Aki Kawazoe

Inhibitory effects of orally administrated liposomal bovine lactoferrin on the LPS-induced osteoclastogenesis

Bovine lactoferrin (bLF) modulates the production of proinflammatory cytokines including tumor necrosis factor (TNF)-α, and may thus control alveolar bone destruction associated with periodontitis. In this study, the effects of bLF on mRNA expression in lipopolysaccharide (LPS)-stimulated osteoblasts (OBs) and on LPS-induced osteoclastogenesis were examined. The inhibitory effects of oral administration of liposomal-bLF (L-bLF), which improved the robustness of bLF to digestive enzymes, on alveolar bone resorption using LPS-induced periodontitis rat model are also reported. Three groups of 7-week-old male Wistar rats were treated with L-bLF (L-bLF group), bLF (bLF group), or the vehicle (control group) in drinking water (n=6 in each group). On day 7, LPS was topically applied into the gingival sulcus. Number of osteoclasts and immunoexpression of TNF-α were analyzed. The bLF inhibited the upregulation of TNF-α-mRNA- and upregulation of receptor activator of NFκB (RANKL)-mRNA expression and eliminated downregulation of osteoprotegerin (OPG)-mRNA expression in LPS-stimulated OBs and reduced LPS-induced osteoclastogenesis in co-culture with primary OBs and bone marrow cells. In the control group, the number of osteoclasts increased after LPS treatment. The number of osteoclasts that appeared along the alveolar bone margin was significantly reduced (P<0.01) in the L-bLF but not in the bLF group. Furthermore, L-bLF suppressed upregulation of TNF-α immunoexpression in periodontal tissue and TNF-α and interleukin (IL)-1β-mRNA level in gingival tissue. The results of this study indicate that oral administration of L-bLF significantly reduces alveolar bone resorption induced by LPS stimulation through inhibition of TNF-α production and modulation of RANKL/OPG balance in OBs. It is suggested that L-bLF could be a potent therapeutic and preventive agent for attenuating alveolar bone destruction in periodontitis patients.

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.

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.

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.

An adolescent patient with multiple impacted teeth.

Multiple impacted permanent teeth is uncommon and rarely reported in the literature. This article reports the treatment of an adolescent patient with multiple impacted teeth without systemic disease. A 9-year 2-month-old boy complained of a delay of eruption of the first molars. All first molars were unerupted, and the left deciduous second molar was a submerged tooth. The panoramic radiograph showed all permanent teeth except the incisors were unerupted and, especially for the first molars, spontaneous eruption was not expected. His medical history was uneventful. A lingual arch appliance and a segmental arch were placed on the mandibular and maxillary dentitions, respectively, to guide eruption of the impacted first molars. After traction of the first molars, eruption of the impacted lower premolars was induced. Furthermore, at 15 years the impacted mandibular second molars were also positioned properly by use of the lingual arch with auxiliary wires. After achieving traction of the impacted teeth, tooth alignment was initiated using multibracket appliances after the bilateral extraction of the second premolars. After 22 months of treatment with multibracket appliances, an acceptable occlusion was achieved with a Class I molar relationship. After 2 years of retention an acceptable occlusion was maintained without any relapse in the occlusion. Since a delay in the treatment of impacted teeth may induce secondary problems such as root dilacerations and ankylosis, it is highly recommended to perform early treatment of multiple impacted teeth during adolescence.

Ultrasound stimulation attenuates root resorption of rat replanted molars and impairs tumor necrosis factor-α signaling in vitro

BACKGROUND AND OBJECTIVE A therapeutic protocol to minimize root resorption induced by tooth replantation has not yet been universally established. In this context, noninvasive modality such as ultrasound therapy have been a focus of increased interest. This study aimed to evaluate the inhibitory effect of ultrasound therapy on root resorption of replanted rat molars. In addition, the study aimed to promote insights into the mechanism through which ultrasound mediates the metabolism of periodontal cells in vitro. MATERIAL AND METHODS An experimental model of tooth replantation in rats, involving luxation and immediate replacement of the maxillary first molars, was used to assess the inhibitory effect of an ultrasound-therapy regimen (15 min of exposure to ultrasound, each day for 21 d) on root resorption. Moreover, the effect of ultrasound on osteoclastogenesis/cementoclastogenesis was examined in vitro using a mouse osteoblastic stromal cell line (ST2) and a mouse cementoblastic cell line (OCCM-30). RESULTS The area of root resorption lacunae was statistically decreased (p < 0.01) in the ultrasound-treated sample. In addition, immunohistochemical staining, using murine TNF-α polyclonal antibody, failed to detect tumor necrosis factor-α (TNF-α) protein in the ultrasound-treated sample compared with the control. An in vitro study showed that the lipopolysaccharide (LPS)-induced expression of Tnfalpha mRNA was significantly reduced by ultrasound therapy in both osteoblastic and cementoblastic cells. Moreover, the TNF-α-induced up-regulation of Rankl mRNA was also inhibited by ultrasound. CONCLUSION Ultrasound may contribute to the reduction of the trauma-induced inflammatory reaction through impairment of the TNF-α signaling pathway. It is therefore suggested that ultrasound shows potential as a therapeutic tool to optimize the regenerative potential of periodontal tissues on replanted teeth.

