High frequency surface reflected bulk wave induced mechanotransduction

The innate ability of cells to detect and respond to mechanical cues in their microenvironment plays a crucial role in the development and fate of the cells. This ability is facilitated by complex internal machinery involving systems to sense the changes in the microenvironment, transmit the signals to other parts of cells and further govern cellular activities. Contrary to simple static mechanostimuli primarily involving constant-force loadings such as compression, tension and shear (or forces applied at very low oscillatory frequencies (≤ 1 Hz) that essentially render their effects quasi-static), dynamic mechanostimuli comprising more complex vibrational forms (e.g., time-dependent, i.e., periodic, forcing) at higher frequencies are less explored. We used acoustic forcing, typically at ultrasonic frequencies (> 20 kHz), particularly 10 MHz, to stimulate mammalian adherent cells to understand the mechanotransduction involved. We reveal that exposing cells to high frequency mechanotimulation in the form of surface reflected bulk wave (SRBW) of MHz frequency can initiate mechanosensing through the cell membrane. Furthermore, we note that SRBW induces changes in cell membrane activating stretch activated channels (SACs). We also identify how mechanical stimuli are transmitted from the cell membrane to the cytoplasm to initiate various signalling cascades. Contrary to the impression that higher frequencies should not significantly affect cells, we demonstrate, for the first time, that exposing a variety of cells to nanometer-amplitude vibrations in the form of surface reflected bulk waves at 10 MHz can govern their transcriptomic behaviour in different ways. Moreover, this high MHz-order mechanostimulation can render cellular responses distinctive to mechanostimulations at lower Hz and kHz frequencies while retaining high cell viability (> 95%) owing to the absence of cavitation- induced heat and stress on the cells. Three studies were conducted to elucidate the mechanotransduction involved in high frequency mechanostimualtions. The first study on mammalian cells investigates connotations of high frequency mechanostimulation-induced increased intracellular calcium. We show that the high frequency mechanostimulation induces a transient change in intracellular calcium flux through membrane aberrations. Such variances initiate the ESCRT-dependent signalling pathway, which, in turn, leads to an eight- to ten-fold enrichment in the number of exosomes that can be produced in just 280 mins, which is equivalent to a yield of approximately 1.7-2.1 fold/hr. We also note that high frequency mechanostimulation induced responses in the cell depend primarily upon extracellular calcium. Meanwhile, short exposure of high frequency MHz-order mechanostimulation in the form of nanoscale amplitude vibrations directs mesenchymal stem cells can induce longterm osteogenic commitment with short stimulations. It is noted that these mechanostimulations, depending on the culture conditions, can initiate different signalling cascades through the activation of SACs like piezo channels. It is also observed that these mechanostimulation preferentially direct stem cells from different tissue sources toward osteogenesis. Similarly, exposing interendothelial junctions in an endothelial monolayer to high frequency mechanostimulation for a short duration generated multiple cellular arrangements depending on the altered cytoskeletal arrangements and adherens junctions. Upon mechanostimulation, responding to altered intracellular calcium, cells exhibited altered cytoskeletal rearrangement and impaired adherens junctions inducing transient endothelial barrier permeabilisation and upon relaxation, the integrity of the endothelial barrier not only recovered but also enhanced considerably, which is characterised by the formation of circumferential actin bundle and mature adherens junctions. Thus we observe this distinct biphasic response, which maintains the enhanced barrier integrity for more than 4 hours. Such an ability to regulate and enhance endothelial barrier capacity is instrumental in developing in vitro barrier models that closely resemble their in vivo counterparts. These studies note that high frequency mechanostimulation induced mechanotransduction involves membrane aberrations that activate SACs like piezo channels altering intracellular calcium. The mechanical cues, thus sensed by cells, are transmitted through intracellular calcium, followed by cytoskeletal rearrangement initiating multiple signalling cascades like ESCRT pathway, Rho-ROCK signalling, Epac1-Rap1 pathway inducing transient and permanent mechanoresponses such as exosome production, stem cell differentiation and endothelial barrier modulation. Finally, we offer perspectives on the possible existence of a universal mechanism common across all forms of acoustically-driven mechanostimuli that underscores the central role of the cell membrane as the key effector, and calcium as the dominant second messenger, in the mechanotransduction process.