Calcium-dependent cAMP mediates the mechanoresponsive behaviour of endothelial cells to high-frequency nanomechanostimulation.

The endothelial junction plays a central role in regulating intravascular and interstitial tissue permeability. The ability to manipulate its integrity therefore not only facilitates an improved understanding of its underlying molecular mechanisms but also provides insight into potential therapeutic solutions. Herein, we explore the effects of short-duration nanometer-amplitude MHz-order mechanostimulation on interendothelial junction stability and hence the barrier capacity of endothelial monolayers. Following an initial transient in which the endothelial barrier is permeabilised due to Rho-ROCK-activated actin stress fibre formation and junction disruption typical of a cell's response to insults, we observe, quite uniquely, the integrity of the endothelial barrier to not only spontaneously recover but also to be enhanced considerably-without the need for additional stimuli or intervention. Central to this peculiar biphasic response, which has not been observed with other stimuli to date, is the role of second messenger calcium and cyclic adenosine monophosphate (cAMP) signalling. We show that intracellular Ca2+, modulated by the high frequency excitation, is responsible for activating reorganisation of the actin cytoskeleton in the barrier recovery phase, in which circumferential actin bundles are formed to stabilise the adherens junctions via a cAMP-mediated Epac1-Rap1 pathway. Despite the short-duration stimulation (8 min), the approximate 4-fold enhancement in the transendothelial electrical resistance (TEER) of endothelial cells from different tissue sources, and the corresponding reduction in paracellular permeability, was found to persist over hours. The effect can further be extended through multiple treatments without resulting in hyperpermeabilisation of the barrier, as found with prolonged use of chemical stimuli, through which only 1.1- to 1.2-fold improvement in TEER has been reported. Such an ability to regulate and enhance endothelial barrier capacity is particularly useful in the development of in vitro barrier models that more closely resemble their in vivo counterparts.

Sonomechanobiology: Vibrational stimulation of cells and its therapeutic implications.

All cells possess an innate ability to respond to a range of mechanical stimuli through their complex internal machinery. This comprises various mechanosensory elements that detect these mechanical cues and diverse cytoskeletal structures that transmit the force to different parts of the cell, where they are transcribed into complex transcriptomic and signaling events that determine their response and fate. In contrast to static (or steady) mechanostimuli primarily involving constant-force loading 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 well understood in comparison. We review the mechanotransductive processes associated with such acoustic forcing, typically at ultrasonic frequencies (>20 kHz), and discuss the various applications that arise from the cellular responses that are generated, particularly for regenerative therapeutics, such as exosome biogenesis, stem cell differentiation, and endothelial barrier modulation. Finally, we offer perspectives on the possible existence of a universal mechanism that is 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.

Short-Duration High Frequency MegaHertz-Order Nanomechanostimulation Drives Early and Persistent Osteogenic Differentiation in Mesenchymal Stem Cells.

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 stimuli inducing long-term osteogenic commitment. More specifically, rapid treatments (10 min daily over 5 days) of the high frequency (10 MHz) mechanostimulation are shown to trigger significant upregulation in early osteogenic markers (RUNX2, COL1A1) and sustained increase in late markers (osteocalcin, osteopontin) through a mechanistic pathway involving piezo channel activation and Rho-associated protein kinase signaling. Given the miniaturizability and low cost of the devices, the possibility for upscaling the platform toward practical bioreactors, to address a pressing need for more efficient stem cell differentiation technologies in the pursuit of translatable regenerative medicine strategies, is ensivaged.

High frequency acoustic cell stimulation promotes exosome generation regulated by a calcium-dependent mechanism.

Exosomes are promising disease diagnostic markers and drug delivery vehicles, although their use in practice is limited by insufficient homogeneous quantities that can be produced. We reveal that exposing cells to high frequency acoustic irradiation stimulates their generation without detriment to cell viability by exploiting their innate membrane repair mechanism, wherein the enhanced recruitment of calcium ions from the extracellular milieu into the cells triggers an ESCRT pathway known to orchestrate exosomal production. Given the high post-irradiation cell viabilities (≈95%), we are able to recycle the cells through iterative irradiation and post-excitation incubation steps, which facilitate high throughput production of a homogeneous population of exosomes-a particular challenge for translating exosome therapy into clinical practice. In particular, we show that approximately eight- to ten-fold enrichment in the number of exosomes produced can be achieved with just 7 cycles over 280 mins, equivalent to a yield of around 1.7-2.1-fold/h.

Collagen synthesis promoting pullulan-PEI-ascorbic acid conjugate as an efficient anti-cancer gene delivery vector.

Cationized pullulan (pullulan-PEI; PP) was synthesized and further modified with an anti-oxidant molecule, ascorbic acid (PPAA) at various ratios. The nanoplexes formed at an optimum ratio of 4:1 was within a size of 150nm and had a zeta potential of 9-14mV. The nanoplexes at this ratio was used for further investigations. The cell internalization and transfection efficiency of these nanoplexes were determined in presence of serum. The internalization and transfection efficiency were found to be unaffected by the presence of fetal bovine serum. Another interesting observation was that this polymer was found to have collagen synthesis promoting property. The collagen synthesis effect of these polymers was quantified and observed that PPAA3 promoted the highest. Transfection efficiency was evaluated by assessing the p53 gene expression in C6 rat glioma cells and cell death was quantified to be 96% by flow cytometry, thus establishing the high efficacy of this polymer.

Betaine conjugated cationic pullulan as effective gene carrier.

Polyethyleneimne (PEI) is a very efficient transfecting agent but is toxic due to high charge density. To generate a vector which is efficient and less cytotoxic, PEI was conjugated with pullulan (PPEI). Further conjugation was done on PPEI with zwitter ionic betaine which possess antifouling property. PEI of molecular weight 1.2, 2, and 10 kDa were used in the study. Buffering capacity of pullulan-PEI-betaine (PPB) conjugates was found to be sufficient enough for the polymers to make endosomal escape. The polymers proved to be less cytotoxic and highly hemocompatible than PEI. Nuclear localization of YOYO tagged DNA was observed with the nanoplexes developed using PPEI and PPBs of PEI 10 kDa. Transfection efficiency was evaluated using p53 expressing gene and the live dead assay demonstrated very high transfection efficiency with PPB conjugates of PEI 10 kDa.