Naoki Shibutani

Effect of sympathetic nervous activity on alveolar bone loss induced by occlusal hypofunction in rats

OBJECTIVE To elucidate the effect of sympathetic nervous activity on alveolar bone loss induced by occlusal hypofunction in rat molars. DESIGN Occlusal hypofunction in the molar area was produced by attaching appliances to rat maxillary and mandibular incisors. In addition, a non-selective β-adrenergic receptor antagonist, propranolol, was administered orally to rats in drinking water to pharmacologically suppress sympathetic nervous activity. After 1 week, alveolar bones in all groups were examined by micro-CT, histomorphometry and histology to determine their trabecular bone phenotypes and histological changes. RESULTS The marrow spaces of the interradicular alveolar bone of rat mandibular first molars (M1) increased in the occlusal hypofunction group (Group H) but not in the control group (Group C), whilst these decreased in rats in the occlusal hypofunction group that were administered propranolol (Group HB). Bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) for interradicular alveolar bone in M1 in Group H were significantly lower than those in Group C, whereas those in Group HB remained as high as those in Group C. The number of TRAP-positive cells in Group H increased compared to that in Group C, whereas it significantly decreased in Group HB. CONCLUSIONS These results suggest that sympathetic nervous activity may influence the alveolar bone loss induced by occlusal hypofunction.

Occlusal hypofunction causes periodontal atrophy and VEGF/VEGFR inhibition in tooth movement

OBJECTIVE To examine changes in microvasculature and the expression of vascular endothelial growth factor A (VEGF-A) and VEGF receptor 2 (VEGFR-2) in rat hypofunctional periodontal ligament (PDL) during experimental tooth movement. MATERIALS AND METHODS Twelve-week-old male Sprague-Dawley rats were divided into normal occlusion and occlusal hypofunction groups. After a 2-week bite-raising period, rat first molar was moved mesially using a 10-gf titanium-nickel alloy closed coil spring in both groups. On days 0, 1, 2, 3, and 7 after tooth movement, histologic changes were examined by micro-computed tomography and immunohistochemistry using CD31, VEGF-A, VEGFR-2, and the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. RESULTS Hypofunctional molars inclined more than normal molars and did not move notably after day 1 of tooth movement. Blood vessels increased on the tension side of the PDL in normal teeth. Immunoreactivities for VEGF-A and VEGFR-2 in normal teeth were greater than those in hypofunctional teeth during tooth movement. Compressive force rapidly caused apoptosis of the PDL and vascular endothelial cells in hypofunctional teeth, but not in normal teeth. CONCLUSIONS Occlusal hypofunction induces vascular constriction through a decrease in the expression of VEGF-A and VEGFR-2, and apoptosis of the PDL and vascular cells occurs during tooth movement.

Influence of Occlusal Stimuli on the Microvasculature in Rat Dental Pulp

OBJECTIVE To examine the influence of occlusal stimuli on the vasculature in the dental pulp, using an occlusal hypofunction model. MATERIALS AND METHODS Twenty 7-week-old male Sprague-Dawley rats were divided into two groups. To produce occlusal hypofunction, the appliances were attached to the maxillary and mandibular incisors. Untreated rats served as controls. Serial horizontal paraffin sections of the mandibular first molar were processed by conventional methods. To evaluate the microvasculature in the dental pulp, sections of each specimen were stained with hematoxylin-eosin. RESULTS In the experimental group, the arterioles in the tooth pulp tissue ran convergently, and their inside diameter was significantly smaller than that of the control group. CONCLUSION This study suggests that occlusal stimuli influence the periodontal ligament throughout the microvasculature of the dental pulp.

Intermittent Hypoxia Influences Alveolar Bone Proper Microstructure via Hypoxia-Inducible Factor and VEGF Expression in Periodontal Ligaments of Growing Rats

