Mandibular Endochondral Growth Is Specifically Augmented by Nutritional Supplementation with Myo-Inositol Even in Rabbits.

Mandibular retrognathism occurs by insufficient mandibular growth and causes several issues, such as respiratory difficulty and diminished masticatory function. At present, functional orthodontic appliances are used for stimulating mandibular growth in pediatric cases. However, the effectiveness of functional appliances is not always stable in daily practices. A more effective, reliable, and safer therapeutic method for mandibular growth promotion would be helpful for growing mandibular retrognathism patients. As we previously discovered that nutritional supplementation of myo-inositol in growing mice specifically increases mandibular endochondral growth, we performed preclinical animal experiments in rabbits in this study. Briefly, six-week-old male Japanese white rabbits were fed with or without myo-inositol supplementation in laboratory chow until 25 weeks old, and 3D image analysis using micro CT data and histological examinations was done. Myo-inositol had no systemic effect, such as femur length, though myo-inositol specifically augmented the mandibular growth. Myo-inositol increased the thickness of mandibular condylar cartilage. We discovered that the nutritional supplementation of myo-inositol during the growth period specifically augmented mandibular growth without any systemic influence, even in rabbits. Our results suggest the possibility of clinical use of myo-inositol for augmentation of the mandibular growth in growing mandibular retrognathism patients in the future.

Orthodontic Management of the Edentulous Space Caused by Surgical Removal of a Large Dentigerous Cyst.

Herein, we report the orthodontic management of a patient with excessive bone and permanent tooth loss after surgical cyst removal. The patient was a 13-year-old Japanese boy who was referred to our department by an oral surgeon. He had an edentulous space with alveolar bone loss and loss of 2 permanent molars in the left mandibular region, following surgical removal of a large dentigerous cyst. We decided to close this space orthodontically. First, we moved the left mandibular second premolar into the edentulous region and autotransplanted the left maxillary lateral incisor in the adjacent distal space. We then performed comprehensive orthodontic treatment to establish stable occlusion. Following treatment, functional and stable occlusion of all permanent teeth was achieved without any spaces. The findings from this case suggest that orthodontic treatment is effective in growing patients with edentulous spaces and alveolar bone loss.

Effects of the Dental Caries Preventive Procedure on the White Spot Lesions during Orthodontic Treatment-An Open Label Randomized Controlled Trial.

(1) Background: The aim of this study was to assess the preventive effect of tooth surface disinfection treatment, in addition to fluoride application, during fixed orthodontic treatment. (2) Methods: An open label randomized control trial for the evaluation of the dental caries preventive procedure was performed for the patients with high caries risk who had been visited at Department of Orthodontics, Tsurumi University Dental Hospital for orthodontics treatment. The follow-up period was six months. White spot lesions (WSLs) were evaluated by quantitative light-induced fluorescence (QLF). Cariogenic bacteria were monitored and evaluated by bacterial culture. In addition, the oral microbiome was evaluated by a next-generation sequence (NGS). (3) Results: By the mixed effect modeling, tooth surface disinfection treatment significantly reduced cariogenic bacteria and all parameters obtained by QLF. (4) Conclusions: Tooth surface disinfection treatment, in addition to PMTC and fluoride application, were effective for dental caries prevention and keeping a healthy microbiome during orthodontic treatment.

Low-intensity pulsed ultrasound enhances the rate of lateral tooth movement and compensatory bone formation in rats.

