Mari Okayasu

Low-density Lipoprotein Receptor Deficiency Causes Impaired Osteoclastogenesis and Increased Bone Mass in Mice because of Defect in Osteoclastic Cell-Cell Fusion

Background: Dyslipidemia is associated with osteoporosis, atherosclerosis, and vascular calcification. Results: Osteoclastogenesis in LDLR-deficient mice was reduced because of impaired cell fusion in preosteoclasts consistent with reduced Atp6v0d2 and DC-STAMP proteins, both of which regulate the fusion in the plasma membrane. Conclusion: LDL uptake via LDLR is essential for osteoclastogenesis. Significance: The findings improve the understanding of the correlation between osteoclast formation and lipids. Osteoporosis is associated with both atherosclerosis and vascular calcification attributed to hyperlipidemia. However, the cellular and molecular mechanisms explaining the parallel progression of these diseases remain unclear. Here, we used low-density lipoprotein receptor knockout (LDLR−/−) mice to elucidate the role of LDLR in regulating the differentiation of osteoclasts, which are responsible for bone resorption. Culturing wild-type osteoclast precursors in medium containing LDL-depleted serum decreased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation, and this defect was additively rescued by simultaneous treatment with native and oxidized LDLs. Osteoclast precursors constitutively expressed LDLR in a RANKL-independent manner. Osteoclast formation from LDLR−/− osteoclast precursors was delayed, and the multinucleated cells formed in culture were smaller and contained fewer nuclei than wild-type cells, implying impaired cell-cell fusion. Despite these findings, RANK signaling, including the activation of Erk and Akt, was normal in LDLR−/− preosteoclasts, and RANKL-induced expression of NFATc1 (a master regulator of osteoclastogenesis), cathepsin K, and tartrate-resistant acid phosphatase was equivalent in LDLR-null and wild-type cells. In contrast, the amounts of the osteoclast fusion-related proteins v-ATPase V0 subunit d2 and dendritic cell-specific transmembrane protein in LDLR−/− plasma membranes were reduced when compared with the wild type, suggesting a correlation with impaired cell-cell fusion, which occurs on the plasma membrane. LDLR−/− mice consistently exhibited increased bone mass in vivo. This change was accompanied by decreases in bone resorption parameters, with no changes in bone formation parameters. These findings provide a novel mechanism for osteoclast differentiation and improve the understanding of the correlation between osteoclast formation and lipids.

Osteocytes produce interferon-β as a negative regulator of osteoclastogenesis

Background: Global interferon-β deficiency causes osteoporosis. Lack of interferon-β production by osteoclast precursors is considered to induce excess osteoclastogenesis. Results: Isolated osteocytes express higher amount of interferon-β mRNA than osteoclast precursors and inhibit osteoclastogenesis partially in interferon-β-dependent manner. Conclusion: Osteocytes produce interferon-β as an inhibitor of osteoclastogenesis. Significance: Osteocytic interferon-β might be involved in the regulation of bone homeostasis. Osteoclastogenesis is controlled by osteocytes; osteocytic osteoclastogenesis regulatory molecules are largely unknown. We searched for such factors using newly developed culture methods. Our culture system mimics the three-dimensional cellular structure of bone, consisting of collagen gel-embedded osteocytic MLO-Y4 cells, stromal ST2 cells on the gel as bone lining cells, and bone marrow cells. The gel-embedded MLO-Y4 cells inhibited the osteoclastogenesis induced by 1,25(OH)2D3 without modulating receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) production by ST2 cells, despite MLO-Y4 cells supported osteoclastogenesis in the absence of ST2 cells. In the bone marrow cell culture, the conditioned medium from MLO-Y4 cells decreased the capability of osteoclastic differentiation from the cells induced by macrophage colony-stimulating factor. This decreased capability was concomitant with an increase in protein kinase R mRNA expression and an inhibition of c-Fos translation. These changes were partially normalized by the simultaneous addition of an anti-interferon (IFN)-β neutralizing antibody to MLO-Y4 cell conditioned medium. To study primary osteocytes, we prepared non-osteocytic cell-free osteocyte-enriched bone fragments (OEBFs). When osteoclast precursors were induced by macrophage colony-stimulating factor in the presence of OEBFs, the generated cells exhibited a diminished capacity for osteoclastogenesis. OEBFs prepared from OPG-knock-out mice exhibited a similar effect, indicating OPG-independent inhibition. The addition of anti-IFN-β neutralizing antibody during the co-culture with OEBFs partially recovered the osteoclastogenic potential of the generated cells. The MLO-Y4 cells and OEBFs expressed IFN-β mRNA. Although osteocytic RANKL is known to be important for osteoclastogenesis, our data suggest that osteocytes also produce IFN-β as an inhibitor of osteoclastogenesis.

Evaluation of Mandibular Hypoplasia in Patients With Hemifacial Microsomia: A Comparison Between Panoramic Radiography and Three-Dimensional Computed Tomography

Objective To compare the accuracy of three-dimensional computed tomography (3D-CT) and panoramic radiography in the evaluation of mandibular hypoplasia in patients with hemifacial microsomia (HFM). Design Retrospective study of imaging data. Setting Images selected from the archives of the University of Tokyo Hospital. Subjects Twenty patients with unilateral HFM who had undergone both panoramic radiography and 3D-CT in the same period. Method Mandibular deformities were classified according to the Pruzansky classification; eight patients had Grade I deformity and 12 patients had Grade II deformity. Ramus heights were measured on both panoramic radiographs and 3D-CT. Main outcome measures Magnification in panoramic radiographs and extent of mandibular asymmetry as estimated by the affected/unaffected side ratio based on two methods were examined. The Pearson product-moment correlation coefficient was used to estimate correlations between parameters. Results The magnification of ramus heights on panoramic radiographs showed large variations in Grade II patients. The affected/unaffected side ratio estimated by the two methods showed a strong correlation in Grade I patients (correlation coefficient 0.99; p < .0001). Conversely, a weak correlation was seen in Grade II patients (correlation coefficient 0.77; p = .0036), and affected/unaffected side ratios from panoramic radiographs were both over- and underestimated. Conclusions The accuracy of evaluation using panoramic radiography was fairly reliable in Grade I patients. Conversely, accuracy was poor in Grade II patients, and evaluation using 3D-CT seems preferable. The combination of two methods with careful consideration is recommended for clinical applications.

