Journal of Hard Tissue Biology
Online ISSN : 1880-828X
Print ISSN : 1341-7649
ISSN-L : 1341-7649
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  • Daisuke Yamaguchi, Kazuo Takeuchi, Atsuko Ueno, Daisuke Kato, Shin Miy ...
    2021 Volume 30 Issue 1 Pages 1-6
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    Drug repositioning (DR) is a strategy to explore new medicinal effects from existing approved drugs whose safety and pharmacokinetics have already been established. We focused on geranylgeranylacetone (GGA), which is known as a heat shock proteins (HSPs) inducing agent. GGA is mainly used as a gastric mucosal protective agent; however, its effects on bone tissues have not been studied. Therefore, we hypothesized that “GGA induces HSPs in osteoblasts thereby promotes cell differentiation”, and administered GGA to MC3T3E-1 cells to examine cell responses. Methods: MC3T3E-1 were cultured in osteogenic medium. After the cultures were established, test cultures were exposed to GGA (GGA group). Cell proliferation, collage synthesis and ALP activity were measured on days 7 and 14 of culture. Alizarin Red S staining was performed on days 21 of culture. Results: On days 14 of culture, cell proliferation and collage synthesis were significantly higher in the GGA group than in the control group (P<0.05). On days 7 and 14 of culture, ALP activity was significantly higher in the GGA group than in the Control group (P<0.05). On days 28 of culture, the Alizarin Red S stained areas were significantly higher in the GGA group than in the control group (P<0.05). Conclusion: GGA promoted the differentiation of MC3T3E-1 in an in vitro cell culture model.

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  • Mayumi Maesako, Takafumi Kishimoto, Taku Horie, Miki Suzuki, Kazuho In ...
    2021 Volume 30 Issue 1 Pages 7-12
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    In this study, we performed alkaline degradation testing to investigate the microstructural properties of a new resin composite composed of a single paste containing no pigments that still has the same basic structure as conventional pigment-containing composites. Our aim was to identify factors that affect the matching of various shades of teeth using structural color technology. The effect of these structural properties on the surface properties of the resin composite was also investigated. We found that the structural properties of the new composite included a wide distribution of many spherical organic fillers of various sizes. In addition, spherical inorganic filler of 260 nm in diameter was uniformly distributed at almost the same density both within the organic filler and within the base resin surrounding it. The organic filler and base resin, which are regarded as difficult to couple, were strongly bound without any gaps forming even after alkaline degradation testing. Although the surface layer of the organic filler was vulnerable to alkaline degradation, the center part exhibited very low degradation similar to the matrix surrounding the organic filler. In the new resin composite, the bonding state between the base resin and the various fillers was significantly improved. Furthermore, the microstructural properties were inferred to be effective factors for producing structural color, including the shape, particle diameter, distribution mode and density of the fillers as well as the properties of the base resin. However, these structural properties were not found to affect surface properties such as the line roughness, surface roughness, gloss, discoloration, and wettability.

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  • Sho Aoki, Kyoko Harada, Saki Kawai, Yoko Abe, Sachiko Nagata, Rie Imat ...
    2021 Volume 30 Issue 1 Pages 13-18
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    Appropriate heat shock results in the production of heat shock proteins (HSPs) whose expression and phosphorylation contribute to repair of damaged proteins, cell proliferation, and cell recovery from shock stimuli. However, there is no information regarding the expression of HSPs in human deciduous dental pulp fibroblast-like cells (hDDPF) in response to mild short-term heat shock. The aim of this study was to investigate the cellular effects of mild short-term heat shock on hDDPF. Cells were subjected to heat shock at 43°C–49°C for 15 min, and cell proliferation was assessed by 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. mRNA and protein expressions of HSP27, 70, and 90 were detected by reverse transcription PCR and western blot analysis, respectively. Phosphorylation of the AKT and ERK signaling pathways of HSP production was evaluated by western blotting. Heat shock at 43°C for 15 min increased the cell proliferation rate and the mRNA expressions of HSP27, 70, and 90 in hDDPF. Moreover, protein expression of HSP70 was significantly enhanced 24 h after heat shock, and the phosphorylation of AKT was also confirmed. Because HSP70 is critical in tissue repair and regeneration, mild short-term heat shock may enhance tissue repair in hDDPF.

