts. During amelogenesis, the secretory ameloblasts were Hsp 25 immunopositive and the enamel free area (EFA) cells showed intense Hsp 25-IR when they developed a ruffled border. Ruffle-ended ameloblasts (RA) also consistently showed intense Hsp 25-IR, but smooth ended ameloblasts (SA) showed weak IR. These data suggest that Hsp 25 is related to the formation and maintenance of the ruffled border of RA and EFA cells.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：QUINTESSENCE PUBLISHING CO INC  

The present study reports on the expression of galanin receptor-1 (GALR1) in rat trigeminal ganglion and pulpal nerves by immunohistochemistry and by in situ hybridization histochemistry. Furthermore, the trigeminal neurons expressing GALR1 were counted by a computer image analyzer. In the rat trigeminal ganglion, GALR1-immunoreactivity was recognizable in about 30% of neurons categorized as small to medium sized, this finding being confirmed by in situ hybridization analysis. One of the antisera to GALR1 could label a small number of pulpal nerve fibers; they were distributed throughout the coronal pulp to terminate near the odontoblast cell layer but never penetrated into the predentin and dentin. Ultrastructurally, immunoreactive products for GALR1 were confined to the axoplasm of unmyelinated nerve fibers, and some, but not all, nerve endings near the odontoblasts were also positive in immunoreaction. The expression of GALR1 in the trigeminal ganglion and dental pulp suggested that galanin/GALR1 might play important roles in transmission and/or modulation of pain sensation in rat teeth.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：QUINTESSENCE PUBLISHING CO INC  

During tooth development, the expression of Msx2 mRNA is spatiotemporally shifted among the several components of the dental epithelium and mesenchyme. Recently, we demonstrated that Msx2-deficient mice display defective tooth, hair follicle, and mammary gland development, in addition to defects of skull ossification and persistent calvarial foramen. Although Msx2-deficient mice have shown the abnormalities of amelogenesis, the detailed phenotypes remain to be clarified. The present study analyzed the abnormalities of tooth development in Msx2-deficient mice. The phenotypes of defective teeth observed in the Msx2(-/-) mice were as follows. The enamel organ showed condensed features, preventing the vascular invasion there from inducing the TUNEL-negative degeneration of ameloblasts. Secretory ameloblasts represented abnormal features such as irregular-shaped Tomes's processes and numerous vacuoles in their distal cytoplasm. The enamel organ contained no TUNEL-positive cells, although the temporary appearance of TUNEL-positive cells occurred in the wild-type mice. Both crown and root in the Msx2(-/-) mice showed the irregular-shaped morphology, concomitant with the abnormal development of Hertwig's epithelial root sheath. The amelogenesis imperfecta was caused by the degeneration of ameloblasts and by the disappearance of the enamel-free area in the cusp area of molars. Our results provide evidence that Msx2 plays crucial roles in amelogenesis and the normal morphology of the tooth.

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Language：English   Publisher：Biomedical Research Press  

