Alterations to Titanium Surface Depending on the Fluorides and Abrasives in Toothpaste.

Fluoride and abrasives in toothpastes may cause corrosion and deterioration of the titanium used for implants and other prostheses. The purpose of this study was to investigate how the presence or absence and types of fluoride and abrasives affected the titanium surface texture. Brushing with toothpastes was performed on pure-titanium discs using an abrasive testing machine. Unprocessed titanium discs without brushing were used as control samples. Surface roughness, color, and gloss of titanium were measured and the differences compared with the control were analyzed. Additionally, titanium surfaces and abrasives in toothpastes were observed using a scanning electron microscope to compare the surface texture of each sample. Some toothpastes (abrasive+) significantly increased the difference in surface roughness, color, and gloss, compared with ultrapure water. Toothpaste (fluoride+/abrasive+) that had many polygonal abrasive particles led to the largest color differences and exhibited notable scratches and a larger number of contaminant- or corrosion-like black spots. In contrast, brushing with toothpaste without fluoride or abrasives (fluoride-/abrasive-) caused little change to the titanium surface. These results suggest that both fluoride and abrasives in toothpaste used for brushing may be factors that affect surface texture and corrosion resistance of titanium.

Porcine Dental Epithelial Cells Differentiated in a Cell Sheet Constructed by Magnetic Nanotechnology.

Magnetic nanoparticles (MNPs) are widely used in medical examinations, treatments, and basic research, including magnetic resonance imaging, drug delivery systems, and tissue engineering. In this study, MNPs with magnetic force were applied to tissue engineering for dental enamel regeneration. The internalization of MNPs into the odontogenic cells was observed by transmission electron microscopy. A combined cell sheet consisting of dental epithelial cells (DECs) and dental mesenchymal cells (DMCs) (CC sheet) was constructed using magnetic force-based tissue engineering technology. The result of the iron staining indicated that MNPs were distributed ubiquitously over the CC sheet. mRNA expression of enamel differentiation and basement membrane markers was examined in the CC sheet. Immunostaining showed Collagen IV expression at the border region between DEC and DMC layers in the CC sheet. These results revealed that epithelial-mesenchymal interactions between DEC and DMC layers were caused by bringing DECs close to DMCs mechanically by magnetic force. Our study suggests that the microenvironment in the CC sheet might be similar to that during the developmental stage of a tooth bud. In conclusion, a CC sheet employing MNPs could be developed as a novel and unique graft for artificially regenerating dental enamel.

Soluble RANKL Cleaved from Activated Lymphocytes by TNF-α-Converting Enzyme Contributes to Osteoclastogenesis in Periodontitis.

Host immune responses play a key role in promoting bone resorption in periodontitis via receptor activator of NF-κB ligand (RANKL)-dependent osteoclastogenesis. Both membrane-bound RANKL (mRANKL) expressed on lymphocytes and soluble RANKL (sRANKL) are found in periodontal lesions. However, the underlying mechanism and cellular source of sRANKL release and its biological role in periodontitis are unclear. TNF-α-converting enzyme (TACE) is reported to cleave the following: 1) precursor TNF-α with release of mature, soluble TNF-α and 2) mRANKL with release of sRANKL. Both soluble TNF-α and sRANKL are found in the periodontitis lesion, leading to the hypothesis that TACE expressed on lymphocytes is engaged in RANKL shedding and that the resulting sRANKL induces osteoclastogenesis. In the current study, upon stimulating PBLs with mitogens in vitro, RANKL expression, sRANKL secretion, and TACE expression were all upregulated. Among the four putative mRANKL sheddases examined in neutralization assays, TACE was the only functional sheddase able to cleave mRANKL expressed on PBL. Moreover, PBL culture supernatant stimulated with mitogens in the presence of anti-TACE Ab or anti-RANKL Ab showed a marked reduction of osteoclastogenesis from osteoclast precursors, indicating that TACE-mediated sRANKL may possess sufficient osteoclastogenic activity. According to double-color confocal microscopy, B cells expressed a more pronounced level of RANKL and TACE expression than T cells or monocytes in periodontally diseased gingiva. Conditioned medium of patients' gingival lymphocyte culture increased in vitro osteoclastogenic activity, which was suppressed by the addition of anti-TACE Ab and anti-RANKL Ab. Therefore, TACE-mediated cleavage of sRANKL from activated lymphocytes, especially B cells, can promote osteoclastogenesis in periodontitis.