INTRODUCTION: Because mechanical stimulation of the periodontal ligament by low-intensity pulsed ultrasound (LIPUS) has been shown to increase the speed of bone remodeling, this study aimed to examine the effects of LIPUS stimulation on the rate of tooth movement and bone remodeling during lateral tooth movement. METHODS: Twelve-week-old Wistar rats were divided into 2 groups. The LIPUS group received experimental tooth movement with LIPUS stimulation, and the tooth movement (TM) group were provided experimental tooth movement without LIPUS. For each group, the upper right first molars were moved buccally with fixed appliances. LIPUS exposure was placed in the region corresponding to the right maxillary first molar. Three days after tooth movement, tartrate-resistant acid phosphatase was examined. Fourteen days after tooth movement, the intermolar width, bone mineral content, and bone volume fraction were analyzed by micro-computed tomography, and newly formed bone was measured histomorphometrically. RESULTS: The number of TRAP-positive cells in the compressed region was higher in the LIPUS group. The intermolar width was significantly higher in the LIPUS group than in the TM group. The alveolar bone around the maxillary first molar showed no differences in bone mineral content and bone volume fraction between the LIPUS and TM groups. The LIPUS group exhibited a more significant amount of newly formed alveolar bone than the TM group. CONCLUSIONS: The present study provides evidence of the beneficial effects of LIPUS on the lateral tooth movement.

Extracellular HSP72 induces proinflammatory cytokines in human periodontal ligament fibroblast cells through the TLR4/NFκB pathway in vitro.

OBJECTIVE: The aim of the present study was to examine the effect of extracellular heat shock protein (HSP) 72 on human periodontal ligament fibroblast cells (hPDLFs) in vitro. DESIGN: hPDLFs were stimulated by recombinant human HSP72 (rhHSP72). TAK-242 was used to inhibit toll-like receptor 4 (TLR4) activity. Interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α mRNA levels were analyzed by real-time PCR and protein levels were analyzed by enzyme-linked immunosorbent assay. p65/RelA phosphorylation was analyzed by western blot. RESULTS: IL-6, IL-8 and TNF-α mRNA and protein levels were significantly increased by rhHSP72 stimulation. These effects were inhibited by TAK-242 treatment. Additionally, p65/RelA phosphorylation was increased after 5-min rhHSP72 stimulation, which was inhibited by TAK-242 treatment. CONCLUSION: Extracellular HSP72 induces proinflammatory cytokines through TLR4/NF-κB in hPDLFs.

Management of open bite that developed during treatment for internal derangement and osteoarthritis of the temporomandibular joint.

This case report describes the orthodontic treatment performed for open bite caused by internal derangement (ID) and osteoarthritis (OA) of the temporomandibular joint (TMJ). A Japanese woman, aged 31 years and 11 months, referred to our department by an oral surgeon had an open bite with clockwise rotation of the mandible and degeneration of the condyle. The overbite was corrected through intrusion of the maxillary and mandibular molars using mini-screw implants to induce counterclockwise rotation of the mandible. Then, the mandibular second premolars were extracted and comprehensive orthodontic treatment was performed to establish a Class I molar relationship with distalization of the maxillary arch and to eliminate anterior crowding. Following treatment, her facial profile improved and a functional and stable occlusion was achieved without recurrence of the TMJ symptoms. These results suggest that orthodontic intrusion of the molars is one of the safer and less stressful alternatives for the management of open bite due to degeneration of the condyles caused by ID and OA of TMJ.

Human periodontal ligament fibroblasts are the optimal cell source for induced pluripotent stem cells.

Among the various kinds of fibroblasts existing in the human body, the periodontal ligament (PDL) fibroblasts have been suggested as multipotent cells. Periodontal ligament fibroblasts are characterized by rapid turnover, a high remodeling capacity and remarkable capacity for renewal and repair. They also differentiate into osteoblasts and cementoblasts. We established iPS cells from human PDL fibroblasts by introducing the ES cell markers Oct3/4, Sox2, Nanog, Klf4 and Lin28 by retrovirus transduction, even without the oncogene c-Myc. The iPS cells established in this study expressed the ES cell markers and formed teratomas in SCID mice. The c-Myc expression level in the PDL fibroblasts was higher than that in the iPS cells by quantitative RT-PCR. Therefore, we have concluded that PDL fibroblasts could be an optimal cell source for iPS cells.

