Characterization of Mechanical and Cellular Effects of Rhythmic Vertical Vibrations on Adherent Cell Cultures

Abstract

This paper presents an innovative experimental setup that employs the principles of audio technology to subject adherent cells to rhythmic vertical vibrations. We employ a novel approach that combines three-axis acceleration measurements and particle tracking velocimetry to evaluate the setup's performance. This allows us to estimate crucial parameters such as root mean square acceleration, fluid flow patterns, and shear stress generated within the cell culture wells when subjected to various vibration types. The experimental conditions consisted of four vibrational modes: No Vibration, Continuous Vibration, Regular Pulse, and Variable Pulse. To evaluate the effects on cells, we utilized fluorescence microscopy and a customized feature extraction algorithm to analyze the F-actin filament structures. Our findings indicate a consistent trend across all vibrated cell cultures, revealing a reduction in size and altered orientation (2D angle) of the filaments. Furthermore, we observed cell accumulations in the G1 cell cycle phase in cells treated with Continuous Vibration and Regular Pulse. Our results demonstrate a negative correlation between the magnitude of mechanical stimuli and the size of F-actin filaments, as well as a positive correlation with the accumulations of cells in the G1 phase of the cell cycle. By unraveling these analyses, this study paves the way for future investigations and provides a compelling framework for comprehending the intricate cellular responses to rhythmic mechanical stimulation.

Keywords: F-actin filament angle; F-actin filament length; F-actin filament thickness; Max/MSP signal generation; acceleration; image feature extraction; mechanobiology; particle tracking velocimetry (PTV); rhythmic vertical vibration; shear stress; sound vibrations.

Figure 1

A schematic diagram of the vertical vibration setup and its components. (a) Side view of the thin 8-well glass-bottom slide. (b) Exploded view of the PMMA baseplate. The black paper is included for better visualization, and the paper is not expected to impact the vibration propagation. (c) The vertical vibration experimental setup, including a PC, amplifier, and vibration generator.

Figure 2

A section from the Max/MSP patch illustrating how the numerical sequences were generated to manage the digital audio signals regularly or irregularly within the Max/MSP environment. The red arrowheads were added to indicate the message or signal flow. The metronome object and a fixed integer argument ( i.e., 1000) are used to vary the signals regularly for the Regular Pulse (RP) condition. The random object that generates a sequence of pseudorandom numbers from 0 and 1 less than a set argument is used as input arguments into the metronome object to vary the signals irregularly for the Variable Pulse (VP) condition.

Figure 3

(a) Visual representations of a pseudorandom number sequence used for the Variable Pulse durations and intervals. (b) A density histogram analysis shows the distribution of the durations and intervals. The two colored lines, the kernel density estimations, illustrate an approximately uniform distribution of the series of numbers within the set range between 1000 and 1500.

Figure 4

The autocorrelation analysis of a pseudorandom number sequence used for the varied (a) pulse durations and (b) pulse intervals. The maximum lag is 360 in this data set. Any data point falling within the blue shaded area ($\alpha$ = 0.05) indicates that it is not significantly correlated with the previous point. The analysis illustrates that the coefficient values of the autocorrelation are close to 0, and the consecutive numbers are mostly not significantly related in the sequence. Therefore, the sequence is most likely produced randomly.

Figure 5

Particle Tracking Velocimetry (PTV) setup with a VWR glass cuvette (10 × 10 mm, the same dimensions as a well of the ibidi glass bottom 8-well slide) secured on the 3D-printed slit holder on the vertical vibration generator. The slit holder and glass cuvette were held down firmly on the baseplate with screws. The laser sheet in the cuvette was created using a CO${}_2$ laser and the slit, which was 0.1 mm in width and 10 mm in height. The glass cuvette was filled with DMEM cell media (350 µL) and microparticles (Ø = 10 µm). A Grasshopper3 USB3 CCD camera was used to capture images. The images were taken using a stroboscopic technique by keeping the framerate of the camera at 25 FPS while the vibration generator produced a 50 Hz sine tone.

