Toshihiro Inubushi

Dynamic Compressive Properties of the Mandibular Condylar Cartilage

The mandibular condylar cartilage plays an important role as a stress absorber during function. However, relatively little information is available on its dynamic properties under compression. We hypothesized that these properties are region-specific and depend on loading frequency. To characterize the viscoelastic properties of the condylar cartilage, we performed dynamic indentation tests over a wide range of loading frequencies. Ten porcine mandibular condyles were used; the articular surface was divided into 4 regions, anteromedial, anterolateral, posteromedial, and posterolateral. The dynamic complex, storage, and loss moduli increased with frequency, and these values were the highest in the anteromedial region. Loss tangent decreased with frequency from 0.68 to 0.17, but a regional difference was not found. The present results suggest that the dynamic compressive modulus is region-specific and is dependent on the loading frequency, which might have important implications for the transmission of load in the temporomandibular joint.

Cementoblast response to low- and high-intensity ultrasound

OBJECTIVE It has been shown that ultrasound stimulation accelerates repair of orthodontically induced root resorption. However, the mechanism of such adaptive change is still unclear. The purpose of this study was to evaluate pulsed ultrasound on the differentiation-potential and cementoblast-mediated osteoclastogenesis using a cementoblastic cell line. DESIGN Cultured cementoblasts (murine cementoblastic cell line, OCCM-30) were subjected to ultrasound exposure (frequency = 1 MHz; pulsed 1:4; spatial average temporal average intensities=30 or 150 mW/cm(2)) or sham exposure for 15 min per day. Expression levels of alkaline phosphatase (ALP), type I collagen (COL-I), osteoprotegerin (OPG) and receptor activator of nuclear factor small ka, CyrillicB ligand (RANKL) mRNAs were analysed by a real-time PCR analysis. Furthermore, ALP activity, collagen synthesis, and protein levels of OPG and RANKL were examined after 6-day ultrasound exposure. RESULTS Real-time PCR analysis indicated that, irrespective of the intensity, single ultrasound exposure increased the expression of transcripts for COL-I and ALP after 24h; the expression of OPG and RANKL also increased after 1 and 4h, respectively. Cultured cementoblasts receiving ultrasound stimulation for 6 days showed a significant (p<0.05 or 0.01) increase in cell number and collagen synthesis. ALP activity and OPG synthesis were also significantly (p<0.05) upregulated by ultrasound stimulation with 150 mW/cm(2). CONCLUSIONS These results demonstrated that ultrasound stimulation especially with 150 mW/cm(2) might be a better candidate as a medical remedy to protect against root resorption and/or accelerate its repair.

Molecular mechanisms of the inhibitory effects of bovine lactoferrin on lipopolysaccharide-mediated osteoclastogenesis.

Background: We previously reported that bovine lactoferrin (bLF) reduces LPS-induced bone resorption. However, it was not clear how bLF inhibits LPS-mediated osteoclastogenesis through osteoblasts. Results: bLF impaired LPS-mediated NFκB activation by interfering with TNF receptor-associated factor 6 (TRAF6) polyubiquitination. Conclusion: bLF is a potent anti-inflammatory agent. Significance: bLF may hold promise as a therapeutic agent for inflammatory diseases associated with bone destruction. Lactoferrin (LF) is an important modulator of the immune response and inflammation. It has also been implicated in the regulation of bone tissue. In our previous study we demonstrated that bovine LF (bLF) reduces LPS-induced bone resorption through a reduction of TNF-α production in vivo. However, it was not known how bLF inhibits LPS-mediated TNF-α and RANKL (receptor activator of nuclear factor κB ligand) production in osteoblasts. In this study we show that bLF impairs LPS-mediated TNF-α and RANKL production. bLF inhibited LPS-mediated osteoclastogenesis via osteoblasts in a co-culture system. Furthermore, bLF pretreatment inhibited LPS-induced NFκB DNA binding activity as well as IκBα and IKKβ (IκB kinase β) phosphorylation. MAP kinase activation was also inhibited by bLF pretreatment. However, bLF pretreatment failed to block the degradation of IRAK1 (interleukin-1 receptor-associated kinase 1), which is an essential event after its activation. Remarkably, we found that bLF pretreatment inhibited LPS-mediated Lys-63-linked polyubiquitination of TNF receptor-associated factor 6 (TRAF6). We also found that bLF is mainly endocytosed through LRP1 (lipoprotein receptor-related protein-1) and intracellular distributed bLF binds to endogenous TRAF6. In addition, bLF inhibited IL-1β- and flagellin-induced TRAF6-dependent activation of the NFκB signaling pathway. Collectively, our findings demonstrate that bLF inhibits NFκB and MAP kinase activation, which play critical roles in chronic inflammatory disease by interfering with the TRAF6 polyubiquitination process. Thus, bLF could be a potent therapeutic agent for inflammatory diseases associated with bone destruction, such as periodontitis and rheumatoid arthritis.

