An Optical Platform to Study Cellular Mechanotransduction in 3D Hydrogels

Introduction: Cells sense and respond to mechanical cues, a phenomenon known as mechanotransduction. Mechanotransduction plays a crucial role in cellular behavior and tissue development, and is implicated in a variety of diseases, from cancer to traumatic brain injury. However, understanding how cells respond to mechanical cues in three-dimensional (3D) environments remains a significant challenge, largely due to the lack of appropriate methodologies to impart temporally-controlled mechanical forces in a system that mimics the complex in vivo environment. Here, we present an optical platform that utilizes laser-generated microcavitation bubbles to probe cellular mechanosensitivity of intracellular calcium signaling in primary dermal fibroblasts (NHDFs) embedded in Collagen-1 and polyvinyl alcohol (SLOPVA) hydrogels. This technique imparts noninvasive, localized, and tunable mechanical stimuli to cells in 3D environments, allowing for the study of mechanotransduction with sub-second temporal resolution and micrometer spatial resolution. In this study, we demonstrate the use of our platform to initiate cellular mechanotransduction in NHDFs and analyze the effect of matrix ultrastructure and cell orientation on cell mechanosensitivity. This technique enables the study of cellular mechanotransduction in 3D model systems to facilitate investigations of disease pathophysiology as well as high throughput screening of cellular mechanotransduction in variable biomaterial scaffolds and in the presence of candidate therapeutic biomolecules.

Materials and

Methods: Microcavitation bubbles were generated using a frequency-doubled Nd:YAG laser (EKSPLA PL299) with a wavelength of 532 nm and pulse duration of 24 ps. The laser pulses were directed through a 10X magnification microscope objective (Olympus XAPO, 0.4 NA) into a sample within a commercial scanning confocal microscope platform (Olympus FV3000). The NHDFs were transfected with calcium indicator GCaMP3 lentivirus, providing a fluorescent readout of intracellular calcium concentration. We define cell mechanosensitivity as the proportion of cells within a defined region that reached a threshold intensity of 110% within 7.5 seconds following bubble generation, as determined by our MATLAB image processing algorithm. Using confocal fluorescence microscopy, we compared NHDF mechanosensitivity at varying radial distances from the center of the microcavitation bubble. The effect of hydrogel ultrastructure and cell orientation on NHDF mechanosensitivity was investigated by performing experiments on two material systems with equivalent elastic moduli but different microarchitecture: Collagen-1 (fibrillar) and SLOPVA (amorphous).

Results, Conclusions, and Discussions: We found that NHDFs exposed to a single 250 micron diameter cavitation bubble embedded in Collagen-1 hydrogels exhibit a greater spatial extent of intracellular calcium signaling than those in SLOPVA hydrogels. We hypothesize that this may be due to force transmission through the fiber network of Collagen-1. Interestingly, we observe an increased cellular mechanosensitivity in cells radially oriented to the bubble compared to cells circumferentially oriented to the bubble. In contrast, NHDFs embedded in SLOPVA did not display orientation-dependent mechanosensitivity. We hypothesize that this finding may be due to the fact that radially oriented cells are associated with stress fibers aligned along the radial axis of the bubble, resulting in compressive stresses and strains as opposed to the tensile stresses and strains experienced by circumferentially oriented cells. Nevertheless, this finding highlights our platform’s capability to distinguish between compressive and tensile forces in a 3D matrix.

In conclusion, we have demonstrated a novel optical method that uses laser-generated microcavitation bubbles to provide impulsive mechanical stimulation to cells cultured in 3D tissue constructs. Our results represent a new application of laser-generated microcavitation bubbles distinct from previous studies describing bubble-mediated targeted cell lysis, ablation, and drug delivery. The optical basis of this method lends itself to integration with biological microscopy and enables both immediate and long-term visualization of cellular responses to impulsive stimulation. Furthermore, our method potentially enables a high-throughput approach for the screening of cell-biomaterial interactions in response to mechanical stimuli. Moreover, the sensitivity of microcavitation bubble dynamics to mechanical properties provides a unique means to noninvasively stimulate cells mechanically, measure local mechanical properties, and visualize resulting cellular responses all within the same platform. The ease of integration of a pulsed laser microbeam into a standard optical microscopy platform supports the broad adoption of this approach for researchers to study cellular mechanotransduction, and the further application of this technique in various disease models may be of significant benefit to the understanding of the role of mechanotransduction in disease pathophysiology.

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