A-Disintegrin and Metalloproteinase (ADAM) 17 Enzymatically Degrades Interferon-gamma.

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that exerts anti-tumor and anti-osteoclastogenic effects. Although transcriptional and post-transcriptional regulation of IFN-γ is well understood, subsequent modifications of secreted IFN-γ are not fully elucidated. Previous research indicates that some cancer cells escape immune surveillance and metastasize into bone tissue by inducing osteoclastic bone resorption. Peptidases of the a-disintegrin and metalloproteinase (ADAM) family are implicated in cancer cell proliferation and tumor progression. We hypothesized that the ADAM enzymes expressed by cancer cells degrades IFN-γ and attenuates IFN-γ-mediated anti-tumorigenic and anti-osteoclastogenic effects. Recombinant ADAM17 degraded IFN-γ into small fragments. The addition of ADAM17 to the culture supernatant of stimulated mouse splenocytes decreased IFN-γ concentration. However, ADAM17 inhibition in the stimulated mouse T-cells prevented IFN-γ degradation. ADAM17-expressing human breast cancer cell lines MCF-7 and MDA-MB-453 also degraded recombinant IFN-γ, but this was attenuated by ADAM17 inhibition. Degraded IFN-γ lost the functionality including the inhibititory effect on osteoclastogenesis. This is the first study to demonstrate the extracellular proteolytic degradation of IFN-γ by ADAM17. These results suggest that ADAM17-mediated degradation of IFN-γ may block the anti-tumorigenic and anti-osteoclastogenic effects of IFN-γ. ADAM17 inhibition may be useful for the treatment of attenuated cancer immune surveillance and/or bone metastases.

Increase in receptor activator of nuclear factor κB ligand/osteoprotegerin ratio in peri-implant gingiva exposed to Porphyromonas gingivalis lipopolysaccharide.

BACKGROUND/PURPOSE: The prevalence of peri-implant diseases, including peri-implant mucositis and peri-implantitis, is increasing. The aim of this study was to elucidate the pathological mechanisms of inflammation and alveolar bone resorption in peri-implant tissues. To do this, we fabricated inflamed gingiva around mini-implants in the palatine processes of rats using lipopolysaccharide derived from Porphyromonas gingivalis (P.g-LPS). MATERIALS AND METHODS: Pure titanium mini-implants were implanted into the palatine processes of rats, and then intermittent injections of P.g-LPS were made into the gingival tissues surrounding the mini-implants. The expression patterns of tumor necrosis factor-α, interleukin-1ß, chemokine (C-C motif) ligand 2, receptor activator of nuclear factor κB ligand (RANKL), and osteoprotegerin (OPG) in the tissues were examined using real-time reverse transcriptase polymerase chain reaction or enzyme-linked immunosorbent assays. Immunohistochemical analysis was also performed to compare the T and B cells expressing RANKL. RESULTS: P.g-LPS increased the expressions of tumor necrosis factor-α, interleukin-1ß, chemokine (C-C motif) ligand 2, and RANKL in the gingival tissues surrounding the mini-implants. In contrast, the expression of OPG in the P.g-LPS samples was decreased. Consequently, the RANKL/OPG ratio was significantly increased. Moreover, cells stained positively for both anti-CD3 and anti-RANKL antibodies were only found in the samples treated with P.g-LPS. CONCLUSION: These data revealed that P.g-LPS injections increased the RANKL/OPG ratio in the gingival tissues surrounding mini-implants in the rat model. In addition, the CD3-positive cells in the gingival tissues injected with P.g-LPS expressed RANKL. This suggests that the activated T cells capable of infiltrating gingival tissues affected by P.g-LPS may be one of the sources of RANKL and may also be involved in the disease progression from peri-implant mucositis to peri-implantitis.

Inhibition of RANKL-dependent cellular fusion in pre-osteoclasts by amiloride and a NHE10-specific monoclonal antibody.