Immunocompetent cells and cytokine expression in the rat periodontal ligament at the initial stage of orthodontic tooth movement.

OBJECTIVE: The aim of the present study is to investigate the involvement of immunocompetent cells, pro-inflammatory cytokines and HSP, to evaluate a change of periodontal ligament during the initial stage of orthodontic tooth movement. DESIGN: In the present study, we investigated the distributional density of immunocompetent cells, the localisation of cytokines, and the expression levels of their mRNA in the periodontal ligament during the initial stage of orthodontic tooth movement, using immunohistochemistry and real-time PCR. RESULTS: Orthodontic tooth movement led to significant recruitment of OX6(+) cells and ED1(+) cells in the rat PDL. Double-immunofluorescence staining showed that some ED1(+) cells expressed pro-inflammatory factors of IL-1ß and TNF-α in the PDL during orthodontic tooth movement. Real-time PCR analysis revealed that the expression levels of IL-1ß (Il1b) and TNF-α (Tnf) mRNA gradually increased following its decrease after 1h of orthodontic tooth movement. These findings suggest that ED1(+) cells are involved in the expression of TNF-α and IL-1ß and the subsequent regulation of bone resorption on pressure side. HSP27 (Hspb1) mRNA levels were significantly increased as compared with the control at 1h of the initial stage of treatment. CONCLUSION: ED1(+) cells involved in the expression of TNF-α and IL-1ß may play an important role in the initial reaction of the PDL and in the induction of the osteoclastic bone resorption during orthodontic tooth movement.

Mechanism of active eruption of molars in adolescent rats.

The mechanism of active eruption of molars was examined in 36 male adolescent Wistar rats. Histological, histochemical [tetracycline (TC) labelling and alkaline phosphatase (ALP) activity], and immunohistochemical [transforming growth factor (TGF)-ß1, -ß2, and -ß3] investigations were conducted of the rat molar areas. Real-time reverse transcription-polymerase chain reaction (RT-PCR) for mRNA of TGF-ß was performed on the periodontal ligament (PDL) dissected out by laser capture microdissection. TC labelling lines showed that a considerable amount of bone formation occurred in the alveolar crest region, apical region, and intraradicular septum, indicating that the maxillary molars had moved downward. However, the periodontal fibres revealed a regular arrangement (alveolar crest, horizontal and oblique fibres) during the experimental period. This suggests that new formation of alveolar crest fibres and rearrangement of the periodontal fibres occurred in the PDL. ALP activity was intense on the bone surface and in the PDL. TGF-ß1 was also detected in osteoblasts and fibroblasts but less so in cementoblasts. Real-time RT-PCR also demonstrated significant expression of mRNA of TGF-ß1 in the PDL, indicating that TGF-ß1 was involved in active eruption. These results suggest that active eruption occurs in adolescent rats and can be managed by TGF-ß1.

HSPA1A is upregulated in periodontal ligament at early stage of tooth movement in rats.

Heat shock proteins (HSPs) are molecular chaperones that maintain intracellular protein homeostasis and ensure survival of cells. Continuous orthodontic force on the tooth is considered to be a type of physical stress loaded to the periodontal ligament (PDL). However, little is known about the role of HSPs during tooth movement. This study was performed to examine the expression of HSPs in the PDL during tooth movement using laser microdissection, microarray analysis, real-time RT-PCR and immunohistochemistry. Gene expression of HSPA1A in the pressure zone of the PDL was higher during 6 h of tooth movement than in the control group. Expression of HSPA1A decreased with time. HSPA1A was also detected in the pressure zone of the PDL at the protein level 24 h after the initial tissue change. These results strongly suggest that expression of HSPA1A in the PDL during early stages of tooth movement is a critical factor for tissue reaction.

Root resorption after experimental tooth movement using superelastic forces in the rat.