Figure 6

Schematic diagram of the image feature extraction algorithm to analyze F-actin filament structures (filament angle, length, and thickness) using fluorescence microscopic images. The two shaded areas in image 4 indicate the zoomed-in positions shown in images 5 and 6. (1) Acquired fluorescence microscopic image used as input; (2)–(6) images filtered through specific steps, and extracted features (filament angle, length, and thickness) were exported for further data analysis.

Figure 7

A comparison of the acceleration measurement of the vertical vibration setup between X, Y, and Z dimensions shows acceleration only in the Z (vertical) direction.

Figure 8

A comparison of measured acceleration between the experimental conditions. Note the continuous (uninterrupted) acceleration measured under Continuous Vibration (CV), rigidly the same pulse duration and interval under Regular Pulse (RP), and varying pulse duration and intervals under Variable Pulse (VP).

Figure 9

Fast Fourier Transform (FFT) analysis of the acceleration measurement of each experimental condition. (Top row) The full range of the FFT analysis shows a few minor peaks around the odd harmonics above the fundamental frequency. (Bottom row) The same FFT analysis zoomed in around 50 Hz to show the fundamental frequency (∼51 Hz) in the measured acceleration.

Figure 10

Visual representation of the parameters between the different vibration conditions. (a) A comparison of the total vibration and silence times between the conditions. (b) A comparison of the highest acceleration peak and RMS acceleration. (c) The crest factors (the ratio between the peak and RMS) show how “peaky” the signal is. The higher the value, the more the peakiness. CV scores show a lesser crest factor than the other two conditions, meaning the acceleration was more stable, and there was less variation between the acceleration peaks and the RMS throughout the sampled period. This corresponds to the RMS acceleration value, where CV scored the highest RMS value. (d) Comparison of the RMS velocity and (e) RMS displacement between different conditions.

Figure 11

Distribution of the acceleration of the vertical vibration under each condition. Under RP and VP, the distribution is dominated by the lowest category (0-1) in (Top row) and around 0 g in (Bottom row), which represent the intervals (silences) between pulses. (Top row) Sub-divisions of absolute values of the measured acceleration. (Bottom row) Histograms of the measured acceleration.

Figure 12

PTV results. The maximum intensity projection images, velocity fields, and shear stress plot at the boundary layer close to the bottom surface of the cuvette are shown according to the experimental conditions (a) CV, (b) RP, and (c) VP. (d) Distribution of estimated shear stress in absolute values, which shows a smoothed out distribution and where the values are concentrated.

Figure 13

Motion analysis of the particles from the images obtained using the PTV setup (Figure 5). The motiongrams (in black background) and the quantity of motion (QoM) plotted over time (1000 frames) reveal the contrasting degrees of regularity in the motion of the particles between CV, RP, and VP conditions.

Figure 14

Analysis of the extracted features from the microscopic images. The results are statistically compared between the experimental conditions (NV, CV, RP, and VP). Red stars indicate the test that compared one of the vibration conditions to NV (control), gray stars indicate the test that compared the in-between vibration conditions, and ns indicates not significant. (a) The boxplot analysis (top) shows the distribution of the data. The box represents 50% of the data points, the green dotted line in the box represents the mean, and the orange line represents the median. The bar plots (bottom) show the differences in median filament lengths, thicknesses, and mean filament angles. (b) Filament angle distribution is presented with a density histogram and polar plots. Note that the angle does not show directions except for the 2D orientation within the collected microscopic images. The red dotted line in the polar plots indicates the mean value. * p < 0.05, *** p < 0.001.

Figure 15

Cell cycle distribution in control (NV) and vibrated (CV, RP, VP) HeLa cells analyzed 24 h after vibration treatment. Red stars indicate a significant difference relative to the control (NV) cells. Flow cytometry dot plots showing the gating strategy are included in Supplementary Figure S1. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 16

Correlation between the extracted features from the microscopic images (median filament length and thickness and mean filament angle) and characterized mechanical parameters (RMS acceleration and estimated shear stress). r is the Pearson correlation coefficient. Markers: NV—star, CV—circle, RP—square, VP—triangle.