DOI： 10.2220/biomedres.23.85

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

The present study reports on a characteristic spatio-temporal propagation of differential fluorescent images in the rat, brain stem slice by a high-speed optical imaging technique. Coronal or sagittal slices were stained with a voltage-sensitive dye RH-414, and the superficial layer of the trigeminal caudalis (Vc) was then electrically stimulated. The marginal layer and substantia gelatinosa showed larger rostrocaudal excitation than coronal, despite a lack of tract stimulation expansion in either direction. A perfusion of 0.5 mu mol/l TTX, not 10 mu mol/l CNQX suppressed these propagations. These findings suggest that the superficial layer of Vc has spatial differences in neuronal excitation propagation, as evidenced by morphological observations that dendrites in the superficial layers extend in the rostrocaudal direction. NeuroReport 12:3985-3988 (C) 2001 Lippincott Williams & Wilkins.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The expression in various cell types of peripheral tissues of glial fibrillary acidic protein (GFAP), first discovered as an intermediate filament specific for astrocytes, remains controversial owing to numerous reports of a wide distribution for GFAP-immunoreactivity in various cells. The present study employed immunohistochemistry to investigate the precise expression of GFAP in the dental pulp and trigeminal ganglion of adult rats and wild-type mice as well as GFAP-knockout mice. The exhibition of GFAP-immunoreactivity in the trigeminal ganglion was further examined by a reverse transcription polymerase chain reaction (RT-PCR) technique, and in situ hybridization histochemistry using a specific cRNA probe prepared by us. The immunoreaction for GFAP was recognizable in the axons, Schwann cells, and the fibroblasts in the dental pulp of rats and wild-type littermate mice. However, mice with null mutations in the GFAP gene remained immunoreactive for GFAP in all these locations. Intense GFAP-immunoreactivity was found in a small number of satellite cells in the trigeminal ganglion in all animals examined in this study. RT-PCR analysis demonstrated bands for the GFAP gene corresponding to the length expected from the primer design in the samples of trigeminal ganglion and dental pulp. In situ hybridization histochemistry also showed intense signals for GFAP mRNA in some satellite cells of the trigeminal ganglion, but never in the neurons. These data suggest that the GFAP-immunoreactive molecules in the pulpal axons and fibroblasts react non-specifically with the polyclonal antibody and are probably a closely related type of intermediate filament.

DOI： 10.1679/aohc.64.503

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

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 cytoplast-nic 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. (C) 2001 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0003-9969(01)00052-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The postnatal expression of heat shock protein (Hsp) 25 during the amelogenesis of rat molars was investigated by immunocytochemistry and confocal microscopy. The localization pattern of Hsp 25-immunoreactivity in the inner enamel epithelium and ameloblast cell layer of the rat molars was almost identical to that in the rat incisors which we have previously reported: an intense Hsp 25-immunoreactivity, which first appeared in the preameloblasts, was recognized in secretory ameloblasts and ruffle-ended ameloblasts with stage-specific immunointensity. Confocal microscopy with Hsp 25-antibody and rhodamine-labeled phalloidin clearly demonstrated the co-localization of Hsp 25 and actin filaments in the ameloblast layer, supporting our hypothesis that this molecule might serve to reinforce the ameloblast layer during enamel formation as well as the formation and maintenance of the ruffled border in ruffle-ended ameloblasts. Interestingly, the enamel free area cells, which essentially lack the ability for enamel formation, showed the Hsp 25-immunoreactivity during 4-11 days when they developed a ruffled border, but decreased in that immunoreactivity after postnatal 15 days following apoptosis. Since Hsp 25 has been shown to be a specific inhibitor of apoptosis, the enamel-free area cells contribute to determine the outline of dentin at the cusped area. These data support our previous hypothesis on the diverse functions of Hsp 25 in amelogenesis.

DOI： 10.1679/aohc.64.369

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The regeneration process of dental pulp following tooth replantation in rat molars was investigated by immuno cyto chemistry for heat shock protein (Hsp) 25 and protein gene product 9.5 (PGP 9.5). In control teeth at postnatal 4 weeks, the odontoblasts showed intense Hsp 25-immunoreactivity in the coronal dental pulp, but little or no immunoreactivity in the root and floor pulp. In contrast, the Hsp, 25-negative odontoblasts in the latter areas displayed immunoreactivity for PGP 9.5. Tooth replantation caused loss of Hsp 25- and PGP 9.5-immunoreactions in the dental pulp during postoperative days 1-3. At postoperative day 5, plump cells with clear nucleoli and several fine processes-presumably newly differentiated odontoblasts-at the pulp-dentin border became immunopositive for Hsp 25. These data suggest that the expression of Hsp 25- and PGP 9.5-immunoreactivity reflects the status of differentiation of the odontoblasts. Furthermore, some pulpal nerve fibers as well as the Schwann cells in the dental pulp, ordinarily negative in Hsp 25-immunoreaction, acquired their immunoreactivity by postoperative day 5, but lost it thereafter, suggesting the involvement of Hsp, 25 in the regeneration of pulpal nerve fibers. In the case of bone-like tissue formation in the pulp space, on the other hand, no Hsp 25-immunoreactive odontoblasts were recognized in the pulp-dentin border. Thus, the alignment of Hsp 25-immunopositive odontoblasts along the pulp-dentin border indicates a decisive factor for inducing the reparative dentin formation after tooth replantation.