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  • Bayarmaa Batzorig, Kenjiro Nakano, Kosei Murata, Mayumi Maesako, Kazuh ...
    2021 Volume 30 Issue 1 Pages 19-26
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    In the treatment of dentin hypersensitivity accompanying tooth substance defects such as wedge-shape defects, hypoesthesia can be achieved by applying a desensitizing agent before carrying out restoration using resin composite. However, almost no research has investigated the adhesion of resin to dentin coated with the latest desensitizing agents. Therefore, this study investigated the effects of various desensitizing agents on the adhesion of resin to dentin in combination with a 1-step self-etch system by using a hypersensitive dentin model in which the dentinal tubules were opened without etching and there was almost no smear layer on the intertubular dentin. Specimens with a #4000 polished dentin flat surface were ultrasonically cleaned for 60 min (15 min × 4 times). Then, the bond strength, failure modes, and micromorphology of surfaces coated with desensitizing agent to which resin was bonded immediately afterward and surfaces coated with desensitizing agent to which the resin was bonded after storage for 7 days in water were compared against a control to which no desensitizing agent was applied. The desensitizing agents used in this research did not promote adhesion of the resin immediately after application, but rather suppressed or completely obstructed it. Although deposits of microparticles and thin film material, which were observed immediately after application, tended to disappear after 7 days of storage in water, some of the desensitizing agents exhibited the same bond strength as the control, whereas other desensitizing agents did not show recovery of adhesion strength. Therefore, care is required when performing resin restoration immediately after application of a desensitizing agent, depending on the agent used, and caution must be exercised in the selection of desensitizing agents in the clinical setting.

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  • Masahiro Murakami, Keiji Nagano, Kouichi Hamaoka, Daisuke Kato, Tatsus ...
    2021 Volume 30 Issue 1 Pages 27-32
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    Ozone water has long been known as a bactericidal disinfectant. However, the bactericidal effect of ozone water on bacteria associated with oral diseases has not been thoroughly examined. Further, although oral bacteria reside in biofilms, few studies have explored the effects of ozone water on biofilms. In this study, we aimed to investigate the bactericidal effect of ozone water on bacteria and bacterial biofilms associated with oral diseases. We examined the bactericidal and cleaning effects of ozone water on pathogenic bacteria associated with oral diseases (Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus mutans, and Porphyromonas gingivalis) under planktonic and biofilm growth conditions. When planktonic bacteria were exposed to 5-ppm ozone water, a remarkable antibacterial activity was observed against all of the tested bacterial species. Contrarily, biofilms showed high resistance to ozone water; the bacterial load only slightly decreased even after repeated exposure to ozone water. However, when ozone water was continuously applied at a low flow rate to the biofilms on polystyrene disks, the number of bacteria on the disks was significantly decreased. Our results have shown that the continuous application of ozone water can eliminate oral disease-related bacteria even in biofilms.

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  • Yoshimasa Makita, Kazuya Masuno, Yasuhiro Imamura, Shin-ichi Fujiwara, ...
    2021 Volume 30 Issue 1 Pages 33-36
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    Cyanoacrylate adhesive is used worldwide as instant glue because of their one-drop-curing properties. Medical cyanoacrylate adhesive exhibits high adhering strength; its safety for use has been established from its clinical application, use in the living body, and oral use. A hydroxyapatite pellet (HAP) coated with cyanoacrylate adhesive was left in pH 4.0 phthalate standard solution and the concentration of eluted calcium ion was measured. The concentration of eluted calcium ion were 0.4 mM (HAP coated with a cyanoacrylate adhesive) and 6.2 mM (control study of HAP), respectively. This result indicates that the coating with a cyanoacrylate adhesive can inhibit the elution of HAP even in acidic conditions. Cyanoacrylate adhesive was applied to the extracted teeth in the presence of artificial saliva, and the thickness of the adhered area was observed. The thickness of the cyanoacrylate adhesive adhered to the extracted tooth has slightly decreased until the 5th day. The thickness has hardly changed until the 31st. This result shows that the cyanoacrylate adhesive was not removed from the tooth surface in the presence of artificial saliva.