The functions of Na(+) /H(+) exchangers (NHEs) during osteoclastic differentiation were investigated using the NHE inhibitor amiloride and a monoclonal antibody (MAb). Compared with sRANKL-stimulated control cells, amiloride decreased the number of large TRAP-positive osteoclast cells (OCs) with ≥10 nuclei and increased the number of small TRAP-positive OCs with ≤10 nuclei during sRANKL-dependent osteoclastic differentiation of RAW264.7 cells. NHE10 mRNA expression and OC differentiation markers were increased by sRANKL stimulation in dose- and time-dependent manners. NHEs 1-9 mRNA expression was not increased by sRANKL stimulation. Similar to amiloride, a rat anti-mouse NHE10 MAb (clone 6B11) decreased the number of large TRAP-positive OCs, but increased the number of small TRAP-positive OCs. These findings suggested that inhibition of NHEs by amiloride or an anti-NHE10 MAb prevented sRANKL-promoted cellular fusion. The anti-NHE10 MAb has the potential for use as an effective inhibitor of bone resorption for targeted bone disease therapy.

Release of titanium ions from an implant surface and their effect on cytokine production related to alveolar bone resorption.

Although interest in peri-implant mucositis and peri-implantitis has recently been increasing, the mechanisms driving these diseases remain unknown. Here, the effects of titanium ions on the inflammation and bone resorption around an implant were investigated. First, the accumulated amount of Ti ions released into gingival and bone tissues from an implant exposed to sodium fluoride solution was measured using inductively coupled plasma mass spectrometry. Next, the cellular responses in gingival and bone tissues to Ti ions and/or Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) were assessed using a rat model. More Ti ions were detected in the gingival tissues around an implant after treatment with sodium fluoride (pH 4.2) than in its absence, which suggests that the fluoride corroded the implant surface under salivary buffering capacity. The injection of Ti ions (9ppm) significantly increased the mRNA expression and protein accumulation of chemokine (C-C motif) ligand 2, as well as the ratio of receptor activator of nuclear factor-κB ligand to osteoprotegerin, in rat gingival tissues exposed to P. gingivalis-LPS in a synergistic manner. In addition, the enhanced localization of toll-like receptor 4, which is an LPS receptor, was observed in gingival epithelium loaded with Ti ions (9ppm). These data suggest that Ti ions may be partly responsible for the infiltration of monocytes and osteoclast differentiation by increasing the sensitivity of gingival epithelial cells to microorganisms in the oral cavity. Therefore, Ti ions may be involved in the deteriorating effects of peri-implant mucositis, which can develop into peri-implantitis accompanied by alveolar bone resorption.

Involvement of ERK and p38 MAPK pathways on Interleukin-33-induced RANKL expression in osteoblastic cells.

The receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) system is a well-known key factor in osteoclast differentiation, and osteoblastic lineage cells are the major sources of RANKL and OPG in local bone tissue. Recently, a new molecule from the interleukin (IL)-1 family, IL-33, was identified. Here, we report the possible involvement of IL-33 in RANKL and OPG expression, and the signaling pathways that are required for maximal IL-33-induced RANKL expression in MC3T3-E1 osteoblastic cells. Stimulation with IL-33 increased the mRNA expression and secretion of RANKL in MC3T3-E1 cells. The IL-33-induced RANKL mRNA expression was inhibited by an anti-IL-33 monoclonal antibody. Furthermore, ERK and p38 MAPK inhibitors, but not a JNK inhibitor, suppressed IL-33-induced RANKL mRNA expression. On the other hand, IL-33 had no effect on OPG mRNA expression and protein secretion. These results taken together suggest that IL-33 stimulates RANKL expression through mechanisms dependent on the ERK and p38 MAPK pathways in MC3T3-E1 cells.

Evaluation of trabecular bone formation in a canine model surrounding a dental implant fixture immobilized with an antimicrobial peptide derived from histatin.

JH8194 induces osteoblast differentiation, although it was originally designed to improve antifungal activity. This suggests that JH8194 is useful for implant treatment. Therefore, the aim of this study was to evaluate the osseointegration capacity of JH8194-modified titanium dental implant fixtures (JH8194-Fi). The implants were randomly implanted into the edentulous ridge of dog mandibles. Healing abutments were inserted immediately after implant placement. Three weeks later, peri-implant bone levels, the first bone-to-implant contact points, and trabecular bone formation surrounding the implants were assessed by histological and digital image analyses based on microcomputed tomography (microCT). The histological analysis revealed an enhancement of mature trabecular bone around the JH8194-Fi compared with untreated fixtures (control-Fi). Similarly, microCT combined with analysis by Zed View™ also showed increased trabecular bone formation surrounding the JH8194-Fi compared with the control-Fi (Student's t-test, P < 0.05). JH8194 may offer an alternative biological modification of titanium surfaces to enhance trabecular bone formation around dental implants, which may contribute to the transient acquirement of osseointegration and the long-term success of implant therapy.

