Effects And Mechanism Of Low-magnitude, High-frequency Compound Vibration On Osteoclast Differentiation

Osteoporosis is a systemic skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue, resulting in an increased risk of fracture. It was estimated that people with osteoporosis had been more than 200 millions in the world. Asia has become one of high incidence of osteoporosis, and there had been more than 90 millions patients in China. Its incidence has leapt to the 7th of common diseases. Fractures and other complications of osteoporosis result in deformity, death and expensive medical costs. These give patients, families and society heavy burden. As the elderly population increases, the situation becomes more prominent. Osteoporosis is not just a medical problem but also a social problem requiring urgent attention. Drugs that inhibit the formation or activity of osteoclasts are extensive used for treating osteoporosis. Drugs that promote bone formation are used less. However, there are no exact clinical effect, and result in some treatment side effects. Wolffs law shows that the bone remodels to adapt to the needs of function and the changes of the mechanical stress. So the mechanical stress loading on osteocytes within the bone is the original power for bone reconstruction. The mechanical vibration signal that is lower than that leading to injury the bone tissue, has a strong osteogenic effect. It not only can prevent bone loss, but also can improve the bone structure and mechanical properties of biology. So vibration is a non-invasive, non-pharmacological and safe treatment for osteoporosis. A certain frequency and intensity are required for vibration inducing osteoblastic bone formation. Too low intensity or frequency is hard to reach the threshold stimulation for bone formation. While too high, the vibration intensity is harmful to human. Bone is very sensitive to vibration. Studies have shown that short-term low-magnitude, high-frequency vibration can promote quantity and quality of bone by reducing bone resorption, increasing trabecular bone mass and formation rate, promoting osteoblast proliferation and differentiation. It has been well-demonstrated that vibration has a positive influence on bone formation. However, the mechanism of vibration is unknown. Currently, there are three hypotheses for the mechanism, such as blood perfusion, bone adaptation and vibration bone conduction. But the exact signaling pathways are unclear. Vibration not only can improve bone mass, but can also prevent muscular atrophy, enhance muscle strength and enhance balance. Currently short-term low-magnitude, high-frequency vibration has been widely used in osteoporosis research. In order to fit human motion characteristics better, we designed the compound vibration apparatus instead of whole-body vibration, and hoping to maintain bone formation while changing the modes of vibration. The characteristics of the compound vibration apparatus are as follows:1, a natural alternating movement pattern, human in an imbalance posture on vibration process must have a change in posture to compensate for the imbalanced posture. This will enable patients to produce an alternate of the pelvic motion exercise training. This alternate of the pelvis up and down motion is the basic action of human walking and running. The vibration apparatus provides a vibration stimulus consistent with the characteristics of human anatomy training.2, the compound vibration increases the muscle contraction through the body repeatedly adjusting the posture to maintain body balance. This strengthens the nerve - muscle system, increases the mechanical stimulation to bone tissue, and enhances the body's balance training.3, the compound vibration, based on the vertical vibration, strengthening the neuromuscular system, gives emphasis on the role of the neuromuscular system in prevention and treatment of osteoporosis. Our earlier researchs got some results to confirm the effect of the compound vibration on osteogenesis. Firstly, different frequencies of vibration had an impact on the osteoblast cycle, proliferation and alkaline phosphatase activity. The appropriate frequency of compound vibration promoting osteoblast proliferation and differentiation was 15-45Hz. Secondly, Compound vibration could increase bone mineral density of ovariectomized rats, improve bone micro structure and improve bone strength. Furthermore, we confirmed that compound vibration not only could improve bone mass, but also could prevent muscular atrophy, enhance muscle strength and improve peripheral nerve function, which benefited balance. It was well-demonstrated that compound vibration had a positive influence on bone formation. But the effect of compound vibration on osteoclast, the only bone resorption cell, has not been studied. In short, the vibration signal is a complex network, and further study will be done for the specific mechanisms.Bone remodeling is a self-renewal process, including absorbing bone by osteoclasts and forming new bone constantly by osteoblasts on the resorption bone. Osteoclasts play an important role in maintaining stable bone mass in the remodeling process after bones mature. More or osteoclast activity increased results in increased bone resorption and bone remodeling imbalance, and even leads to the occurrence of many bone diseases such as:OP, rheumatoid arthritis, metastatic bone tumor. Multinucleated macrophages form osteoclasts by fusion of the mononuclear macrophages of hematopoietic precursors. Osteoclasts with bone resorption functions mainly lie in the bone surface and the bone