DOI： 10.1679/aohc.64.425

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The temporal and spatial expression pattern of Fos protein in spinal dorsal horn neurons was examined by immunohistochemistry in rats with chronic constriction injury (M) to the sciatic nerve. In normal animals, a few Fos-immunoreactive (-IR) neurons were detected in the dorsal horn of the lumbar spinal cord, Following induction of M, a very large number of Fos-IR neurons appeared in the spinal dorsal horn, but a significant number of Fos-IR neurons were also observed in the contralateral dorsal horn where primary afferents of the injured sciatic nerve rarely project. Sham-operated animals also had a significant number of Fos-IR neurons in the dorsal horn bilaterally. The number of Fos-IR neurons reached its maximal level I day following placement of the ligatures (PO ld). The ratio of the number of Fos-IR neurons in the ipsilateral dorsal horn to the contralateral dorsal horn, however, had its peak level 3 days following CCI (3.1-fold increase compared to the contralateral dorsal horn). The number of Fos-IR neurons in the dorsal horn gradually decreased, but increased again around PO 15d. On PO 30d, the number of Fos-IR neurons decreased and became comparable to that in normal animals. The present results indicate that the induction of Fos-IR neurons in the dorsal horn caused by CCI is biphasic and reaches its maximal level on PO 3d, near the time of hyperalgesia onset. (C) 2001 Elsevier Science BY. All rights reserved.

DOI： 10.1016/S0006-8993(01)02783-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The present study was undertaken to reveal spatio-temporal changes in the distribution of Fos-like immunoreactive (-IR) neurons in the parabrachial nucleus (PBN), one of the important relay nuclei for processing autonomic and somatosensory information from the oro-facial regions, following the induction of experimental tooth movement in rat upper molars. The experimental tooth movement was induced by the insertion of elastic rubber between the first and second upper molars. In normal animals, the PBN contained a smaller number of Fos-IR neurons. Following experimental tooth movement, the Fos-IR neurons increased in number significantly on both the ipsilateral and contralateral PBN, reaching a maximum at 4 h (about 10 times that of normal animals), and then decreased gradually. However, a significant number of Fos-IR neurons remained at 24 h post-operation. Remarkable side-by-side differences in the number of Fos-IR neurons were recognized at 1 to 4 h following the experimental tooth movement. Their number returned to normal (basal) levels at 5 days post. All subnuclei of PBN showed similar temporal changes in the number of Fos-IR neurons, this being particularly apparent in lateral PBN. Administrations of morphine (3 and 10 mg/kg, i.p.) drastically reduced the induction of Fos-IR neurons in all subnuclei of both the ipsilateral and contralateral PBN in a dose-dependent manner, and its effect was antagonized by pretreatment with naloxone (2 mg/kg, i.p.). The reduction of Fos-IR neurons by morphine pretreatment suggests that the appearance of Fos-IR neurons in the PBN may be partly due to the noxious stimulation and/or stress arising from tooth movement. The bilateral expression of Fos-IR neurons in the PBN indicates that the experimental tooth movement causes the activation of PBN neurons for the processing of somatosensory as well as autonomic information. The prolonged expression of Fos-IR neurons in all the subnuclei of bilateral PBN reflects clinical features of the transient discomfort and/or abnormal sensations, which many patients often complain about during orthodontic treatment. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0006-8993(01)02639-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0006-8993(01)02458-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

