Publications (4)10.22 Total impact
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ABSTRACT: Fatigue loading causes a spatial distribution of osteocyte apoptosis co-localized with bone resorption spaces peaking around microdamage sites. Since osteocytes have been shown to regulate osteoclast formation and activity, we hypothesize that osteocyte apoptosis regulates osteoclastogenesis. In this study, we used serum-starvation to mimic reduced nutrient transport in microdamaged bone and induce apoptosis in MLO-Y4 osteocyte-like cells; conditioned medium was used to apply soluble factors released by apoptotic osteocytes (aOCY) to healthy non-apoptotic MLO-Y4 cells. Osteoclast precursor (RAW264.7 monocyte) migration and differentiation were assessed in the presence of conditioned media (CM) from: (A) aOCY, (B) osteocytes treated with apoptosis conditioned medium (i.e., healthy osteocytes in the presence of apoptosis cues; apoptosis CM-treated osteocytes (atOCY)), and (C) osteocytes treated with non-apoptosis conditioned medium (i.e., healthy osteocytes in the absence of apoptosis cues; non-apoptosis CM-treated osteocytes (natOCY)). Receptor activator for nuclear factor-κB ligand (RANKL), macrophage colony stimulating factor (M-CSF), vascular endothelial growth factor (VEGF) and osteoprotegerin (OPG) mRNA, and protein expression were measured. Our findings indicate that soluble factors released by aOCY and atOCY promoted osteoclast precursor migration (up to 64% and 24% increase, respectively) and osteoclast formation (up to 450% and 265% increase, respectively). Osteoclast size increased up to 233% in the presence of aOCY and atOCY CM. Recruitment, formation and size were unaltered by natOCY. RANKL mRNA and protein expression were upregulated only in aOCY, while M-CSF and VEGF increased in atOCY. Addition of RANKL-blocking antibody abolished aOCY-induced osteoclast precursor migration and osteoclast formation. VEGF and M-CSF blocking antibodies abolished atOCY-induced osteoclastogenesis. These findings suggest that aOCY directly and indirectly (through atOCY) initiate targeted bone resorption by regulating osteoclast precursor recruitment and differentiation. J. Cell. Biochem. 112: 2412–2423, 2011. © 2011 Wiley-Liss, Inc.
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ABSTRACT: Whole body vibration (WBV), consisting of a low-magnitude, high-frequency (LMHF) signal, is anabolic to bone in vivo and may act through alteration of the lineage commitment of mesenchymal stromal cells (MSC). We investigated the effect of LMHF vibration on rat bone marrow-derived MSCs (rMSCs) in an in vitro system. We subjected rMSCs to repeated (six) bouts of 1-h vibration at 0.3g and 60 Hz in the presence of osteogenic (OS) induction medium. The OS differentiation of rMSCs under the loaded and non-loaded conditions was assessed by examining cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of various osteoblast-associated markers [ALP, Runx2, osterix (Osx), collagen type I alpha 1 (COL1A1), bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN)], and matrix mineralization. LMHF vibration did not enhance the OS differentiation of rMSCs. Surprisingly, the mRNA level of Osx, a transcription factor necessary for osteoblast formation, was decreased, and matrix mineralization was inhibited. Our findings suggest that LMHF vibration may exert its anabolic effects in vivo via mechanosensing of a cell type different from MSCs.
Conference Paper: Biological Responses of Cementoblasts to Mechanical Vibration in Vitro[Show abstract] [Hide abstract]
ABSTRACT: Objective: To study the effect of low magnitude high frequency (LMHF) mechanical vibration on cementoblasts in vitro Methods: OCCM-30 cementoblasts were seeded in 6-well plates and grew up to 100% confluency. After 24 hours of starvation, the plate wells were filled with starving media, tightly sealed, and placed onto a custom vibration plate attached to a vertically oriented shaker. The cells in the experimental groups were subjected to 30, 60, or 90 Hz of sinusoidal vibrations at 0.3g for 1 hour; while the non-vibration groups were kept stationary. Upon completion of vibration, the medium in each well was replaced with fresh starving medium and post incubated for 1 or 6 hours. The conditioned medium was collected for immunoassay to test released OPG and RANKL, while cell lysate collected for western blot to test cellular OPG, RANKL and sclerostin (SOST - a negative bone formation regulator). One-way ANOVA was used to compare means of more than three groups, followed by Tukey post-hoc test (p=0.05). Results: After 1 hour vibration followed by 1 hour post incubation, the released OPG levels were significantly reduced (p<0.01) in 30 and 60 Hz groups. No significant change of the released OPG was found after 6 hours of post incubation. After 1 hour of post incubation, intracellular RANKL level was increased (p=0.002) in 60 Hz group. Upon 6 hours of post incubation, RANKL level in 30 Hz group was increased significantly (p=0.038), while 60 Hz almost remained at the level as observed after 1 hour of post incubation. SOST was found significantly decreased in 30 and 90 Hz groups (p=0.005) only after 6 hours of post incubation. Conclusion: LMHF vibration actively regulates OPG, RANKL and SOST. The cementoblastic responses to LMHF vibration are frequency-specific, and the regulations of OPG-RANKL axis and SOST are time phase specific.
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ABSTRACT: Osteocytes are well evidenced to be the major mechanosensor in bone, responsible for sending signals to the effector cells (osteoblasts and osteoclasts) that carry out bone formation and resorption. Consistent with this hypothesis, it has been shown that osteocytes release various soluble factors (e.g. transforming growth factor-beta, nitric oxide, and prostaglandins) that influence osteoblastic and osteoclastic activities when subjected to a variety of mechanical stimuli, including fluid flow, hydrostatic pressure, and mechanical stretching. Recently, low-magnitude, high-frequency (LMHF) vibration (e.g., acceleration less than <1 x g, where g=9.81m/s(2), at 20-90 Hz) has gained much interest as studies have shown that such mechanical stimulation can positively influence skeletal homeostasis in animals and humans. Although the anabolic and anti-resorptive potential of LMHF vibration is becoming apparent, the signaling pathways that mediate bone adaptation to LMHF vibration are unknown. We hypothesize that osteocytes are the mechanosensor responsible for detecting the vibration stimulation and producing soluble factors that modulate the activity of effector cells. Hence, we applied low-magnitude (0.3 x g) vibrations to osteocyte-like MLO-Y4 cells at various frequencies (30, 60, 90 Hz) for 1h. We found that osteocytes were sensitive to this vibration stimulus at the transcriptional level: COX-2 maximally increased by 344% at 90Hz, while RANKL decreased most significantly (-55%, p<0.01) at 60Hz. Conditioned medium collected from the vibrated MLO-Y4 cells attenuated the formation of large osteoclasts (> or =10 nuclei) by 36% (p<0.05) and the amount of osteoclastic resorption by 20% (p=0.07). The amount of soluble RANKL (sRANKL) in the conditioned medium was found to be 53% lower in the vibrated group (p<0.01), while PGE(2) release was also significantly decreased (-61%, p<0.01). We conclude that osteocytes are able to sense LMHF vibration and respond by producing soluble factors that inhibit osteoclast formation.
University of Toronto
Toronto, Ontario, Canada
- Institute of Biomaterials and Biomedical Engineering