The purpose of this study was to assess the rate of root resorption in relation to different magnitudes of continuous force during experimental tooth movement using nickel-titanium (NiTi) alloy wire. Four force magnitudes of 0.8, 1.6, 4, and 8 g were applied to the upper first molars of 75 male Wistar rats (300-320 g, 10-week-old) for 1, 7, 14, 21, and 28 days and compared with a control group without an orthodontic appliance. Light microscopic images of the compressed periodontal ligament (PDL) were processed by computer, and the ratio of the root resorption lacuna length to root surface length without the lacuna was analysed and statistically compared using Tukey-Kramer multiple comparison honestly significant difference test. The experimental groups with 4 and 8 g force showed undermining bone resorption with degenerating tissue and marked root resorption, the 1.6 g group showed only root resorption, while the 0.8 g group was similar to the control. Comparison of the ratios showed that the 0.8 g group was similar to the control with no significant difference. The ratio on day 28 in the 1.6 g group was larger than that in the 0.8 g and control groups, while on days 14, 21, and 28, the ratios in the 4 and 8 g groups were larger than those in the control (P < 0.01); these two experimental groups showed the same significant differences. It is suggested that significant root resorption occurs when the force magnitude exceeds 1.6 g in the rat upper first molar during tipping tooth movement by continuous force, and the amount of root resorption increases with serial force magnitudes from 0.8 to 4 g.

Time-lapse observation of rat periodontal ligament during function and tooth movement, using microcomputed tomography.

The aim of this study was to observe the time-lapse changes in the rat periodontal ligament (PDL) during function and tooth movement. Under Nembutal anaesthesia, time-lapse changes in the thickness of the PDL of the first molars were investigated in five 12-week-old adolescent rats with microcomputed tomography. Three-dimensional (3D) images were reconstructed from the data. Histological observation was also performed, using undecalcified frozen sections of the maxillary first molar area. The PDL appeared as a radiolucent furrow on the 3D images. A slight change in the thickness of the PDL was observed 1 hour after initiation of orthodontic force loading, which became significant after 6 hours, with the appearance of pressure-tension zones during the tooth movement. These changes were more significant 3 days after orthodontic loading. Histological observation of the lingual cervical PDL (pressure zone) in nine 12- to 13-week-old rats demonstrated that the periodontal space had become narrow and the cellular elements appeared to be densely packed in the narrowed PDL 6 hours after orthodontic loading. Degeneration of tissues appeared 3 days after loading. Observation of the buccal cervical PDL (tension zone) demonstrated that the PDL was extended 6 hours after orthodontic force loading, and the extension continued for up to 3 days. Alkaline phosphatase activity was distributed in the PDL, except for the degenerating tissues in the pressure zone 3 days after loading. The results suggest that the periodontal reaction was initiated within 6 hours after orthodontic force loading, which was related to the structural changes of the PDL. The changes probably induced an early response in individual cells of the PDL.

Rho-kinase inhibitor prevents hepatocyte damage in acute liver injury induced by carbon tetrachloride in rats.

A protective effect of Rho-kinase inhibitor on various organ injuries is gaining attention. Regarding liver injury, Rho-kinase inhibitor is reported to prevent carbon tetrachloride (CCl4)- or dimethylnitrosamine-induced liver fibrosis and hepatic ischemia-reperfusion injury in rats. Because Rho-kinase inhibitor not only improved liver fibrosis but also reduced serum alanine aminotransferase (ALT) level in CCl4-induced liver fibrosis, we wondered whether Rho-kinase inhibitor might exert a direct hepatocyte-protective effect. We examined this possibility in acute CCl4 intoxication in rats. Rho-kinase inhibitor, HA-1077, reduced serum alanine ALT level in rats with acute liver injury induced by CCl4 with the improvement of histological damage and the reduction of the number of apoptotic cells. In cultured rat hepatocytes in serum-free condition, HA-1077 reduced apoptosis evaluated by quantitative determination of cytoplasmic histone-associated DNA oligonucleosome fragments with the reduction of caspase-3 activity and the enhancement of Bcl-2 expression. HA-1077 stimulated phosphorylation of Akt, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, abrogated the reduction of hepatocyte apoptosis by HA-1077 in vitro. Furthermore, wortmannin abrogated the reduction of serum ALT level by HA-1077 in rats with acute liver injury induced by CCl4, suggesting that the activation of PI3-kinase/Akt pathway may be involved in the hepatocyte-protective effect by Rho-kinase inhibitor in vivo. In conclusion, Rho-kinase inhibitor prevented hepatocyte damage in acute liver injury induced by CCl4 in rats and merits consideration as a hepatocyte-protective agent in liver injury, considering its direct antiapoptotic effect on hepatocytes in vitro.