Dental Infection of Porphyromonas gingivalis Induces Preterm Birth in Mice.

Background Epidemiological studies have revealed a link between dental infection and preterm birth or low birth weight (PTB/LBW), however, the underlying mechanisms remain unclear. Progress in understanding the associated mechanisms has been limited in part by lack of an animal model for chronic infection-induced PTB/LBW, mimicking pregnancy under conditions of periodontitis. We aimed to establish a mouse model of chronic periodontitis in order to investigate the link between periodontitis and PTB/LBW. Methods To establish chronic inflammation beginning with dental infection, we surgically opened mouse (female, 8 weeks old) 1st molar pulp chambers and directly infected with w83 strain Porphyromonas gingivalis (P.g.), a keystone periodontal pathogen. Mating was initiated at 6 wks post-infection, by which time dental granuloma tissue had developed and live P.g. was cultured from extracted tooth root, which serves as a persistent source of P.g. The gestational day (gd) and birth weight were recorded during for P.g.-infected and control mice, and serum and placental tissues were collected at gd 15 to evaluate the systemic and local conditions during pregnancy. Results Dental infection with P.g. significantly increased circulating TNF-α (2.5-fold), IL-17 (2-fold), IL-6 (2-fold) and IL-1β (2-fold). The P.g.-infected group delivered at gd 18.25 vs. gd 20.45 in the non-infected control (NC) group (p < 0.01), and pups exhibited LBW compared to controls (p < 0.01). P.g. was localized to placental tissues by immunohistochemistry and PCR, and defects in placental tissues of P.g. infected mice included premature rupture of membrane, placental detachment, degenerative changes in trophoblasts and endothelial cells, including necrotic areas. P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2. Further placental tissue damage was indicated in P.g. infected mice by decreased CD-31 in endothelial cells, increased expression of 8OHdG, an indicator of oxidative DNA damage, and cleaved caspase-3, a marker of apoptosis. In vitro, P.g. lipopolysaccharide significantly increased expression of COX-2, IL-8 and TNF-α, in HTR-8 trophoblasts in an NF-κB-dependent fashion. Conclusions Our novel mouse model supports previous epidemiological studies signifying dental infection as predisposing factor for PTB/LBW. We demonstrate PTB and LBW in infected mice, translocation of P.g to placental tissues, increased circulating and local pro-inflammatory markers, and the capability of P.g. LPS to directly induce cytokine production in trophoblasts, in vitro. These findings further underscore the importance of local and systemic infections and inflammation during pregnancy and suggest that prevention and/or elimination of dental infections such as marginal or periapical periodontitis before pregnancy may have a beneficial effect on PTB/LBW.

Infection with Porphyromonas gingivalis exacerbates endothelial injury in obese mice.

Background A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg), on pathogenesis of atherosclerosis in obesity. Methods In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD) or normal chow diet (CD), as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1) were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA) induced endothelial cells apoptosis and regulation of cytokine gene expression. Results Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate. Conclusions Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury.