Responses of immunocompetent cells to tooth replantation during the regeneration process of the dental pulp in rat molars were investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6 antibody), monocyte/macrophage lineage cells (ED1 antibody) and protein gene product 9.5 (PGP 9.5), as well as by histochemical reaction for periodic acid-Schiff (PAS). Tooth replantation caused an increase in both the number of OX6- and ED1-positive cells and their immunointensity in the replanted pulp, but almost all PGP 9.5-immunoreactive nerves diminished in the initial stages. By postoperative day 3, many OX6- and ED1-immunopositive cells had accumulated along the pulp-dentin border to extend their cytoplasmic processes into the dentinal tubules in successful cases. Once reparative dentin formation had begun after postoperative day 7, OX6- and ED1-immmunopositive cells became scattered in the odontoblast layer, while reinnervation was found in the coronal pulp. The temporal appearance of these immunocompetent cells at the pulp-dentin border suggests their participation in odontoblast differentiation as well as in initial defense reactions during the pulpal regeneration process. On postoperative day 14, the replanted pulp showed three regeneration patterns: (1) reparative dentin, (2) bone-like tissue formation, and (3) an intermediate form between these. In all cases, PAS-reactive cells such as polymorphonuclear leukocytes (PML) and mesenchymal cells occurred in the pulp space. However, the prolonged stagnation of inflammatory cells was also discernible in the latter two cases. Thus, the findings on PAS reaction suggest that the migration of the dental follicle-derived cells into the pulp space and the subsequent total death of the proper pulpal cells are decisive factors for eliciting bone-like tissue formation in the replanted pulp.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

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.

DOI： 10.1679/aohc.63.381

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE AMERICA INC  

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans(1,2). Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen, This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina(3) (PFM), Msx2(-/-) mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis, Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.

DOI： 10.1038/74231

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ACAD PERIODONTOLOGY  

Background: Guided bone regeneration (GBR) has been widely utilized for the promotion of bone augmentation in bone loss areas. However, little information has been available regarding chronological changes in newly formed bone and alterations in the nature of newly formed bone after removal of a barrier membrane. The present study attempted to establish a GBR model for rat maxillae. We also examined the effects of membrane application periods on newly formed bone and its remodeling process after removal of the membrane in this experimental model.
Methods: Thirty-five Wistar rats were divided into 2 groups: a membrane application group and a membrane removal group. The chronological changes of newly formed bone were evaluated histologically and statistically.
Results: At 2 weeks after the GBR procedure, bony cavities had completely filled the newly formed bone in the experimental side. In the control side, corticalization on the surface of the newly formed bone proceeded with a decrease in the bone marrow cavity, whereas the bone marrow space had enlarged by 12 weeks post-surgery in the experimental side. In the membrane removal group, the osteoblasts appeared on newly formed bone at 1 week after membrane removal. Comparatively thick compact bone had formed on the surface of the newly formed bone at 4 weeks after membrane removal, and corticalization proceeded later.
Conclusions: The long-term application of a barrier membrane induces the enlargement of the bone marrow spaces. We suggest that PTFE membrane removal in adequate time promotes the corticalization and maturation of the newly formed bone by the GBR technique.

DOI： 10.1902/jop.2000.71.3.341

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The postnatal development of the terminal Schwann cell, an analogue of the lamellar cell in cutaneous sensory receptors, was examined by histochemistry for non-specific cholinesterase and immunohistochemistry for S-100 protein in the periodontal Ruffini endings of the rat incisor. Double immunohistochemistry for S-100 protein and protein gene product 9.5 (PGP 9.5) was also performed to examine the relationship between terminal Schwann cells and axons. Histochemistry for non-specific cholinesterase was able to demonstrate the age-related development of the terminal Schwann cells. the morphology and distribution of the developing terminal Schwann cells became almost identical to those in adults during postnatal days 15-18. Axons showing PGP 9.5-like immunoreactivity elongated and expanded after arrangement of terminal Schwann cells in the alveolus-related part. This suggests that the terminal Schwann cell is important in the development and maturation of the periodontal Ruffini endings. (C) 2000 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0006-8993(99)02463-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ACAD PERIODONTOLOGY  