Blocking of sodium and potassium ion-dependent adenosine triphosphatase-α1 with ouabain and vanadate suppresses cell-cell fusion during RANKL-mediated osteoclastogenesis.

To examine the possible enrolment of Na(+)/K(+)-ATPase during osteoclast differentiation, Na(+)/K(+)-ATPase inhibitors, including ouabain and vanadate, were used in this study. These inhibitors significantly inhibited cell-cell fusion of RAW264.7 cells and bone marrow cells induced by RANKL. Interestingly, in response to RANKL-stimulation, ouabain and vanadate decreased the number of large TRAP+ osteoclasts in the culture of RAW264.7 cells, as well as bone marrow cells. In contrast, the number of small TRAP+ osteoclasts either increased in RAW264.7 cells or were otherwise less affected in bone marrow cells than large TRAP+ osteoclasts. Large TRAP+ osteoclasts are defined as having ≥ 10 nuclei/cell and having more potency in bone resorption than small multinuclear osteoclasts with <9 nuclei/cell. Na(+)/K(+)-ATPase α1 and ß2 mRNAs were detected in sRANKL-stimulated RAW264.7 cells. Moreover, real-time quantitative PCR showed that ouabain and vanadate suppressed the RANKL-dependent induction of the osteoclast fusion-promotion molecule DC-STAMP at the mRNA level. Finally, and importantly, RNAi-mediated suppression of Na(+)/K(+)-ATPase α1 resulted in a diminished number of large TRAP+ osteoclasts in the sRANKL-stimulated RAW264.7 cells, along with the decreased level of DC-STAMP mRNA expression. These findings strongly suggest that blockage of the Na(+)/K(+)-ATPase α1 subunit by ouabain or vanadate caused the inhibition of RANKL-induced cell-cell fusion, resulting in the generation of large osteoclasts through suppression of DC-STAMP expression. Thus, in addition to its known function of sodium and potassium ion exchange during bone resorption by mature osteoclasts, this study has revealed a novel molecular role of the Na(+)/K(+)-ATPase α1 subunit in osteoclastogenesis.

Aggregatibacter actinomycetemcomitans Omp29 is associated with bacterial entry to gingival epithelial cells by F-actin rearrangement.

The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa) into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29), a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29(+)/OmpA(-) E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9) than Omp29(-)/OmpA(-) E. coli. While the entry of Aa and Omp29(+)/OmpA(-) E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK), a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin) and Rho GTPases inhibitor (EDIN), both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway.

Bovine milk fermented with Lactobacillus rhamnosus L8020 decreases the oral carriage of mutans streptococci and the burden of periodontal pathogens.

AIM: The aim of this study was to find the oral isolate of lactobacilli, which has the potential to inhibit either periodontal, cariogenic, or fungal pathogens in vitro, and to examine the effects of bovine milk fermented with the isolate on the oral carriage of cariogenic and periodontal pathogens. METHODS: The inhibitory effects of the supernatant of Man-Rogosa-Sharpe broth, in which each of 42 oral isolates of lactobacilli grown, was examined. One isolate, Lactobacillus rhamnosus L8020, that showed the potential to inhibit either periodontal, cariogenic, or fungal pathogens in vitro, was used to examine the effects of fermented milk on the oral carriage of cariogenic and periodontal pathogens, which was examined by a placebo-controlled and cohort trial using 50 participants. RESULTS: Edible yogurt containing Lactobacillus rhamnosus L8020 significantly reduced the oral carriage of mutans streptococci (P < 0.01) and four periodontal pathogens examined: Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, and Fusobacterium spp. (P < 0.01), but the phenomenon were not observed with the placebo yogurt (P > 0.05). CONCLUSION: These results suggest that yogurt with Lactobacillus rhamnosus L8020 could reduce the risk of dental caries and periodontal disease.

Titanium ion induces necrosis and sensitivity to lipopolysaccharide in gingival epithelial-like cells.