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  • Maki Nagasaki, Kumiko Nakai, Hideki Tanaka, Manami Ozaki, Kengo Kato, ...
    2021 Volume 30 Issue 1 Pages 37-44
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    Preexisting diseases, such as diabetes and chronic inflammation in periodontal tissue, are risk factors associated with bisphosphonate-related osteonecrosis of the jaw. Osteoblasts produce prostaglandin (PG)E2 via cyclooxygenases (COX), and the autocrine action of PGE2 impacts the function of osteoblasts, including receptor activator of NF-kappa B ligand (RANKL) and osteoprotegerin (OPG) production. This study assessed the effects of the stimulation of zoledronate in the presence of lipopolysaccharide (LPS) and high concentrations of glucose on the expression of COX-2, RANKL, and OPG, in addition to PGE2 production in osteoblasts. MG-63 cells were cultured in medium containing 1 µg/ml LPS, 25 mM glucose (high glucose), and/or zoledronate (1×10-8, 1×10-7, 1×10-6, 5×10-6, or 1×10-5 M). The mRNA expression of COX-2, RANKL, and OPG genes was determined by real-time polymerase chain reaction. The concentrations of RANKL and OPG protein and PGE2 in the culture supernatant were examined by enzyme-linked immunosorbent assay. Zoledronate at a concentration of 5×10-6 M overwhelmingly increased COX-2 mRNA expression. The expression levels of RANKL and OPG as well as PGE2 production was significantly increased in cells stimulated with 5×10-6 M zoledronate in the presence of LPS and high glucose than in the unstimulated cells (control). NS398, a specific inhibitor of COX-2, blocked the stimulatory effects of zoledronate (in the presence of LPS and high glucose) on PGE2 production and the protein expression levels of RANKL and OPG. The ratio of RANKL/OPG was also increased following zolendronate stimulation. In addition, a significant difference was observed not in the stimulation with zoledronate alone, but by the stimulation of zoledronate in the presence of LPS and high glucose as compared that in controls. These results suggest that LPS and high concentrations of glucose enhances zoledronate-induced increase in RANKL/OPG ratio via the autocrine action of NS398-blocked PGE2 in osteoblasts.

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  • Zhen Li, Ming Dong, Wen Li, Ling Li, Xinxin Yu, Ying Kong, Hui Kong
    2021 Volume 30 Issue 1 Pages 45-52
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    The sinonasal inverted papilloma is one of the more common benign tumors of the nasal cavity and sinus. It originates from the schneiderian membrane. It has the characteristics of easy recurrence and malignant transformation. This experiment found that Cytokeratin 8 expression increased in the sinonasal inverted papilloma. To better detect the role of Cytokeratin 8 in the proliferation and deterioration of the sinonasal inverted papilloma, we divided the sinonasal inverted papilloma into NIPN, NIPAP, and NIPAH by histological. The expression of Cytokeratin 8 in the sinonasal inverted papilloma was detected by immunohistochemistry. We found that the increased expression of Cytokeratin 8 was consistent with the invasion of sinonasal squamous cell carcinoma. In vitro, it was found that after Cytokeratin 8 interference, the proliferative and invasion of head and neck squamous cells were decreased. Cytokeratin 8 played an important role of sinonasal inverted papilloma malignant transformation to sinonasal squamous cell carcinoma. Inhibition of Cytokeratin 8 could diminish the proliferation and block the invasion of head and neck squamous cells.

