Keisuke Tsutsui

Monoallelic BUB1B mutations and defective mitotic-spindle checkpoint in seven families with premature chromatid separation (PCS) syndrome.

Cancer‐prone syndrome of premature chromatid separation (PCS syndrome) with mosaic variegated aneuploidy (MVA) is a rare autosomal recessive disorder characterized by growth retardation, microcephaly, childhood cancer, premature chromatid separation of all chromosomes, and mosaicism for various trisomies and monosomies. Biallelic BUB1B mutations were recently reported in five of eight families with MVA syndrome (probably identical to the PCS syndrome). We here describe molecular analysis of BUB1B (encoding BubR1) in seven Japanese families with the PCS syndrome. Monoallelic BUB1B mutations were found in all seven families studied: a single‐base deletion (1833delT) in four families; and a splice site mutation, a nonsense mutation, and a missense mutation in one family each. Transcripts derived from the patients with the 1833delT mutation and the splice site mutation were significantly reduced, probably due to nonsense‐mediated mRNA decay. No mutation was found in the second alleles in the seven families studied, but RT‐PCR of BUB1B and Western blot analysis of BubR1 indicated a modest decrease of their transcripts. BubR1 in the cells from two patients showed both reduced protein expression and diminished kinetochore localization. Their expression level of p55cdc, a specific activator of anaphase‐promoting complex, was normal but its kinetochore association was abolished. Microcell‐mediated transfer of chromosome 15 (containing BUB1B) into the cells restored normal BubR1 levels, kinetochore localization of p55cdc, and the normal responses to colcemid treatment. These findings indicate the involvement of BubR1 in p55cdc‐mediated mitotic checkpoint signaling, and suggest that >50% decrease in expression (or activity) of BubR1 is involved in the PCS syndrome.

Effects of Vascular Endothelial Growth Factor on Osteoclast Induction during Tooth Movement in Mice

For orthodontic tooth movement, remodeling of the alveolar bone is maintained by a repeated process of bone resorption and new bone formation, controlled, respectively, by osteoclasts and osteoblasts. Recently, we have found that recombinant human vascular endothelial growth factor (rhVEGF) acts as a macrophage colony-stimulating factor in osteoclast induction in osteopetrotic (op/op) mice. The purpose of this study was to investigate whether rhVEGF stimulates osteoclast differentiation during experimental tooth movement. Purified rhVEGF was injected once into the buccal gingival groove around the incisors. An experimental appliance with a helical loop was bonded onto the upper incisors, and an initial force of 1.0 g was applied for three days. The number of osteoclasts appearing in the periodontal ligament space on the pressure side of the alveolar bone was increased markedly. These results suggest that local administration of rhVEGF enhances the number of osteoclasts, and may increase the rate of orthodontic tooth movement. Abbreviations: recombinant human vascular endothelial growth factor (rhVEGF), macrophage colony-stimulating factor (M-CSF), osteopetrotic mice (op/op mice),fms-like tyrosine kinase (Flt-1), periodontal ligament (PDL), phosphate-buffered saline (PBS), tartrate-resistant acid phosphatase (TRAP), and analysis of variance (ANOVA).

Expression of Vascular Endothelial Growth Factor and the Effects on Bone Remodeling during Experimental Tooth Movement

Vascular endothelial growth factor (VEGF) has an ability to induce functional osteoclasts as well as neovascularization. We recently reported that the number of osteoclasts was enhanced by the injection of recombinant human VEGF (rhVEGF) with the application of mechanical force for experimental tooth movement. In this study, the expression of VEGF was detected in osteoblasts on the tension side of the alveolar bone. Moreover, the rate of tooth movement was significantly increased in the rhVEGF injection groups compared with the controls. These results suggested that VEGF, highly expressed by mechanical stimuli, enhances the number of osteoclasts as a paracrine factor, and that the amount of tooth movement is accelerated by both endogenous VEGF and injected rhVEGF.

Effects of Sex Hormone Disturbances on Craniofacial Growth in Newborn Mice

It is well-known that sex hormones influence bone metabolism. However, it remains unclear as to how sex hormones affect bone growth in newborn mice. In this study, we performed orchiectomy (ORX) and ovariectomy (OVX) on newborn mice, and examined the effects on craniofacial growth morphometrically. ORX and OVX were performed on five-day-old C57BL/6J mice. Four weeks after surgery, lateral cephalograms were taken of all of the mice, with the use of a rat and mouse cephalometer. Cephalometric analysis of the craniofacial skeleton was performed by means of a personal computer. Inhibition of craniofacial growth was found in the experimental groups but not in the sham-operated groups. In the nasomaxillary bone and mandible, the amount of growth was significantly reduced. These results suggest that craniofacial growth is inhibited by sex hormone disturbances not only in puberty but also immediately after birth.