vascular channels. They play an important role bone resorption of bone remodeling in physiological and pathological. However, the study on osteoclasts which are terminal cells was limited. Not only osteoclasts separation and purification were difficult, but also osteoclasts could not differentiate and subculture. RAW 264.7 cells are mouse mononuclear macrophage derived from a tumor induced by Abelson murine leukemia virus. RAW264.7 cells are equivalent to an early phase in osteoclast differentiation. We understand the physiological role of osteoclasts more by the finding of receptor activator of nuclear factor-kB ligand (RANKL)/RANK/osteoprotegerin (OPG) regulatory axis. With in-depth understanding of the regulatory mechanisms of osteoclast differentiation, RANKL got more attention in osteoclast differentiation, maturation and activation. RANKL bound and activated the RANK, which are expression on osteoclastic precursor cells for regulated osteoclastogenesis and bone resorption. RAW264.7 cells can be induced osteoclast differentiation by RANKL, so it is widely used in the study of osteoclast differentiation.The effect of low-magnitude, high-frequency compound vibration (LMHFCV) on bone formation is the result of osteoblasts or osteoclasts? The study of vibration on osteoclast differentiation is better able to elaborate the vibration mechanism to prevent osteoporosis. In this work, we investigated osteoclast differentiation in RAW cells under mechanical stress using LMHFCV, which will give some new insight into the mechanism of LMHFCV on anti-osteoporosis.Objectives1. To observe the effect of LMHFCV on the RANKL-induced osteoclast differentiation of RAW264.7 cells.2. To observe the effect of LMHFCV on the expression of osteoclast-specific genes including cathepsin K (CATK), matrix metallopeptidase 9 (MMP-9) and tartrate resistant acid phosphatase (TRAP).3. To observe the effect of LMHFCV on the c-Fos protein expression.Methods1. The effect of LMHFCV on the RANKL-induced osteoclast differentiation of RAW264.7 cells.Cells were incubated in 96-well cell culture clusters with 200μl complete medium at a concentration of 3×103 cells/well for 4 days. The next day the medium was shifted to 10% FBS-CS medium. 1μl RANKL (10μg/ml) was added to RANKL group and LMHFCV group at a concentration of 50ng/ml. The medium was replaced every 3 days. RAW264.7 cells in LMHFCV group were subjected to LMHFCV at 45 Hz,0.3 g for 15 min per day, once a day. After 4 days, cells were stained using TRAP kit according to the manufacturer's instructions. Then stained cells were observed and photographed under inverted microscope.2. The effect of LMHFCV on the expression of osteoclast-specific genes.Cells were incubated in 6-well cell culture clusters with 2 ml complete medium at a concentration of 1×105 cells/well for 3 days. The next day the medium was shifted to 10% FBS-CS medium.10μl RANKL (10μg/ml) was added to RANKL group and LMHFCV group at a concentration of 50ng/ml. RAW264.7 cells in LMHFCV group were subjected to LMHFCV at 45 Hz,0.3 g for 15 min per day, once a day. After 3 days, we monitored the expression of osteoclast-specific genes including CATK, MMP-9 and TRAP by real-time RT-PCR. Total RNA was extracted from the induced cells using Trizol. The total RNA was reverse transcribed into cDNA. Real-time RT-PCR amplification and detection was performed using SYBR(?) Premix Ex TaqTM it in the ABI7500 according to the manufacturer's instructions. We calculated CATK, MMP-9 and TRAP mRNA by normalizing them to the endogenous housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH).3. The effect of LMHFCV on the c-Fos protein expression.Cells were incubated in 3.5cm cell culture clusters with 2 ml complete medium at a concentration of 1×105 cells/well for 3 days. The next day the medium was shifted to 10% FBS-CS medium.10μl RANKL (10μg/ml) was added to RANKL group and LMHFCV group at a concentration of 50ng/ml. RAW264.7 cells in LMHFCV group were subjected to LMHFCV at 45 Hz,0.3 g for 15 min per day, once a day. After 3 days, cells were harvested and extracted the total protein. c-Fos protein expression was determined using western blot by normalizing it to GAPDH.Results1. LMHFCV inhibited RANKL-induced osteoclast differentiation of RAW264.7 cells.After 4 days, cells were stained using TRAP kit. Few TRAP-positive multinucleated cells (MNCs) were observed in control group. RANKL increased TRAP-positive MNCs (≥3 nuclei), osteoclasts (P<0.001). However, RANKL-induced TRAP-positive MNCs were significantly inhibited by LMHFCV (P=0.002). Furthermore, we also found that the number of large TRAP-positive MNCs (≧10 nuclei) was significantly reduced compared with RANKL group (P<0.001).2. LMHFCV decreased the expression of osteoclast-specific genes.After 3 days, we monitored the expression of osteoclast-specific genes including CATK, MMP-9 and TRAP by real-time RT-PCR. RANKL significantly increased genes CATK, MMP-9 and TRAP expression compared to the control group (P<0.001). However, LMHFCV significantly depressed the RANKL-induced up-regulation of CATK, MMP-9 and TRAP expression (P<0.01).3. LMHFCV down-regulates the expression of c-Fos protein.After 3 days, we examined whether LMHFCV modulated the transcription factors in RANKL-stimulated RAW264.7 cells using Western blot analysis. Compared to the control group, RANKL significantly enhanced the expression of c-Fos protein (P=0.027). LMHFCV significantly decreased the RANKL-induced up-regulation of c-Fos (P= 0.017).ConclusionsLMHFCV inhibited RANKL-induced osteoclast differentiation of RAW264.7 cells through c-Fos pathway.