Gingival epithelial-like cells (GE-1) were cultured and used to examine the cellular responses of gingival tissues to varying concentrations of titanium (Ti) ions. Titanium ions at concentrations of more than 13 ppm significantly decreased the viability of GE-1 cells and increased LDH release from the cells into the supernatant, but had no significant effect on their caspase 3 activity. These data suggest that a high concentration of Ti ions induced necrosis of the GE-1 cells. Titanium ions at a concentration of 5 ppm significantly increased the level of CCL2 mRNA expression in GE-1 cells exposed to lipopolysaccharide derived from Porphyromonas gingivalis in a synergistic manner. Moreover, the mRNA expression levels of TLR-4 and ICAM-1 in GE-1 cells loaded with Ti ions at 9 ppm were significantly enhanced as compared with those in GE-1 cells without Ti stimulation. We suggest that Ti ions are in part responsible for monocyte infiltration in the oral cavity by elevating the sensitivity of gingival epithelial cells to microorganisms. Taken together, these data indicate that Ti ions may be involved in cytotoxicity and inflammation at the interfaces of dental implants and gingival tissue.

Titanium immobilized with an antimicrobial peptide derived from histatin accelerates the differentiation of osteoblastic cell line, MC3T3-E1.

The objective of this study was to evaluate the effect of titanium immobilized with a cationic antimicrobial peptide (JH8194) derived from histatin on the biofilm formation of Porphyromonas gingivalis and differentiation of osteoblastic cells (MC3T3-E1). The titanium specimens (Ti) were immobilized with JH8194, according to the method previously described. The colonization of P. gingivalis on JH8194-Ti was significantly lower than that on control- and blocking-Ti. JH8194-Ti enhanced the mRNA expressions of Runx2 and OPN, and ALPase activity in the MC3T3-E1, as compared with those of control- and blocking-Ti. These results, taken together, suggested the possibility that JH8194-Ti may be a potential aid to shorten the period of acquiring osseointegration.

Immobilized-OPG-Fc on a titanium surface inhibits RANKL-dependent osteoclast differentiation in vitro.

The purpose of the present study was to examine the effect of osteoprotegerin (OPG)-Fc fusion protein immobilized on a titanium surface on the initial differentiation of osteoclast precursor RAW264.7 cells. These cells were cultured on titanium specimens over which OPG-Fc was immobilized. The enhancement of tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expression in RAW264.7 cells exposed to receptor activator of NF-kappaB ligand (RANKL) stimulation on OPG-Fc-coated titanium was significantly lower than that in RAW264.7 cells exposed to RANKL on titanium specimens without immobilized OPG-Fc (ANOVA, P < 0.01). Preincubation of OPG-Fc-coated titanium, in a medium supplemented with 10% fetal bovine serum at 37 degrees C for two days before the cells were seeded, had no significant effect on the decrease in mRNA expression (ANOVA, P < 0.01). Taken together, these results indicate that OPG-Fc immobilized on a titanium surface blocks the differentiation of RAW264.7 cells induced by RANKL stimulation.

Impact of titanium ions on osteoblast-, osteoclast- and gingival epithelial-like cells.

PURPOSE: To investigate the effects of titanium (Ti) ions on the cell viability, the cell differentiation and the gene expressions related to bone resorption including Receptor Activator of NF-kappaB Ligand (RANKL) and Osteoprotegerin (OPG) in the tissues around dental implants, the osteoblast-, osteoclast-, and gingival epithelial-like cells were exposed to Ti ions. METHODS: An MTS assay was carried out to evaluate the viabilities of osteoblast-like MC3T3-E1, osteoclast-like RAW264.7 and epithelial cell-like GE-1 cells. The gene expressions in these cells were analyzed by the use of RT-PCR and real-time quantitative RT-PCR. RESULTS: Ti ions in the concentration range 1-9 ppm had little effect on the viabilities of MC3T3-E1, RAW264.7 and GE-1, whereas 20 ppm Ti ions significantly decreased the viabilities of all cells. Analyses of RT-PCR and real-time quantitative RT-PCR data revealed that Ti ions at 9 ppm remarkably inhibited the expressions of Runx2, Osterix and type I collagen in MC3T3-E1. In RAW264.7, Ti ions showed no effects on the levels of mRNAs for TRAP and cathepsin K enhanced by RANKL. Ti ions at the range of 1-9 ppm showed no effects on the levels of mRNAs for RANKL and OPG in GE-1, while Ti ions at 9 ppm enhanced the expression of these genes in MC3T3-E1. CONCLUSIONS: These results, taken together, suggested that Ti ions show the biological effects, both on the viabilities of osteoblast and osteoclast and on the differentiation of either the osteoblastic or osteoclastic cells, which may influence the prognosis of dental implants.