Intermittent hypoxia (IH) recapitulates morphological changes in the maxillofacial bones in children with obstructive sleep apnea (OSA). Recently, we found that IH increased bone mineral density (BMD) in the inter-radicular alveolar bone (reflecting enhanced osteogenesis) in the mandibular first molar (M1) region in the growing rats, but the underlying mechanism remains unknown. In this study, we focused on the hypoxia-inducible factor (HIF) pathway to assess the effect of IH by testing the null hypothesis of no significant differences in the mRNA-expression levels of relevant factors associated with the HIF pathway, between control rats and growing rats with IH. To test the null hypothesis, we investigated how IH enhances mandibular osteogenesis in the alveolar bone proper with respect to HIF-1α and vascular endothelial growth factor (VEGF) in periodontal ligament (PDL) tissues. Seven-week-old male Sprague–Dawley rats were exposed to IH for 3 weeks. The microstructure and BMD in the alveolar bone proper of the distal root of the mandibular M1 were evaluated using micro-computed tomography (micro-CT). Expression of HIF-1α and VEGF mRNA in PDL tissues were measured, whereas osteogenesis was evaluated by measuring mRNA levels for alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2). The null hypothesis was rejected: we found an increase in the expression of all of these markers after IH exposure. The results provided the first indication that IH enhanced osteogenesis of the mandibular M1 region in association with PDL angiogenesis during growth via HIF-1α in an animal model.

U-shaped Osteotomy Around the Descending Palatine Artery to Prevent Posterior Osseous Interference for Superior/Posterior Repositioning of the Maxilla in Le Fort I Osteotomy.

In maxillary orthognathic surgery, superior repositioning of the maxilla is sometimes difficult, and removal of bony interference, especially around the descending palatine artery (DPA), is very time-consuming in cases of severe maxillary impaction. Posterior repositioning of the maxilla for removal of bony interference between the posterior maxilla and the pterygoid process is also technically difficult. Because the most common site of hemorrhage in Le Fort I osteotomy is the posterior maxilla, this bone removal is a source of frustration for surgeons in DPA injury. When the DPA is injured during bone removal and ligation is performed, aseptic necrosis of the maxilla may occur. Therefore, a simple and safe method for maxillary superior/posterior repositioning is required to remove osseous interference around the DPA. The authors describe here U-shaped osteotomy around the DPA to prevent posterior osseous interference for superior/posterior repositioning of the maxilla in Le Fort I osteotomy.

Alveolar bone loss induced by the orthodontic tooth movement under hypofunctional conditions in rats

Abstract Purpose To examine the effect of occlusal hypofunctional conditions on orthodontic tooth movement and its relation to the structure and quality of alveolar bone using the rat model. Materials and methods Twelve-week-old male Sprague-Dawley rats were divided into 4 groups of 8 animals each: normal occlusion (N) group, normal occlusion with tooth movement (M) group, occlusal hypofunction (H) group, and occlusal hypofunction with tooth movement (HM) group. In H and HM groups, the anterior bite plate and metal cap were attached to the maxillary and mandibular incisors using a light-curing composite resin to induce the occlusal hypofunctional condition. In M and HM groups, an orthodontic force was applied in a palatal direction to the buccal surface of the maxillary first molar (M1) using a nickel–titanium alloy wire. Micro-CT imaging and histomorphometric analysis using fluorescent bone labeling of the alveolar bone surrounding the M1s were performed in each group. Results Tooth movement of M1 in HM group, was rather accelerated with enhanced tipping than in M group. Micro-CT analysis revealed significant decrease in bone volume fraction, bone mineral density and trabecular thickness of the interradicular bone in HM group among the experimental groups. The fluorescent labeling lines in the interradicular bone were decreased in number in H and M groups compared with N group. A few discontinuous irregular dotted lines-like labeling was observed in HM group. Conclusion The occlusal hypofunctional condition accelerates orthodontic tooth movement of the respective teeth, while it results in severe bone loss in the surrounding alveolar bone.

Low-intensity pulsed ultrasound reduces periodontal atrophy in occlusal hypofunctional teeth

OBJECTIVE To clarify whether low-intensity pulsed ultrasound (LIPUS) exposure has recovery effects on the hypofunctional periodontal ligament (PDL) and interradicular alveolar bone (IRAB). MATERIALS AND METHODS Twelve-week-old male Sprague-Dawley rats were divided into three groups (n = 5 each): a normal occlusion (C) group, an occlusal hypofunction (H) group, and an occlusal hypofunction group subjected to LIPUS (HL) treatment. Hypofunctional occlusion of the maxillary first molar (M1) of the H and HL groups was induced by the bite-raising technique. Only the HL group was irradiated with LIPUS for 5 days. The IRAB and PDL of M1 were examined by microcomputed tomography (micro-CT) analysis. To quantify mRNA expression of cytokines involved in PDL proliferation and development, real-time reverse transcription quantitative PCR (qRT-PCR) was performed for twist family bHLH transcription factor 1 (Twist1), periostin, and connective tissue growth factor (CTGF) in the PDL samples. RESULTS Micro-CT analysis showed that the PDL volume was decreased in the H group compared with that of the C and HL groups. Both bone volume per tissue volume (BV/TV) of IRAB was decreased in the H group compared with that in the C group. LIPUS exposure restored BV/TV in the IRAB of the HL group. qRT-PCR analysis showed that Twist1, periostin, and CTGF mRNA levels were decreased in the H group and increased in the HL group. CONCLUSION LIPUS exposure reduced the atrophic changes of alveolar bone by inducing the upregulation of periostin and CTGF expression to promote PDL healing after induction of occlusal hypofunction.