Background: The detailed mechanism of osseointegration, the most appropriate implant-bone interface, remains unclear in jaw tissues at the ultrastructural level in contrast to the many reports using long bones. The present study reports on tissue response to titanium-implantation on an animal model using rat maxilla.
Methods: Animals were sacrificed at 1 to 28 days postimplantation and prepared tissue specimens, freed from implants by a cryofracture technique, were processed for transmission electron microscopy and histochemistry for tartrate resistant acid phosphatase activity (TRAPase).
Results: Different patterns in bone formation were recognized between lateral and base areas of implant cavities. In the lateral area with narrow gaps, bone deposition took place from the pre-existing bone towards the implant after active bone resorption by osteoclasts reactive to TRAPase. However, no distinct bone formation appeared in the lateral area where the implant had been installed close to the osteotomy margin. On the other hand, new bone formation was found at the base area without any apparent bone resorption. Interestingly, mononuclear cells reactive to TRAPase, presumably preosteoblasts, frequently occurred near preosteoblasts. Osseointegration around the implants was obtained in this model by 28 days post-implantation except for the lateral area with complete contact with implants, where the thin layer remained in contact with the implant surface.
Conclusions: These findings indicate that ossification proceeds at different modes around the titanium implant in rat maxilla, depending on the nature of the recipient bones and the dimension of the gap between the implant and osteotomy margin.

DOI： 10.1902/jop.2000.71.2.287

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Histological Documentation  

The present study reports on different regeneration patterns of axons and Schwann cells in the periodontal ligament of the rat incisor using immunohistochemistry of protein gene product 9.5 (PGP 9.5) and S-100 protein. Three kinds of injury (transection, crush and segmental resection) were applied to the inferior alveolar nerve. In normal animals, PGP 9.5- and S- 100-immunoreactivities were detected in the axons and Schwann cell elements of periodontal Ruffini endings, respectively. They were restricted to the alveolus-related part, occurring only rarely in the tooth-related part and in the shear zone (the border between the alveolus-related and tooth-related parts). Both transection and segmental resection caused the complete disappearance of PGP 9.5-immunoreactive nerve fibers in the periodontal ligament, while a small number of them could be found following the crush injury. Regenerating PGP 9.5-reactive nerve fibers appeared at 5 days and 21 days following the transection and segmental resection, respectively. The regeneration of periodontal nerve fibers completed in a period of 21-28 days and 14-21 days following the transection and crush, respectively, but was not completed even at 56 days following the segmental resection. The behavior of Schwann cells during regeneration was similar after the different nerve injuries
spindle-shaped S-100-immunoreactive cells, presumably Schwann cells, appeared in the shear zone and the tooth-related part. These cells disappeared 5-7 days prior to the complection of the regeneration of axonal elements of the periodontal ligament following the transection and crush. Following the segmental resection, in contrast, spindle-shaped S-100-positive cells disappeared from the tooth-related part at 42 days, although the axonal regeneration of periodontal Ruffini endings proceeded even until 56 days. We thus conclude that the duration of the migration of Schwann cells depends on the state of the regeneration of axons.

DOI： 10.1679/aohc.63.43

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Anatomists  

The periodontal ligament, a dense collagenous tissue between tooth and alveolar bone, receives a rich sensory nerve supply, and contains tsvo kinds of sensory receptors
nociceptor and mechanoreceptor. The mechanical stimuli for teeth can evoke various oral reflexes, which facilitate mastication via the periodontal mechanorecepors. In spite of many reports on the periodontal sensory receptors, recent studies have revealed that the Ruffini endings, categorized as low-threshold slowly adapting type II (SA II) stretch receptors, are primary mechanoreceptors in the periodontal ligament. This paper summarized recent findings on the morphological features and developmental aspects of the periodontal Ruffini endings.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：S. Karger AG  