Correction of severe open bite using miniscrew anchorage.

This report describes the treatment of a case of severe open bite with posterior crossbite. While treating open bite, the outcome may not always be successful with orthodontic therapy alone. In such cases, surgical therapy is often chosen to gain a stable occlusion. Skeletal anchorage systems such as miniscrews are now frequently used for correcting severe malocclusion. In this report, we treated an open bite by intruding the molars with miniscrews placed bilaterally in the interdental space between both the upper and lower posterior teeth. The active treatment period was 36 months and the patients teeth continued to be stable after a retention period of 36 months.

Orally Administered Liposomal Lactoferrin Inhibits Inflammation-Related Bone Breakdown Without Interrupting Orthodontic Tooth Movement

BACKGROUND Bovine lactoferrin (bLF) modulates the production of tumor necrosis factor-alpha (TNF-α) and inhibits alveolar bone breakdown associated with periodontitis. This study is designed to examine the effects of orally administered liposomal bLF (LbLF) on orthodontic force (OF)-induced alveolar bone remodeling during experimental tooth movement. METHODS Two groups of male Wistar rats were treated with either LbLF or control solution in drinking water 7 days before OF application. Lipopolysaccharide (LPS) was injected into the gingival sulcus in half the rats in each group. Thus, four groups: OF, OF+LbLF, OF+LPS, and OF+LPS+LbLF were established. RESULTS Orally administered LbLF significantly reduced apical migration of junctional epithelium in the OF and OF+LPS groups. In OF+LPS, osteoclast number in the alveolar crestal area was increased by LPS treatment, whereas osteoclast number was significantly reduced in OF+LPS+LbLF through suppression of TNF-α production. Osteoclastic induction in the middle part, mainly from OF application, was not affected by LbLF administration. Inhibition of tooth movement was not induced by LbLF. CONCLUSIONS Orally administered LbLF significantly inhibits LPS-induced alveolar bone resorption but not OF-induced bone remodeling. LbLF could be a potent therapeutic and preventive agent to control periodontal inflammation in patients undergoing orthodontic treatment.

Treatment of a Case of Skeletal Class II Malocclusion with Temporomandibular Joint Disorder Using Miniscrew Anchorage

Abstract At the present time, there are no reports in the literature on the treatment of temporomandibular joint disorder (TMD) by intrusion of molars using mini-screws. This case report describes the treatment for a female patient, aged 19 years seven months, with a TMD and an excessive lower anterior facial height. Overjet and overbite were +5.0 mm and +0.5 mm, respectively. The patient had a history of orthodontic treatment in which her first premolars were all extracted. During the first orthodontic treatment, a clockwise mandibular rotation was observed as a result of the increase of posterior dentoalveolar height. She had temporomandibular joint (TMJ) pain during mouth opening and complained of difficulty in eating due to masticatory dysfunction. The pretreatment Schüller views of both TMJ showed a posterior condyle position. In order to correct the overjet, molar relationship and the mandibular condyle position, a miniscrew was inserted into the palatal region of the upper first molar to intrude the upper posterior teeth. As the upper molars were intruded, the overjet was decreased, and a class I molar relationship was achieved by a counterclockwise mandibular rotation. After one year of treatment, an acceptable occlusion was achieved, and the condyle moved into centric position in the glenoid fossa. The patient’s teeth continued to be stable, and she had no pain in TMJ after a retention period of three years. The result of this treatment showed that molar intrusion using miniscrew anchorage is effective for treatment of a TMD patient with a posterior condyle position.