Effects of Cyclic Tensile Forces on the Expression of Vascular Endothelial Growth Factor (VEGF) and Macrophage-colony-stimulating Factor (M-CSF) in Murine Osteoblastic MC3T3-E1 Cells

It has been reported that vascular endothelial growth factor (VEGF), expressed by osteoblasts, can induce osteoclast recruitment and thus affects bone remodeling. The purpose of this study was to investigate the effects of cyclic tensile forces on the expression of VEGF and macrophage-colony-stimulating factor (M-CSF) in osteoblastic MC3T3-E1 cells. VEGF and M-CSF gene expression and protein concentration were determined by real-time PCR and enzyme-linked immunoassay. The expression of VEGF and M-CSF mRNA in the experimental group was higher than in the control group. The increase in the concentration of VEGF and M-CSF protein in the experimental group was time-dependent. Moreover, gadolinium (an S-A channel inhibitor), but not nifedipine (L-Type Ca2+ channel blocker), treatment reduced the concentration of VEGF and M-CSF mRNA and protein in the experimental groups. These findings suggest that cyclic tensile forces increase the expression of VEGF and M-CSF in osteoblastic MC3T3-E1 cells via a stretch-activated channel (S-A channel).

Neutralizing effects of an anti-vascular endothelial growth factor antibody on tooth movement.

Our recent studies demonstrated that local administration of recombinant human vascular endothelial growth factor (rhVEGF) during experimental tooth movement enhanced the number of osteoclasts and the rate of tooth movement. The purpose of this study was to examine the effect of anti-VEGF polyclonal antibody on osteoclastic differentiation, the amount of tooth movement, and the degree of tooth relapse in 30-day-old mice. First, these mice were subjected to various doses of anti-VEGF polyclonal antibody, with tooth movement for three days. In the next study, daily injections of 10-microg antibody were administered for 18 days during the experimental tooth movement. The amount of tooth movement was measured as in our previous study. Furthermore, in the third study, we administered daily injection of 10-microg antibody and measured tooth relapse after the experimental tooth movement for 45 days. The osteoclasts number in 10- and 50-microg antibody two-time injection group was significantly smaller than that in the controls (P < .05). The number of osteoclasts was decreased more substantially by daily injection of 10-microg antibody, showing more significant differences from the controls (P < .01). The amount of tooth movement was significantly less in the experimental group than in the controls on days 15 and 18 (P < .05). Furthermore, the amount of relapse in the experimental group was significantly less than that in the controls on days 9 and 11 after removal of the appliance (P < .05). These results show that the treatment of anti-VEGF polyclonal antibody markedly reduced the osteoclasts number and inhibited the amount of tooth movement and relapse of moved teeth.

Amyloid β Protein Deposition in Osteopetrotic (op/op) Mice Is Reduced by Injections of Macrophage Colony Stimulating Factor

The deposition of amyloid β (Aβ) protein is a neuropathological change that characterizes Alzheimers disease. Animals with the osteopetrosis (op/op) mutation suffer from a general skeletal sclerosis, a significantly reduced number of macrophages and osteoclasts in various tissues, and have no systemic macrophage colony stimulating factor (M-CSF). This study examined the effect that M-CSF injections had on Aβ deposition and microglial cell distribution in the brains of normal and op/op mice. Aβ-positive plaques were detected in the cerebral cortex of op/op mice, but not in normal mice. M-CSF reduced the numbers of Aβ-positive plaques in op/op mice. The microglial cell population was reduced in op/op mice compared with normal mice, and M-CSF increased the numbers to 65.8% of that observed in normal mice. Our results suggest that a clearer understanding of the role that microglial cells play in Aβ deposition may help determine the mechanisms involved in the pathogenesis of Alzheimers disease.

Effects of insulin-like growth factor-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture under different masticatory loading conditions in growing mice

It is well accepted that mechanical loading inhibits bone resorption and increases in vivo bone formation. It is also known that cyclic mechanical loading, in particular, can enhance bone formation significantly. These findings suggest a significant role for mechanical stimuli in bone remodelling mediated by various local growth factors including insulin-like growth factor-I (IGF-I). Earlier studies showed that the nasal bone length and premaxillary bone width were significantly greater in mice fed a solid diet rather than a granulated diet, and that these dimensions increased significantly in a solid-diet group treated with IGF-I. The present study sought to examine the effect of IGF-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture subjected to different masticatory loadings. For the solid-diet groups, the numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells and osteoblasts were significantly greater in the group injected with IGF-I than in the animals injected with physiological saline. In the groups fed a granulated diet, no significant differences in the numbers of TRAP-positive osteoclastic cells and osteoblasts were found over the entire experimental period between mice injected with either IGF-I or physiological saline. It is shown that IGF-I significantly induces the expression of osteoclasts and osteoblasts and the subsequent bone remodelling, and that the effect may be additive as compared to that of mechanical masticatory loading, which seems to be more important in bone remodelling in terms of the numbers of osteoclasts and osteoblasts.

Transplantation of new autologous biomaterials into jaw cleft

This study compared bone-marrow grafting using chondroid or fibula bone grafts transplanted into simulated alveolar bone defects in mice. The osteogenic procedure was also investigated. As an experimental model of the maxillary alveolar bone cleft, suitable for testing bone-inductive materials, a surgical trephine with a low-speed dental engine was used to form critical-sized defects in the pre-maxillary bones of male mice. Distraction osteogenesis was performed using an external fixation device. The osteotomy site was surrounded by an external callus, consisting of hyaline cartilage, that contained a large quantity of chondroid bone. Transplanted bone within chondroid bone was characterized by bone formation and remodelling 30 days post-transplantation, and bone adhesion following chondroid bone grafting was better than adhesion following fibula grafting. The present findings are the first to demonstrate the potential of chondroid bone transplantation as a new therapeutic system of bone grafting, suitable for bone substitution in craniofacial bone defects.