Maxillary Protraction in Patients With Cleft Lip and Palate in Mixed Dentition: Cephalometric Evaluation After Completion of Growth

Objective To clarify the short- and long-term effects of maxillary protraction (MP) in mixed dentition in patients with unilateral cleft lip and palate (UCLP). Design Retrospective study. Setting University of Tokyo Hospital. Patients and Intervention Eleven Japanese patients with UCLP in mixed dentition were treated with MP and followed up until the completion of growth. Multibracket treatment had been performed after MP treatment in all patients. Main Outcome Measure Lateral cephalograms taken before and after MP and after completion of growth were used. Posterior and anterior vertical reference lines (PV, AV) were used to measure the horizontal movements of point A, pogonion, and maxillary first molar (U6). SNA, SNB, ANB, maxillary and mandibular length, mandibular plane angle, Wits value, upper incisor inclination, overjet, and overbite were also measured. Results Large variation was found in the effects of MP, and five patients eventually required orthognathic surgery. In average change with MP, the maxilla showed favorable forward growth. Point A had moved forward from PV but not AV. The mandible rotated backward. However, ANB and the Wits value did not improve. U6 moved forward, and the overjet improved. After MP, the skeletal Class III relationship became severe. Conclusions MP was effective as an early treatment for UCLP patients. However, its effects showed large variation and were in conflict with facial growth. Conscientious explanation of the expected effects and associated problems should be given to the patients/parents before its application.

Lectin-like oxidized low-density lipoprotein receptor-1 abrogation causes resistance to inflammatory bone destruction in mice, despite promoting osteoclastogenesis in the steady state

Inflammatory bone diseases have been attributed to increased bone resorption by augmented and activated bone-resorbing osteoclasts in response to inflammation. Although the production of diverse proinflammatory cytokines is induced at the inflamed sites, the inflammation also generates reactive oxygen species that modify many biological compounds, including lipids. Among the oxidized low-density lipoprotein (LDL) receptors, lectin-like oxidized LDL receptor-1 (LOX-1), which is a key molecule in the pathogenesis of multifactorial inflammatory atherosclerosis, was downregulated with osteoclast differentiation. Here, we demonstrate that LOX-1 negatively regulates osteoclast differentiation by basically suppressing the cell-cell fusion of preosteoclasts. The LOX-1-deleted (LOX-1(-/-)) mice consistently decreased the trabecular bone mass because of elevated bone resorption during the growing phase. In contrast, when the calvaria was inflamed by a local lipopolysaccharide-injection, the inflammation-induced bone destruction accompanied by the elevated expression of osteoclastogenesis-related genes was reduced by LOX-1 deficiency. Moreover, the expression of receptor activator of NF-κB ligand (RANKL), a trigger molecule for osteoclast differentiation, evoked by the inflammation was also abrogated in the LOX-1(-/-) mice. Osteoblasts, the major producers of RANKL, also expressed LOX-1 in response to proinflammatory agents, interleukin-1β and prostaglandin E2. In the co-culture of LOX-1(-/-) osteoblasts and wild-type osteoclast precursors, the osteoclastogenesis induced by interleukin-1β and prostaglandin E2 decreased; this process occurred in parallel with the downregulation of osteoblastic RANKL expression. Collectively, LOX-1 abrogation results in resistance to inflammatory bone destruction, despite promoting osteoclastogenesis in the steady state. Our findings indicate the novel involvement of LOX-1 in physiological bone homeostasis and inflammatory bone diseases.

Questionnaire investigation after orthodontic treatment in patients with unilateral cleft lip and palate: An inter-clinic collaborative study

Abstract Purpose This six-clinic collaborative study collected patient opinions after orthodontic treatment, for quality improvement. Materials and methods Participants comprised 477 patients with unilateral cleft lip and palate (UCLP) who had undergone orthodontic treatment at orthodontic clinics in five university hospitals and one private office in Japan. The questionnaire sent to patients contained 33 questions in five categories, about the respondent, before, during and after orthodontic treatment, and overall treatment. Questionnaires were returned by mail anonymously. Results Responses were received from 183 patients (response rate, 38.4%). Patients tended to anticipate esthetic improvements in anterior teeth and facial appearance, and reported great improvements with orthodontic treatment. Masticatory problems also improved and orthodontic treatment had positive effects on speech. Almost all respondents were satisfied with orthodontic treatment. Problems with overall treatment were long treatment period, difficulty attending clinics and maintenance of oral hygiene during treatment. Conclusion This study clarified the subjective effects and problems with orthodontic treatment in patients with UCLP in Japan. Satisfaction after treatment was high. Anticipated esthetic improvement was accomplished and masticatory problems improved. Orthodontic treatment also positively affected speech. Efficient orthodontic treatment to minimize treatment duration and improved treatment systems for easier access are needed to reduce patient burdens in Japan.