Effects of vascular endothelial growth factor-C and -D on osteoclast differentiation and function in human peripheral blood mononuclear cells.

OBJECTIVE: The purpose of this study was to clarify the interaction of vascular endothelial growth factors (VEGFs)-C and -D with cell surface foetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-4 (Flt-4) receptors in the induction and activity of osteoclasts in cultured human peripheral blood mononuclear cells (PBMCs). DESIGN: PBMCs were cultured on chamber slides or on ivory discs for 2 or 3 weeks in the presence of macrophage-colony stimulating factor (M-CSF), VEGF-A, -C or -D, or placental growth factor (PlGF) with or without receptor activator of nuclear factor kappa-B ligand (RANKL). The number of osteoclasts in each group was counted and the area of ivory resorption was measured. In addition, osteoclast differentiation was further analysed under the same conditions, but with the addition of specific neutralizing antibodies against Flk-1 and Flt-4. RESULTS: RANKL was essential for the induction of osteoclasts in PBMCs. However, significant differences were found in the number of osteoclasts induced by VEGF-A, -C, -D or M-CSF with RANKL compared with control groups lacking or containing RANKL. Blocking of either Flk-1 or Flt-4 resulted in a reduction in the enhancement of osteoclast differentiation in PBMCs by VEGF-C or -D with RANKL. The osteoclasts induced by VEGF-A, -C, -D or M-CSF with RANKL formed significantly larger resorption lacunae than those formed by osteoclasts induced by RANKL alone. CONCLUSIONS: This study showed that VEGF-C and -D play a role in the induction of osteoclast differentiation through both Flk-1 and Flt-4 receptors and influence the area of the ivory resorption in PBMCs.

Age-related production of osteoclasts and the changes of serum levels of vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor (NF)-κB ligand (RANKL) in osteopetrotic (op/op) mice.

OBJECTIVE: Expression of osteoclasts in osteopetrotic (op/op) mice is substantially reduced by the absence of functional macrophage colony-stimulating factor (M-CSF). However, it has been reported that osteoclasts do gradually appear in the bones of op/op mice and spontaneously correct the osteopetrosis. DESIGN: Age-related production of osteoclasts and the changes of serum levels of vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor (NF)-κB ligand (RANKL) in op/op mice were examined. RESULTS: The number of femoral osteoclasts, and the serum levels of VEGF, both gradually increased in op/op mice after birth and reached a peak in 120- and 60-day-old mice, respectively. However, the serum levels of RANKL showed an inverse relationship to osteoclast number. CONCLUSIONS: These findings suggest that the appearance of osteoclasts may be influenced by the serum levels of VEGF and that the serum levels of RANKL may be influenced by the appearance of osteoclasts.

Expression of Sox 9 and type II and X collagens in regenerated condyle.

The present study was designed to examine the expression of Sox 9 and type II and X collagens in regenerated condyle resulting from the use of a functional appliance. Ninety, 3-week-old, mice were divided equally into the following groups: two experimental groups (condylectomy group and condylectomy with functional appliance group) and the corresponding control group. In the condylectomy group, a unilateral condylectomy was performed on the right side. In the condylectomy with appliance group, the mandible was repositioned in a forward direction using a functional appliance after unilateral condylectomy. The expression of Sox 9 and type II and X collagens in the condyle was determined immunohistochemically 4 weeks after surgery. In mice with a condylectomy, the expression was minimal. On the other hand, these factors were highly expressed in the condylectomized side with the appliance. It is thus speculated that cartilaginous regeneration is due to the expression of chondrogenic factors, such as Sox 9 and type II and X collagens. It is also suggested that condyle regeneration results from an optimal intra-articular environment with appropriate joint spaces achieved by condylar repositioning.

A newly developed snack effective for enhancing bone volume.

