Yasuhiro Shimizu

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

OBJECTIVEnTo elucidate the effect of sympathetic nervous activity on alveolar bone loss induced by occlusal hypofunction in rat molars.nnnDESIGNnOcclusal 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnThese results suggest that sympathetic nervous activity may influence the alveolar bone loss induced by occlusal hypofunction.

Soft diet causes greater alveolar osteopenia in the mandible than in the maxilla

OBJECTIVEnTo investigate changes in the bony microstructure of the upper and lower alveolar bone during masticatory loading induced by soft diet feeding in growing rats.nnnDESIGNnThree-week-old male Wistar rats were randomly divided into two groups. Rats were fed with either pellets [control group (n=6)] or a soft diet [experimental group (n=6)] for nine weeks. 3D-microstructure of the alveolar bone of the first molar region (M1) was examined by micro-CT analysis.nnnRESULTSnMicro-CT images showed increased marrow spaces of the inter-radicular alveolar bone around the rat mandibular M1 in the experimental group compared with that in the control group. The bone volume/tissue volume ratio, trabecular thickness, trabecular number, mean intercept length, trabecular width and trabecular star volume for the mandibular M1 inter-radicular alveolar bone were lower in the experimental group than in the control group. Marrow space star volume was increased in the experimental group compared with the control group.nnnCONCLUSIONSnThese results suggest that alveolar osteopenia is more extensive in the mandible than the maxilla in rats that experience low masticatory loading during growth.

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

OBJECTIVEnTo 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.nnnMATERIALS AND METHODSnTwelve-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.nnnRESULTSnHypofunctional 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.nnnCONCLUSIONSnOcclusal 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.

The inhibitory effects of a RANKL-binding peptide on articular and periarticular bone loss in a murine model of collagen-induced arthritis: a bone histomorphometric study

IntroductionWe designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis.MethodsTwenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed.ResultsThe OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites.ConclusionThe peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.

Micro-computed tomography analysis of changes in the periodontal ligament and alveolar bone proper induced by occlusal hypofunction of rat molars

Objective To three-dimensionally elucidate the effects of occlusal hypofunction on the periodontal ligament and alveolar bone proper of rat molars by micro-computed tomography (micro-CT). Methods Occlusal function in the molar area was restricted by attaching an anterior bite plate on the maxillary incisors and a metal cap on the mandibular incisors of 5-week-old male Wistar rats for 1 week. The periodontal ligament space and alveolar bone proper around roots of the mandibular first molar were assessed by histology and micro-CT. Results The periodontal ligament space was narrower and the alveolar bone proper was sparser and less continuous in the hypofunction group than in the control group. Further, both the volume of the periodontal ligament and the volumetric ratio of the alveolar bone proper to the total tissue in the region of interest were significantly lower in the hypofunction group (p < 0.05). Conclusions Occlusal hypofunction induces atrophic changes in the periodontal ligament and alveolar bone proper of rat molars.

Intermittent hypoxia induces disturbances in craniofacial growth and defects in craniofacial morphology.

OBJECTIVESnTo investigate intermittent hypoxia (IH) induced changes in craniofacial morphology and bone mineral density (BMD) in the mandible of growing rats.nnnDESIGNnSeven-week-old male Sprague-Dawley rats were exposed to IH for 4 days or 3 weeks. Sham-operated rats simultaneously breathed room air. Lateral and transverse cephalometric radiographs of the craniofacial region were obtained, and the linear distances between cephalometric landmarks were statistically analyzed. BMD and bone microstructure of the mandible were evaluated using micro-computed tomography (micro-CT).nnnRESULTSnCephalometric analyses demonstrated that exposure to IH only in the two groups for 3 weeks decreased the size of the mandibular and viscerocranial bones, but not that of the neurocranial bones, in early adolescent rats. These findings are consistent with upper airway narrowing and obstructive sleep apnea (OSA). Micro-CT showed that IH increased the BMD in the cancellous bone of the mandibular condyle and the inter-radicular alveolar bone in the mandibular first molar (M1) region.nnnCONCLUSIONSnThis study is the first to identify growth retardation of the craniofacial bones in an animal model of sleep apnea. Notably, 3 weeks of IH can induce multiple changes in the bones around the upper airway in pubertal rats, which can enhance upper airway narrowing and the development of OSA. The reproducibility of these results supports the validity and usefulness of this model. These findings also emphasize the critical importance of morphometric evaluation of patients with OSA.

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.

Impairment of nasal airway under intermittent hypoxia during growth period in rats

OBJECTIVEnTo clarify the influences of intermittent hypoxia (IH) on the growth and development of the midfacial area, including the nasal cavity, in growing rats.nnnDESIGNnSeven-week-old male Sprague-Dawley rats were divided into two groups: the experimental group (n=5), which was exposed to IH for 8h during light periods at a rate of 20 cycles/h (nadir, 4% O₂ to peak, 21% O₂ with 0% CO₂), and the control group (n=5), which was exposed to room air. After 3 weeks, the maxillofacial structures in both groups were evaluated with respect to the height, width, length, surface area, cross-sectional area, and volume of the nasal cavity using soft X-ray and micro-CT.nnnRESULTSnThe experimental group showed a significantly smaller cross-sectional area and volume than did the control group. The surface area exhibited no significant differences between the two groups, although it tended to be smaller in the experimental group than in the control group. The nasal volume divided by the length of the tibia (for comparison with whole-body growth) was significantly smaller in the experimental group than in the control group.nnnCONCLUSIONSnThese data suggest that IH exposure suppresses growth and development of the nasal cavity and may result in nasal breathing disturbance.

A new approach to transfect NF-|[kappa]|B decoy oligodeoxynucleotides into the periodontal tissue using the ultrasound-microbubble method

The objective of this study is to investigate the effect of the ultrasound-microbubble technique in nuclear factor kappa B (NF-κB) decoy oligodeoxynucleotide (ODN) transfection in the gingival tissue in mice. The 6-FAM-labeled scrambled decoy ODN with microbubbles was applied to the periodontal tissue in 8-week-old male C57BL/6J mice by ultrasound radiation at low (LUM-Sc) and high (HUM-Sc) intensities to optimize the transfection condition of the ultrasound-microbubble method. Histological inspections were performed two hours after transfection to compare the expression with that in the sham-operated group without ultrasound radiation (A-Sc). Then, an NF-κB decoy was transfected into the periodontal tissue using the high-intensity ultrasound-microbubble (HUM-NF) technique to examine the anti-inflammatory effects of the decoy ODN. Western blot analysis was performed to investigate the expression of interleukin(IL)-1β, IL-6 and intercellular adhesion molecule-1 (ICAM-1) in the gingival tissues in the HUM-Sc, the HUM-NF and control groups. The fluorescence microscopy results showed that the fluorescent intensity in the periodontal tissues in the LUM-Sc and HUM-Sc groups was significantly higher than that in the A-Sc and the control groups. The fluorescent intensity in the HUM-Sc group, especially in the gingival connective tissue, was the highest of all groups. Western blot analysis indicated that the protein expression levels of IL-1β, IL-6 and ICAM-1 in the HUM-NF group were significantly lower than those in the HUM-Sc and the control groups. These findings suggest that the high-intensity ultrasound-microbubble technique is an effective tool for decoy transfection into the periodontal tissue.

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.