Localization of leucine-rich repeat-containing G-protein-coupled receptor 5- and Ki67-positive periodontal cells expressing runt-related transcription factor 2 during tooth movement

Abstract Purpose Mechanical stimuli induce the proliferation and osteogenic differentiation of periodontal ligament (PDL) progenitor cells. However, the localization of formative and proliferative cells in response to orthodontic force in the PDL is unknown. We aimed to elucidate the expression patterns and localization of the cell proliferation- and osteogenesis-associated markers in the PDL during tooth movement. Materials and methods Five-week-old male Sprague–Dawley rats had their right maxillary first molars (rM1s) moved mesially using a 10-gf titanium-nickel alloy closed-coil spring. Left maxillary first molars (lM1s) served as controls. Immunohistochemical analyses of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), Ki67, and runt-related transcription factor 2 (RUNX2) were performed in the periapical region of M1. mRNA levels of Ki67, Runx2, and β-catenin were quantified by real-time reverse transcription polymerase chain reaction (qRT-PCR). In vitro human PDL stem cells (hPDLSCs) were cyclically stretched for 24 h, and MTT assay and western blot analysis of LGR5 were performed. Results Immunohistochemistry showed that LGR5 and Ki67 were partially co-expressed with RUNX2 in the periapical PDL. Immunoreactivities of LGR5, Ki67, and RUNX2 were significantly greater in the PDL of the rM1 than in that of the lM1 on day 3. qRT-PCR showed that Ki67 and Runx2 mRNA levels, but not β-catenin levels, increased on the mesial side of the periapical PDL on day 3. Western blot analysis revealed that 24 h of cyclic strain stimulated LGR5 expression in hPDLSCs. Conclusion Our findings suggested that orthodontic force induced LGR-5- and Ki67-positive cells expressing RUNX2 in the PDL during tooth movement.

The chemokine receptor type 4 antagonist, AMD3100, interrupts experimental tooth movement in rats

OBJECTIVE The aim of this study was to clarify the role of the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis in osteoclast accumulation, and the influence of orthodontic tooth movement (OTM) under mechanical force application to periodontal tissues, by administration of the CXCR4 antagonist AMD3100. DESIGN The upper right first molar (M1) of rats was moved mesially with a 10-g force titanium-nickel closed coil spring. Rats were treated with phosphate-buffered saline or AMD3100 (5mg/kg), which is a SDF-1 antagonist. After 0, 1, 3, and 7days, alveolar bones in all groups were examined at each time point by micro-computed tomography and histological analysis. RESULTS Tooth movement was decreased significantly in the AMD3100-treated group at 1, 3, and 7days after beginning OTM. The numbers of tartrate-resistant acid phosphatase-positive multinucleated cells in the periodontal ligament around the maxillary M1 were decreased significantly in the treated as compared to the control group on Days 1 and 3. CONCLUSION Administration of AMD3100 decreases OTM and osteoclast accumulation in rat molars under orthodontic force application. These findings suggest that the SDF-1/CXCR4 axis plays an important role in alveolar bone metabolism during OTM.

Vital staining of palatal soft tissue in horseshoe Le Fort I osteotomy for superior repositioning of the maxilla.

In maxillary orthognathic surgery, superior repositioning of the maxilla is sometimes difficult, and removal of the bony interference, especially around the descending palatine artery, is very time-consuming in cases of severe maxillary impaction. A useful method introduced for superior repositioning of the maxilla is horseshoe-shaped osteotomy combined with Le Fort I osteotomy (horseshoe Le Fort I osteotomy). However, injury to the palatal soft tissue during horseshoe-shaped osteotomy may cause aseptic complications of the maxilla. Therefore, a safe method is required to prevent such injury to reduce the risk for aseptic necrosis. We describe here vital staining of palatal soft tissue in horseshoe Le Fort I osteotomy for safer superior repositioning of the maxilla.