BACKGROUND: The incidence of primary osteoporosis is higher in Japan than in USA and European countries. Recently, the importance of preventive medicine has been gradually recognized in the field of orthopaedic surgery with a concept that peak bone mass should be increased in childhood as much as possible for the prevention of osteoporosis. Under such background, we have developed a new bean snack with an aim to improve bone volume loss. In this study, we examined the effects of a newly developed snack on bone volume and density in osteoporosis model mice. METHODS: Orchiectomy (ORX) and ovariectomy (OVX) were performed for C57BL/6J mice of twelve-week-old (Jackson Laboratory, Bar Harbar, ME, USA) were used in this experiment. We prepared and given three types of powder diet e.g.: normal calcium diet (NCD, Ca: 0.9%, Clea Japan Co., Tokyo, Japan), low calcium diet (LCD, Ca: 0.63%, Clea Japan Co.,) and special diet (SCD, Ca: 0.9%). Eighteen weeks after surgery, all the animals were sacrified and prepared for histomorphometric analysis to quantify bone density and bone mineral content. RESULTS: As a result of histomorphometric examination, SCD was revealed to enhance bone volume irrespective of age and sex. The bone density was increased significantly in osteoporosis model mice fed the newly developmental snack as compared with the control mice. The bone mineral content was also enhanced significantly. These phenomena were revealed in both sexes. CONCLUSION: It is shown that the newly developed bean snack is highly effective for the improvement of bone volume loss irrespective of sex. We demonstrated that newly developmental snack supplements may be a useful preventive measure for Japanese whose bone mineral density values are less than the ideal condition.

Fms-like tyrosine kinase (Flt)-4 signaling participates in osteoclast differentiation in osteopetrotic (op/op) mice.

Vascular endothelial growth factor (VEGF) induces osteoclast differentiation as well as neovascularization by binding to the fms-like tyrosine kinase (Flt)-1 and fetal liver kinase (Flk)-1 receptors. The Flt-4 receptor also plays an important role in angiogenesis and lymphangiogenesis. The purpose of this study was to investigate the functions of Flt-4 in the signaling pathway of osteoclast differentiation. We examined the expression of Flt-4 on osteoclast precursor cells (OCPs), and the ability of recombinant human (rh) VEGF-D, one of the ligands of Flt-4, to stimulate the phosphorylation of extracellular-regulated kinase1/2 (ERK1/2) and to activate the nuclear factor-kappa B (NF-kappaB) pathway in OCPs. The number of osteoclasts induced by injection of rhVEGF-D in osteopetrotic (op/op) mice was also evaluated in the absence or presence of neutralizing antibodies to Flt-4. Flt-4 expression was detected on OCPs at both gene and protein levels and stimulation of Flt-4 by rhVEGF-D might induce activation of mitogen-activated protein kinase (MAPK) and NF-kappaB pathways for induction of osteoclast differentiation. Moreover, the number of osteoclasts in op/op mice increased after injection of rhVEGF-D, but was significantly reduced by the injection of Flt-4 neutralizing antibodies. We have therefore shown that Flt-4 expressed on OCPs, might activate MAPK and NF-kappaB pathways and played an important role in osteoclast differentiation.

VEGF and M-CSF levels in periodontal tissue during tooth movement.

It has been reported that both vascular endothelial growth factor (VEGF) and macrophage colonystimulating factor (M-CSF) can induce osteoclast recruitment. Thus, VEGF and M-CSF are considered to be closely involved in the bone remodeling process. The purpose of this study was to evaluate changes in VEGF and M-CSF expression during orthodontic treatment. The expression of VEGF and M-CSF mRNA in osteoblasts and fibroblasts was detected by in situ hybridization during experimental tooth movement in mice. Furthermore, the canine retraction side and the control side of orthodontic patients were compared, revealing a statistically significant increase in both VEGF and M-CSF concentrations in gingival crevicular fluid. These results suggest that orthodontic tooth movement causes an increase in VEGF and M-CSF levels. These factors may induce bone remodeling via osteoclastic bone resorption.