[Functional model of the middle ear ossicles].

In students' dissection practice, it is very difficult to teach students the structures and functions of the middle ear ossicles. The middle ear ossicles are too small to explain their structures and functions. Models are useful in explaining these points, but there have been no models that accurately explain the movements of the middle ear ossicles and the functions of the muscles in the middle ear. This time, we have made a model of middle ear ossicles. Our ear ossicles are made of paper-mache with metal in it. The incudomalleolar and incudostapedial articulations are made of rubber. The tensor tympani and the stapedius muscles are made of wire and the two wires can be fixed by cord stoppers. Our model explains clearly the following mechanisms of the middle ear ossicles. 1. The mechanism of sound conduction system. When the sound vibrates the tympanic membrane, malleus and incus rotate together. The long process of the incus pushes the head of the stapes. The sound is amplified by leverage. 2. Attenuation of sound by contractions of tensor tympani and stapedius muscles. When a loud sound is transmitted through the ossicular system, the tensor tympani muscle pulls the malleus inward while the stapedius muscle pulls the stapes outward. These two forces oppose each other and increase rigidity of the ossicular system, thus reducing the ossicular conduction. 3. The mechanism of how paralysis of stapedius muscle, caused by an injury to the facial nerve, results in hyperacusis. 4. This model also suggests a possible reason why the pars lucida of the tympanic membrane exists.

Research projects related to complete dentures published in 2008 by members of the Japan Prosthodontic Society.

This article reviews the findings of the original papers related to complete dentures published in the Journal of the Japan Prosthodontic Society (Nippon Hotetsu Shika Gakkai Zasshi), Vol. 52, 2008. A total of six articles focused on complete dentures or materials related to complete dentures were selected and summarized. A variety of subjects in relation to removable prosthodontics were discussed in the articles, including denture plaque control, resilient denture lining materials or denture adhesives, and assessment and/or anatomical analysis of prognosis of complete dentures.

Impact of the microgravity environment in a 3-dimensional clinostat on osteoblast- and osteoclast-like cells.

Mechanical unloading conditions result in decreases in bone mineral density and quantity, which may be partly attributed to an imbalance in bone formation and resorption. To investigate the effect of mechanical unloading on osteoblast and osteoclast differentiation, and the expression of RANKL and OPG genes in osteoblasts, we used a three-dimensional (3D) clinostat system simulating microgravity to culture MC3T3-E1 and RAW264.7 cells. Long-term exposure (7 days) of MC3T3-E1 cells to microgravity in the 3D clinostat inhibited the expression of Runx2, Osterix, type I collagen alphaI chain, RANKL and OPG genes. Similarly, 3D clinostat exposure inhibited the enhancement of beta3-integrin gene expression, which normally induced by sRANKL stimulation in RAW264.7 cells. These results, taken together, demonstrate that long-term 3D clinostat exposure inhibits the differentiation of MC3T3-E1 cells together with suppression of RANKL and OPG gene expression, as well as the RANKL-dependent cellular fusion of RAW264.7 cells, suggesting that long-term mechanical unloading suppresses bone formation and resorption.

[Model for the functional instruction of swallowing].

It is difficult to teach students about the mechanism of swallowing. There are three phases of swallowing; oral phase, pharyngeal phase and esophageal phase. The bolus of food is propelled to back of mouth by the tongue and the swallowing reflex happens. After nasopharynx and mouth closure, the glottal closure occurs, then hyoid and larynx are lifted by the contractions of suprahyoid and thyrohyoid muscles. As for the epiglottis, it is compressed by the tongue and inclines downward. As the larynx is lifted upward and anteriorly, slight vacuum is caused in the lower pharynx and upper esophagus at the same time, and pharyngeal constrictor compress bolus, therefore, the bolus passes the piriform fossa, and is inhaled into the esophagus. This time, we made a model in order to explain this complicated mechanism. The mandible is made of paper clay by using a metallic plate in it. The tongue, the soft palate, and the epiglottis are made by using the EVA (Ethylene Vinyl Acetate) sheet. Styloglossus, suprahyoid, thyrohyoid muscles are made with the wire. Moreover, a movable wooden chip represents the contraction of the pharyngeal constrictor muscles. The spring is put in the trachea in order to lift the larynx. The upper part of esophageal constrictor is made with spring plates.