Risa Usumi-Fujita

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.

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

OBJECTIVES To investigate intermittent hypoxia (IH) induced changes in craniofacial morphology and bone mineral density (BMD) in the mandible of growing rats. DESIGN Seven-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). RESULTS Cephalometric 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. CONCLUSIONS This 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

OBJECTIVE To clarify the influences of intermittent hypoxia (IH) on the growth and development of the midfacial area, including the nasal cavity, in growing rats. DESIGN Seven-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. RESULTS The 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. CONCLUSIONS These 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.

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.

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.

Collaborative treatment for a case of condylar hyperplastic facial asymmetry

Facial asymmetry can be caused by unilateral condylar hyperplasia. In such cases, it may be difficult to achieve symmetry since there is dentoalveolar compensation on the affected side, and the occlusal cant does not correspond to the frontal mandibular deviation. In the case presented, surgical orthodontic treatment and orthognathic surgery planning was accomplished for a patient with facial asymmetry due to condylar hyperplasia. The surgical plan was devised with particular attention to the severe dentoalveolar compensation. In this case, prior to the two-jaw surgery, the occlusal cant and frontal mandibular plane inclination was corrected through impaction of the left molar region by segmental osteotomy. Facial asymmetry and severe dentoalveolar compensation were successfully corrected after a unilateral segmental osteotomy and two-jaw surgery, resulting in a stable occlusal relationship and facial symmetry as well as good jaw function. Collaboration between the orthodontists and maxillofacial surgeons was essential for the successful treatment of the patient.

Ultrasound microbubble-mediated transfection of NF-κB decoy oligodeoxynucleotide into gingival tissues inhibits periodontitis in rats in vivo

Periodontitis is a chronic infectious disease for which the fundamental treatment is to reduce the load of subgingival pathogenic bacteria by debridement. However, previous investigators attempted to implement a nuclear factor kappa B (NF-κB) decoy oligodeoxynucleotide (ODN) as a suppressor of periodontitis progression. Although we recently reported the effectiveness of the ultrasound-microbubble method as a tool for transfecting the NF-κB decoy ODN into healthy rodent gingival tissue, this technique has not yet been applied to the pathological gingiva of periodontitis animal models. Therefore, the aim of this study was to investigate the effectiveness of the technique in transfecting the NF-κB decoy ODN into rats with ligature-induced periodontitis. Micro computed tomography (micro-CT) analysis demonstrated a significant reduction in alveolar bone loss following treatment with the NF-κB decoy ODN in the experimental group. RT-PCR showed that NF-κB decoy ODN treatment resulted in significantly reduced expression of inflammatory cytokine transcripts within rat gingival tissues. Thus, we established a transcutaneous transfection model of NF-κB decoy ODN treatment of periodontal tissues using the ultrasound-microbubble technique. Our findings suggest that the NF-κB decoy ODN could be used as a significant suppressor of gingival inflammation and periodontal disease progression.