Although it is known that mechanical stress to osteoblast and periodontal ligament cells suppresses osteoclast differentiation, little is known about the direct effect of mechanical stress on osteoclast differentiation. In this study, we examined the role of mechanical stress on osteoclast differentiation using murine pre-osteoclastic RAW264.7 cells treated with receptor activator of nuclear factor-κB ligand (RANKL). RAW cells were cultured with RANKL, and mechanical stress was applied for a given period. We counted the number of osteoclast cells which were tartrate-resistant acid phosphatase (TRAP)-positive and multinucleated (2 nuclei or more), and measured mRNA by RT-PCR. There was a decrease in the number of osteoclasts under mechanical stress compared with the number under no mechanical stress. The number of nuclei per osteoclast also decreased compared to the number of nuclei per osteoclast cultured with the application of mechanical stress. As the cells were cultured for a period of 1-7 days and/or for different periods of mechanical stress application, osteoclast differentiation decreased with mechanical stress and increased after removing mechanical stress. Expression of mRNA for the osteoclast-specific genes, TRAP, matrix metalloproteinase-9, cathepsin-K and calcitonin receptor, decreased with mechanical stress and was associated with the number of osteoclasts. Inducible nitric oxide synthase mRNA which inhibits osteoclast differentiation, increased with mechanical stress. In spite of the decrease in osteoclast number with mechanical stress, nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and NFATc2 mRNA expression increased with mechanical stress. These findings indicate that mechanical stress directly suppresses osteoclast differentiation and increases NFATc1 and NFATc2 suggesting delayed differentiation.

ABSTRACT-Alkaline phosphatase (ALP) is supposed to be important for bone formation; however, its role is not clear. In this study, we examined the importance of enzymatic activity of ALP and anchoring of ALP protein to the cells for mineralization of an osteoblastic cell line, MC3T3-E1. While we cultured the cells in the presence of tetramisole, an inhibitor of ALP activity, ALP protein was expressed at a similar level to that in the control. Although tetramisole showed no effect on cell growth and increased hydroxyproline accumulation, it decreased the osteocalcin production and the accumulation of calcium and phosphate in the matrices. Tetramisole also inhibited mineralized nodule formation, which was observed by optical microscopy and detected by Von Kossa staining. On the other hand, when ALP protein was released from the cell membranes with the use of phosphatidylinositol-specific phospholipase C, no marked changes were detected in hydroxyproline, calcium and phosphate accumulations in the matrices at late calcification stage, which was consistent with the morphological findings. These results clearly show that enzymatic activity of ALP is necessary for mineralization of MC3T3-E1 cells, but not the presence of ALP protein or anchoring of ALP to the cells.Keywords: Alkaline phosphatase, Mineralization, Phosphatidylinositol-specific phospholipase C, Osteoblastic cell, TetramisoleBone-type alkaline phosphatase (ALP) is supposed to play a key role in the formation and calcification of hard tissues from accumulated circumstantial evidence (1). Although a number of theories have been proposed, its precise function is not well understood yet (2 -6). It is not even clear whether ALP is really indispensable to mineralization.ALP is attached to the external surfaces of plasma membranes by phosphoethanolamine bound to oligosaccharide, which is, in turn, covalently linked to the polar head group of phosphatidylinositol (7 -9). ALP is released from plasma membranes by phosphatidylinositol-specific phospholipase C (PIPLC) (10 -13).We previously reported that ALPs of MC3T3-E1 cells were released into the medium during cell culture and the time course of the increase of ALP in the medium was consistent with the progress of mineralization (14). We assumed that matured and released ALP might be closely related to the mineralization. An alternative report demonstrated that ALP attached covalently to the external surface of plasma membranes was involved in the mineralization of hard tissues (15).Our aim in this study was to clarify the importance of enzymatic activity of ALP and anchoring of ALP to the cells for mineralization. We used an osteoblastic cell line, MC3T3-E1 cells, which were established from fetal murine calvalial cells (16). The cells differentiate into osteoblasts and then produce ALP and process procollagens to collagens; finally the cells form mineralized tissues in vitro. We studied the effects of tetramisole, an inhibitor of ALP activity, and PIPLC additions to culture medium on mineralization of MC3T3-E1 c...

Abstract.Recent research has shown that platinum nanoparticles (nano-Pt) efficiently quench reactive oxygen species (ROS) as a reducing catalyst. ROS have been suggested to regulate receptor activator of NF-κB ligand (RANKL)-stimulated osteoclast differentiation. In the present study, we examined the direct effects of platinum nano-Pt on RANKL-induced osteoclast differentiation of murine pre-osteoclastic RAW 264.7 cells. The effect of the nano-Pt on the number of osteoclasts was measured and their effect on the mRNA expression for osteoclast differentiation was assayed using real-time PCR. Nano-Pt appeared to have a ROS-scavenging activity. Nano-Pt decreased the number of osteoclasts (2+ nuclei) and large osteoclasts (8+ nuclei) in a dose-dependent manner without affecting cell viability. In addition, this agent significantly blocked RANKL-induced mRNA expression of osteoclastic differentiation genes such as c-fms, NFATc1, NFATc2, and DC-STAMP as well as that of osteoclast-specific marker genes including MMP-9, Cath-K, CLC7, ATP6i, CTR, and TRAP. Although nano-Pt attenuated expression of the ROS-producing NOXfamily oxidases, Nox1 and Nox4, they up-regulated expression of Nox2, the major Nox enzyme in macrophages. These findings suggest that the nano-Pt inhibit RANKL-stimulated osteoclast differentiation via their ROS scavenging property. The use of nano-Pt as scavengers of ROS that is generated by RANKL may be a novel and innovative therapy for bone diseases.

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Abstract. Matrix metalloproteinases (MMPs) play important roles in the invasion and metastasis to soft tissues of carcinomas including, oral squamous cell carcinomas (SCCs). Although, osteoclastic bone resorption is an important step in bone involvement in a variety of malignancies, the mechanism of bone involvement of oral SCC remains unclear. Once cancer cells arrest in bone, the bone is a storehouse of a variety of cytokines and growth factors and thus provides an extremely fertile environment for cell growth. The bone-invasive oral cancer cell line, BHY, transcriptionally expressed detectable levels of TGF-ß, IL-1ß, IL-8, parathyroid hormone-related protein (PTHrP) and vascular endothelial growth factor (VEGF) mRNAs and failed to express GM-CSF, IL-6, and TNF-α. Furthermore, the BHY-conditioned medium greatly upregulated IL-6 and RANKL/ODF mRNA expression in osteoblasts, suggesting a potential indirect stimulation of osteoclastogenesis via the osteogenic lineage. Seven out of eleven patients with carcinomas of the lower alveolus and gingiva showing infiltrative bone involvement expressed PTHrP mRNA. These data suggest that the occurrence of PTHrP may be an indication of developing oral malignant carcinomas.