or active stimulation of the cell through the imposition of stress and strain in various forms. [12] Besides being critical to homeostasis and the primary function of the cell, [13,14] mechanotransduction processes have also been implicated in cellular differentiation, [8,11,[15][16][17][18][19] and are therefore of considerable interest given their therapeutic potential for tissue engineering and regenerative medicine. [20] For instance, there have been a myriad of studies involving externally-imposed dynamic mechanical stimuli through the application of laminar shear or cyclic stretching/pressure on cells particularly for osteogenic differentiation, although these have primarily been conducted at low frequencies-usually several Hz (see, for example, refs. [16] and [21]), which are characteristic of the physiological frequencies associated with the motion typically experienced by cells in their local environment, for example, in the human body (e.g., walking and running). Higher frequency (1-kHz-order) nanoscale amplitude vibrations generated using a bulk piezoelectric actuator have more recently been employed to trigger osteogenic differentiation in mesenchymal stem cells (MSCs), [22][23][24][25] wherein it was suggested that these frequencies and amplitudes are respectively commensurate with various cellular time and length scales (particularly those associated with cell membrane undulations), [26,27] and potentially related to that at which collagen becomes piezoelectric in hydrated bone. [28] These previous studies nevertheless reported little upregulation in osteogenic markers beyond 1 kHz, asserting this frequency to be optimal for inducing osteogenesis. [23] In the same way that MHz-order frequencies have led to new avenues in the manipulation of materials and fluids beyond conventional ultrasonic and sonochemical (typically kHz-order) processes, [29] we show here that it is not only possible to induce similar genotypic and phenotypic alterations in human MSCs (hMSCs) at frequencies that are three orders of magnitude higher (i.e., 10 MHz), [30] but also to induce persistent long-term osteogenic lineage commitment considerably earlier.It is important to note that the high MHz-order frequency vibrational excitation in the form of surface reflected bulk waves Stem cell fate can be directed through the application of various external physical stimuli, enabling a controlled approach to targeted differentiation. Studies involving the use of dynamic mechanical cues driven by vibrational excitation to date have, however, been limited to low frequency (Hz to kHz) forcing over extended durations (typically continuous treatment for >7 days). Contrary to previous assertions that there is little benefit in applying frequencies beyond 1 kHz, we show here that high frequency MHz-order mechanostimulation in the form of nanoscale amplitude surface reflected bulk waves are capable of triggering differentiation of human mesenchymal stem cells from various donor sources toward an osteoblast lineage, with early, short time st...