ULTRASOUND STIMULATION INDUCES PGE2 SYNTHESIS PROMOTING CEMENTOBLASTIC DIFFERENTIATION THROUGH EP2/EP4 RECEPTOR PATHWAY

The present study aims to provide insights into how ultrasound treatment (US) can affect the regenerative response of cementum by evaluating the role of prostaglandin E(2) induced by ultrasound stimulation on cementoblastic differentiation. The mouse cementoblast cell line OCCM-30 was exposed to low-intensity ultrasound and the cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin E(2) (PGE(2)) production were quantified. The role of the US-induced PGE(2) in mineralization was examined using COX-2 inhibitor and prostaglandin receptors (EP-receptors) agonists and antagonists. In addition, gene expression of differentiation markers related to mineral metabolism was evaluated. Ultrasound significantly enhanced COX-2 mRNA expression and PGE(2) production. PGE(2) induced by US mediated mineral nodule formation, whereas COX-2 inhibitor treatment eliminated the enhancement of mineralization induced by US stimulation. Mineral deposition was also inhibited by treatment with EP2 or EP4 antagonist. Moreover, up-regulation of differentiation markers induced by US was suppressed by treatment with COX-2 inhibitor. The present findings provide evidence that US stimulation has a positive effect on mineralization ability of cementoblasts through the activation of EP2/EP4 pathway, suggesting that US can be a promising therapeutic tool for cementum repair.

Effect of PGE2 induced by compressive and tensile stresses on cementoblast differentiation in vitro

OBJECTIVE The aim of the study was to clarify the mechanisms underlying orthodontically induced root resorption by characterizing the role of PGE(2) induced by compressive stress (CS) and tensile stress (TS) on cementoblast metabolism in vitro. DESIGN Mouse cementoblast cell line OCCM-30 was continuously stimulated with 0.2 KPa CS or 5.0 KPa TS. COX-2 mRNA expression and PGE(2) production were thus quantified. In addition, cells were treated with COX-2 inhibitor and the role of PGE(2) induced by CS or TS on the expression of genes related to cementoblast differentiation was examined. PGE(2) receptors mRNA expression induced by CS or TS was also evaluated. Moreover, cells were treated with exogenous PGE(2) and the role of PGE(2) concentration on matrix mineralization was verified. RESULTS CS and TS enhanced COX-2 mRNA expression and PGE(2) production. PGE(2) synthesis, however, was markedly induced by CS. Gene expression of bone morphogenetic protein 2 (BMP-2), osteocalcin (OCN) and receptor activator for nuclear factor kappaB ligand (RANKL) was enhanced by CS on an endogenous PGE(2)-mediated manner. Osteoprotegerin (OPG) expression was not affected by CS. Meanwhile, TS up-regulated the expression of BMP-2 and alkaline phosphatase (ALP) on an endogenous PGE(2)-mediated manner. TS down-regulated RANKL mRNA expression, whilst OPG expression was not affected. Moreover, EP4 mRNA expression was considerably enhanced by TS. Regarding PGE(2) concentration, only cells treated with low concentration presented anabolic response. CONCLUSIONS Gene expression was differentially regulated according to the type of mechanical stimulation applied to cementoblasts. In addition, it is shown that PGE(2) plays an important role on mediating cementoblast mechanosensitivity.

Effects of low-intensity pulsed ultrasound on the expression of cyclooxygenase-2 in mandibular condylar chondrocytes.

