Cytoskeletal Configuration Modulates Mechanically Induced Changes in Mesenchymal Stem Cell Osteogenesis, Morphology, and Stiffness

Mesenchymal stem cells (MSC) responding to mechanical cues generated by physical activity is critical for skeletal development and remodeling. Here, we utilized low intensity vibrations (LIV) as a physiologically relevant mechanical signal and hypothesized that the confined cytoskeletal configuration imposed by 2D culture will enable human bone marrow MSCs (hBMSC) to respond more robustly when LIV is applied in-plane (horizontal-LIV) rather than out-of-plane (vertical-LIV). All LIV signals enhanced hBMSC proliferation, osteogenic differentiation, and upregulated genes associated with cytoskeletal structure. The cellular response was more pronounced at higher frequencies (100 Hz vs 30 Hz) and when applied in the horizontal plane. Horizontal but not vertical LIV realigned the cell cytoskeleton, culminating in increased cell stiffness. Our results show that applying very small oscillatory motions within the primary cell attachment plane, rather than perpendicular to it, amplifies the cell’s response to LIV, ostensibly facilitating a more effective transfer of intracellular forces. Transcriptional and structural changes in particular with horizontal LIV, together with the strong frequency dependency of the signal, emphasize the importance of intracellular cytoskeletal configuration in sensing and responding to high-frequency mechanical signals at low intensities

Gap Junctional Communication in Osteocytes Is Amplified by Low Intensity Vibrations In Vitro

The physical mechanism by which cells sense high-frequency mechanical signals of small magnitude is unknown. During exposure to vibrations, cell populations within a bone are subjected not only to acceleratory motions but also to fluid shear as a result of fluid-cell interactions. We explored displacements of the cell nucleus during exposure to vibrations with a finite element (FE) model and tested in vitro whether vibrations can affect osteocyte communication independent of fluid shear. Osteocyte like MLO-Y4 cells were subjected to vibrations at acceleration magnitudes of 0.15 g and 1 g and frequencies of 30 Hz and 100 Hz. Gap junctional intracellular communication (GJIC) in response to these four individual vibration regimes was investigated. The FE model demonstrated that vibration induced dynamic accelerations caused larger relative nuclear displacement than fluid shear. Across the four regimes, vibrations significantly increased GJIC between osteocytes by 25%. Enhanced GJIC was independent of vibration induced fluid shear; there were no differences in GJIC between the four different vibration regimes even though differences in fluid shear generated by the four regimes varied 23-fold. Vibration induced increases in GJIC were not associated with altered connexin 43 (Cx43) mRNA or protein levels, but were dependent on Akt activation. Combined, the in silico and in vitro experiments suggest that externally applied vibrations caused nuclear motions and that large differences in fluid shear did not influence nuclear motion (<1%) or GJIC, perhaps indicating that vibration induced nuclear motions may directly increase GJIC. Whether the increase in GJIC is instrumental in modulating anabolic and anti-catabolic processes associated with the application of vibrations remains to be determined

Differential Efficacy of 2 Vibrating Orthodontic Devices to Alter the Cellular Response in Osteoblasts, Fibroblasts, and Osteoclasts

Modalities that increase the rate of tooth movement have received considerable attention, but direct comparisons between devices are rare. Here, we contrasted 2 mechanical vibratory devices designed to directly transfer vibrations into alveolar bone as a means to influence bone remodeling. To this end, 3 cells types intimately involved in modulating tooth movements—osteoblasts, periodontal ligament fibroblasts, and osteoclasts—were subjected to in vitro vibrations at bout durations prescribed by the manufacturers. As quantified by an accelerometer, vibration frequency and peak accelerations were 400% and 70% greater in the VPro5 (Propel Orthodontics) than in the AcceleDent (OrthoAccel Technologies) device. Both devices caused increased cell proliferation and gene expression in osteoblasts and fibroblasts, but the response to VPro5 treatment was greater than for the AcceleDent. In contrast, the ability to increase osteoclast activity was device independent. These data present an important first step in determining how specific cell types important for facilitating tooth movement respond to different vibration profiles. The device that engendered a higher vibration frequency and larger acceleration (VPro5) was superior in stimulating osteoblast and fibroblast cell proliferation/gene expression, although the duration of each treatment bout was 75% shorter than for the AcceleDent

Nuclear displacements induced by either vibration induced accelerations or vibration induced fluid shear.

<p>Nuclear displacements induced by either vibration induced accelerations or vibration induced fluid shear.</p

Vibration induced calcein transference is gap junction specific.

<p>When gap junction function was blocked with 75 µM of 18α-GA, GJIC+ cell number was significantly reduced compared to non-blocked groups (Normal) and none of the four vibration frequency/acceleration combinations increased the number of GJIC+ cells. ***: p<0.001 against control.</p

Finite element model of an adherent cell to identify vibration induced nuclear displacements.

<p>The elastic FE model comprised the cell membrane, cytoplasm and nucleus (top). Vibration induced fluid shear and accelerations were evaluated in separate dynamic simulations. Fluid shear was simulated by applying dynamically oscillating forces to the cell membrane (bottom left). Oscillatory accelerations were applied directly to the cell contact surface (bottom right).</p

Vibration induced GJIC is controlled by Akt signaling.

<p>One hour after vibration treatment, (A) Cx43 mRNA levels remained unchanged. (B) Cx43 protein levels were also unaffected by vibrations (0.15 g–100 Hz) without and with Akt inhibition but (C) vibrations increased Akt phosphorylation (ser 473) 2.4-fold. (D) Inhibition of Akt activation also inhibited the vibration induced increase in calcein transference. ***: p<0.001 against control.</p