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  • Takenori Kanai, Yoshihiko Sawa, Yoshiaki Sato
    2021 Volume 30 Issue 1 Pages 53-62
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    This study aims to investigate the effect of CLEC-2 on calcification in cultured mouse osteoblasts. In the RT-PCR and cell ELISA analysis, it was confirmed that osteoblasts express podoplanin, osteopontin, osteocalcin and sclerostin in culture, and that expressions of osteopontin and osteocalcin increased in calcification medium. The expression of podoplanin, osteopontin, osteocalcin and sclerostin did not change in osteoblasts with CLEC-2, indicating that CLEC-2 does not affect the expression of these bone proteins in osteoblasts. However, the amounts of calcified nodules and alkaline phosphatase activity were significantly suppressed in cultured osteoblasts by CLEC-2. The quantitative analysis showed that both the calcified nodule amount and alkaline phosphatase activity decreased with CLEC-2 while there was no influence in the cell viability with CLEC-2. Further, the expression of RUNX2 was observed in cytoplasm and in nucleus of cultured mouse osteoblasts while the expression decreased with CLEC-2. In Matrigel-based three-dimensional culture a significant cell process elongation of osteoblasts was observed and the elongation was strongly suppressed with CLEC-2. Considering these, CLEC-2 may have an ability to cancel the calcification of osteoblasts by blocking the maturation of osteoblast via interaction with CLEC-2 receptor podoplanin without any involvements of bone-associated protein production.

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  • Kazuko T Goto, Hiroshi Kajiya, Takashi Tsutsumi, Munehisa Maeshiba, Ta ...
    2021 Volume 30 Issue 1 Pages 63-68
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    Mechanical stress (MS) during hyperocclusion results in elimination of the alveolar hard line, enhancement of bone resorption, and shedding of a tooth, resulting in trauma of occlusion. Several studies have indicated that MS induces cytokine and chemokine expression during alveolar bone absorption in periodontitis and orthodontic treatment. However, it remains unknown regarding the effect of hyperocclusal MS on maintenance of alveolar bone metabolism. Using in vivo and in vitro hyperocclusion models, we investigated the effect of MS on relationships between the expression of stress-dependent and osteoblastogenesis-associated chemokines. In the in vitro model with 6-weeks old mice, MS upregulated the expression of stromal cell-derived factor-1 (SDF-1) in periodontal tissues, a self-renewal chemokine in bone marrow stem cell day 4 after MS. In contrast, in the in vivo model with 30-weeks old mice, SDF-1 expression was significantly upregulated at day 1 after MS in periodontal tissues. MS induced the expression of SDF-1 in the PDL root branch and of the SDF-1 receptor CXCR4 in the dental pulp and bone marrow on day 4 in an in vivo mice model. MS upregulated the expression of SDF-1 in PDL cells. SDF-1 binds to CXCR4, resulting in MS-resistant alveolar bone protection during occlusal traumatism.

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  • Hiroyuki Nakano, Sho Mizobuchi, Kei Suzuki, Kazuya Inoue, Naofumi Yama ...
    2021 Volume 30 Issue 1 Pages 69-72
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    The morphology of the mandible using homologous modeling and principal component analysis, and the accuracy of sex determination based on mandibular morphology were examined. The computed tomography (CT) scans of 84 subjects (44 males, 40 females; mean age, 42.4 ± 15.4 years) were selected for this study. To avoid any effect on the morphology of the mandible, the scans of subjects with fewer than 14 remaining teeth were excluded. Homologous modeling and principal component analysis were performed using mHBM (Digital Human Techbology, Tokyo, Japan) and HBM-Rugle software (Medic Engineering, Kyoto, Japan), respectively. The contribution of the first principal component was 20.8% and that of the second principal component was 11.4%. There was a significant difference between male and female in the first principal component (Wilcoxon test, p < 0.05). Subjects with a negative first principal component value were considered more likely to be female, and those with positive values were more likely to be male (accuracy rate, 61.9%). ROC analysis of this method revealed AUC of 0.62, sensitivity of 0.48, and specificity of 0.78. Multivariate analysis was performed using all principal component values, and ROC analysis performed based on these results revealed AUC of 0.85, sensitivity of 0.82, and specificity of 0.85. Analysis using only the first principal component had lower sensitivity and specificity than reported previously, but the results using all principal component values were similar to those in past reports. This method was considered to be useful for sex determination based on mandibular morphology.