AIMS To determine the effect of low-intensity pulsed ultrasound (LIPUS) on cyclooxygenase-2 (COX-2) expression and related mechanisms by using cultured articular chondrocytes derived from porcine mandibular condyles after treatment with interleukin-1 beta (IL-1β). METHODS Chondrocytes were derived from porcine mandibular condylar cartilage and cultured. The cells were treated with or without 10 ng/mL IL-1β. At the same time, the cells were exposed to LIPUS for 20 minutes. After LIPUS exposure, the conditioned medium was changed to a fresh one without IL-1β, and the cells were incubated for 0 to 24 hours. The effects of LIPUS on IL-1β-treated chondrocytes were examined in terms of the expression of p-integrin β1, COX-2, and phosphorylated extracellular signal-related kinase (p-ERK) 1/2 by real-time polymerase chain reaction (PCR) and Western blot analyses. Differences in the means among multiple groups were examined by one-way analysis of variance (ANOVA) for all groups at each time point, followed by a Scheffé multiple comparison test as a post-hoc test; Student t test was also used. RESULTS COX-2 mRNA level was upregulated by the treatment with IL-1β and was suppressed significantly (P < .01) by LIPUS exposure. Furthermore, LIPUS enhanced gene expression and phosphorylation of integrin β, and it inhibited the expression of p-ERK 1/2. CONCLUSION LIPUS exposure inhibited IL-1β-induced COX-2 expression through the integrin β1 receptor followed by the phosphorylation of ERK 1/2. Despite the restricted duration of its effect, LIPUS is suggested to be a potential candidate as a preventive and auxiliary treatment to suppress the degradation of articular chondrocytes in temporomandibular joint osteoarthritis.

P2X7 receptor in the trigeminal sensory nuclear complex contributes to tactile allodynia/hyperalgesia following trigeminal nerve injury

The present study directly addresses the roles of the P2X7 receptor (P2X7R), an ionotropic adenosine triphosphate (ATP) receptor, and cytokines in the induction of orofacial pain following chronic constriction injury (CCI) of the infraorbital nerve (IoN).

Ultrasound stimulation attenuates resorption of tooth root induced by experimental force application

Root resorption is an adverse outcome of orthodontic tooth movement. However, there have been no available approaches for the protection and repair of root resorption. The aim of this study was to evaluate the effects of low-intensity pulsed ultrasound (LIPUS) on root resorption during experimental tooth movement and the effects of LIPUS in the RANKL/OPG mechanism in osteoblasts and cementoblasts in vitro. Twenty four Wistar strain male rats of 12-week-old were used in this study. The upper first molars were subjected to experimental movement in the mesial direction for 1-3weeks. Through the experimental periods, the right upper first maxillary molar was exposed to LIPUS (LIPUS group) every day for 1, 2 or 3weeks. The nature of root resorption was observed and then quantified by histomorphometric analysis. In the 2weeks period, significantly greater amount of tooth movement was observed in the LIPUS group (p<0.05). In addition, LIPUS group showed less root resorption lacunae and lower number of odontoclasts. In the period of 3weeks, LIPUS group presented significantly shorter length of root resorption lacunae and smaller amount of root resorption area (p<0.01). The number of odontoclasts and osteoclasts was also significantly lower in the LIPUS group (p<0.01 and p<0.05, respectively). However, no significant differences could be found regarding the amount of tooth movement. It is shown that LIPUS exposure significantly reduced the degree of root resorption during tooth movement without interrupting tooth movement. In vitro experiments showed that MC3T3-1 constitutively expressed higher levels of RANKL and RANTES mRNA comparing to OCCM-30. However, OPG mRNA expression was much higher in OCCM-30. LIPUS stimulation significantly increased the mRNA expression of RANKL in MC3T3-E1 at 4 (p<0.01) and 12h (p<0.05), although OPG mRNA expression was not affected by LIPUS. In contrast, the expression of RANKL and OPG mRNAs were both significantly increased by LIPUS in OCCM-30 at 12h (p<0.01). Moreover, LIPUS application suppressed the up-regulation of RANKL mRNA induced by compression force in OCCM-30, but no similar effect could be observed in MC3T3-E1. In conclusion, it is suggested that LIPUS exposure significantly reduces root resorption by the suppression of cementoclastogenesis by altering OPG/RANKL ratio during orthodontic tooth movement without interfering tooth movement. LIPUS may be an effective tool to prevent root resorption during tooth movement and is applicable to clinical use in near future.