Effects of clenbuterol and cyclosporin A on the myosin heavy chain mRNA level and the muscle mass in rat masseter.

To gain more insight into the molecular mechanism of muscle growth and fiber-type transformations, we analyzed the effects of beta(2)-adrenergic agonist clenbuterol (CB) and/or cyclosporin A (CsA), a potent inhibitor of calcineurin (CaN), on the muscle mass as well as on the mRNA levels of myosin heavy chains (MHC I, IIa, IId/x, IIb), using a real-time RT-PCR with specific primers in rat masseter. In comparison with control, the CB treatment significantly decreased the MHC I mRNA level (p < 0.01), but increased the MHC IId/x mRNA level (p < 0.01), and the CsA treatment significantly decreased the MHC I mRNA level (p < 0.05) in association with the significant decrease in MHC IIb mRNA level (p < 0.05). The CB+CsA treatment significantly decreased the levels of MHC I (p < 0.01) and IIa mRNAs (p < 0.05), but increased the MHC IId/x mRNA level (p < 0.001) in association with a significant decrease in MHC IIb mRNA level (p < 0.01), in comparison with control. The masseter muscle mass was significantly (p < 0.001) increased by either the CB or the CB + CsA treatment, but decreased with the CsA treatment (p < 0.01). These results suggest that in rat masseter muscle, CB has an anabolic action accompanying MHC mRNA I IIa IId/x sequence transition independently of CaN-signaling pathways, and CaN is involved in the type I fiber gene expression and the muscle mass maintenance of type IIb fiber.

Effects of bite-opening and cyclosporin A on the mRNA levels of myosin heavy chain and the muscle mass in rat masseter.

To gain more insight into the mechanism of muscle plasticity in response to mechanical overload, we analyzed the effects of bite -opening (BO, 3 mm increase in the vertical dimension for 2 weeks) and/or a calcineurin (CaN) inhibitor, cyclosporin A (CsA, 10 mg/kg body weight, once daily for 2 weeks, ip) treatment on the myosin heavy chain (MHC I, IIa, IId/x, IIb) mRNA levels, using real-time RT-PCR with specific primers and on the muscle mass in rat masseter. As compared with normal control (n = 6), the BO treatment (n = 6) significantly increased the MHC I (p < 0.05) and the IIa mRNA levels (p < 0.01), and the CsA treatment (n = 6) significantly decreased the MHC I mRNA level (p < 0.01) in association with the significant decrease in the MHC IIb mRNA level (p < 0.05). The BO + CsA treatment (n = 6) significantly increased the MHC IIa mRNA level (p < 0.01) in association with the significant decrease in the MHC IIb mRNA level (p < 0.01), as compared with control. The masseter muscle mass was significantly decreased by either the CsA (p < 0.05) or the BO + CsA treatment (p < 0.001), but slightly increased by the BO treatment. These results suggest that in rat masseter the BO treatment produces not only the up-regulation of MHC IIa mRNA independently of CaN-signaling pathways, but also the MHC mRNA transition from IIa to I and the muscle mass maintenance mainly of type IIb fiber through the CaN-signaling pathways.