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  • Jing Zhang, Da-peng Xu, Jiang Shang, Xin-jie Liang, Xue-bin Zhang
    2021 Volume 30 Issue 1 Pages 73-78
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    The aim of the present study was to investigate the induction of osteogenic activity and osteogenic differentiation of canine bone marrow stromal cells (BMSCs) by naringin. BMSCs were separated and cultured in vitro and identified by flow cytometry. Then, different concentrations of naringin (1×10-5, 1×10-6, 1×10-7, 1×10-8, and 1×10-9 mol/l) were added to BMSCs cultured in DMEM to induce their differentiation. The effects of naringin on BMSCs were evaluated by CCK-8 assay and by measuring the activity of alkaline phosphatase (ALP). Calcium nodules, a marker of osteogenesis, were detected by alizarin red staining. The results of cell-surface marker analysis showed that the cells were negative for CD34 and CD45 expression, with values of 0.126% and 0.075%, respectively, and positive for CD90 expression, a value of 95.4%. Naringin at a concentration of 10-6 mol/l obviously promoted cell proliferation, among the concentrations, this concentration achieved the best effects on proliferation and osteogenic differentiation. Calcium nodule staining was positive. The findings demonstrate that naringin can induce the differentiation of bone marrow stromal cells into osteoblasts and that naringin at a concentration of 10-6 mol/l can enhance the proliferation and osteogenic differentiation of BMSCs.

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  • Ning-Ning Sun, Dong-Mei He, Chi Yang, Qing Zhou
    2021 Volume 30 Issue 1 Pages 79-84
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    The present study investigated the changes in Indian hedgehog (Ihh), periarticular cell-derived parathyroid hormone-related protein (PTHrP), and runt-related transcription factor 2 (Runx2) in the temporomandibular joint (TMJ) cartilage with posttraumatic osteoarthritis (PTOA). The miniature pigs were randomly divided into two groups: the experimental group (EG) (n=8) and the control group (CG) (n=4). The left side of EG had type B intracapsular fractures with anterior disc displacement (ICF+DD), while the right side only had type B intracapsular fractures (ICF), and the CG was a blank control. The production of Ihh, PTHrP, and Runx2 was detected by immunohistochemistry staining and real-time polymerase chain reaction (PCR) at weeks 4 and 12 post-surgery. The expression of Ihh, PTHLH /PTHrP, and Runx2 in the EG was significantly lower than that in the CG at 4 and 12 weeks after the operation (P<0.05). Moreover, significant differences were detected between ICF+DD and ICF (P<0.05). Ihh, PTHHL/PTHrP, and Runx2 proteins affect the endochondral osteogenesis of TMJ and play a significant role in PTOA. Our findings suggested that the interaction mechanism among Ihh, PTHLH/PTHrP, and Runx2 is activated when posttraumatic osteoarthritis (PTOA) occurs, but how they regulate each other remains to be investigated.

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Clinical Note
  • Xu-hui Fan, Lei Yue, Peng-Fei Qu, Wei Yang, Ji-Lun Liu, Yao-Qiang Liu, ...
    2021 Volume 30 Issue 1 Pages 85-92
    Published: 2021
    Released: January 27, 2021
    JOURNALS FREE ACCESS

    The present study aimed to establish a strategy to accurately remove teeth adjacent to mesially impacted wisdom teeth. Geometric principles were applied to analyze the resistance of teeth adjacent to mesially impacted wisdom teeth before extraction, and the resistance of adjacent teeth was relieved. The traditional method used to extract mesially impacted wisdom teeth is the chisel technique, which has been gradually replaced by minimally invasive extraction because of its great degree of trauma. This study determined the cutting method, cutting line position, and cutting direction of mesially impacted wisdom teeth based on imaging data from panoramic radiographs under geometric guidance. In the case of a short cutting line, cutting and separation were completed within one attempt, the resistance of adjacent teeth was alleviated, and the surgical duration was shortened.

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