Expression of calbindin D28k (CB)-like immunoreactivity (-LI) was compared with that of protein gene product 9.5 (PGP 9.5), a general neuronal marker, in the periodontal ligament of the rat lower incisor following resection of the inferior alveolar nerve (IAN). In normal animals, the periodontal nerve fibers showing PGP 9.5-LI formed either Ruffini endings with expanded arborization or thin free nerve endings in the alveolar half of the ligament. Thick CB-like immunoreactive (-IR) nerve fibers terminated in a dendritic fashion in the same region, but thin CB-IR nerve fibers were rarely detected. During the 3 days following resection of the IAN, most of the PGP 9.5-IR and all CB-IR nerve fibers disappeared. Regenerated PGP 9.5-IR nerve fibers appeared around 7 days after resection, in contrast to the very small number of regenerated CB-IR nerve fibers. Around 21-28 days following resection, the number and terminal morphology of regenerated PGP 9.5-IR nerve fibers were comparable to those observed in normal animals, but the number of regenerated CB-IR nerve fibers was still smaller. The terminal morphologies of these regenerated CB-IR nerve fibers showed less expansion compared with normal animals at these post-injured periods. The number of regenerated CB-IR nerve fibers increased gradually to return to normal by 56 days following injury. The delayed expression of CB in the regenerated periodontal Ruffini endings suggests that the functional recovery of periodontal Ruffini endings occurred after the regeneration of periodontal Ruffini endings had been completed. Copyright (C) 2000 S. Karger AG, Basel.

DOI： 10.1159/000016791

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The present study employed immunohistochemistry for the detection of S-100 proteins to reveal the alteration in the distribution of Schwann cells in the periodontal ligament of the rat incisor following resection of the inferior alveolar nerve (IAN). In normal animals, S-100-immunostaining demonstrated the profiles of Ruffini endings, primary mechanoreceptors in the periodontal ligament, in the alveolus-related part of the ligament. Under the electron microscope, S-100-like immunoreactivity (-LI) was observed in the cytoplasm of the terminal Schwann cell elements and in some axon profiles of the Ruffini endings. During the regeneration, S-100-like immunoreactive (-IR) terminal Schwann cells in the alveolus-related part of the ligament gradually decreased in number. In contrast, S-100-LI was found in the spindle-shaped cells at the shear zone (the border between alveolus-related and tooth-related parts) and in the tooth-related part, where S-100-LI was rarely detected in normal animals. Immunoelectron microscopic observations revealed that some S-100-IR spindle-shaped cells contained fibrous long spacing (FLS) fibers, suggesting that they were Schwann cells. Some regenerating axons were observed at the shear zone, but were rarely found in the tooth-related part. With the progress of the regeneration of the periodontal Ruffini endings, S-100-IR terminal Schwann cells became rearranged in the alveolus-related part by 42-56 days post injury, whereas the S-100-IR spindle-shaped Schwann cells in the shear zone and tooth-related part disappeared when the regeneration was complete. Copyright (C) 1999 Elsevier Science B.V.

DOI： 10.1016/S0006-8993(99)02153-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

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 immune-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 Hsp 25 might be involved in the inhibition of apoptosis of those cells.

DOI： 10.1679/aohc.62.483

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT SOC HISTOLOGY & CYTOLOGY  

The present immunocytochemical study examined the localization of Trk B, a high affinity neurotrophin receptor, in the neural elements of the periodontal ligament of the rat incisor. In light microscopy, the immunoreactivity was demonstrated in dendritic profiles in the alveolar half of the periodontal ligament. Their location and morphological features indicated that they were periodontal Ruffini endings. Occasional rounded cells associated with periodontal Ruffini endings, which had immunonegative kidney-shaped nuclei, were immunoreactive; these were judged to be terminal Schwann cells. Immunoelectron microscopy revealed the heterogeneous localization of Trk B among individual Ruffini endings. Some terminal Schwann cells contained immunoreactive products for Trk B in the cytoplasm, while others did not. Similarly, a part of the Schwann sheaths covering the axon terminals showed Trk B immunoreactivity. Most axon terminals associated with periodontal Ruffini endings were immunopositive for Trk B, though a few of them were immunonegative. The ordinary Schwann cells did not contain Trk B immunoreactive products. These findings imply that Trk B is required for the maintenance of periodontal Ruffini endings, The different expression pattern of Trk B suggests that neuronal and glial elements comprising individual periodontal Ruffini endings are subject to heterogeneous conditions with regard to the requirement of Trk B.

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