Mechanical Stimulation of Adhesion Receptors Using Light-Responsive Nanoparticle Actuators Enhances Myogenesis

Abstract

The application of cyclic strain is known to enhance myoblast differentiation and muscle growth in vitro and in vivo. However, current techniques apply strain to full tissues or cell monolayers, making it difficult to evaluate whether mechanical stimulation at the subcellular or single-cell scales would drive myoblast differentiation. Here, we report the use of optomechanical actuator (OMA) particles, comprised of a ∼0.6 μm responsive hydrogel coating a gold nanorod (100 × 20 nm) core, to mechanically stimulate the integrin receptors in myoblasts. When illuminated with near-infrared (NIR) light, OMA nanoparticles rapidly collapse, exerting mechanical forces to cell receptors bound to immobilized particles. Using a pulsed illumination pattern, we applied cyclic integrin forces to C2C12 myoblasts cultured on a monolayer of OMA particles and then measured the cellular response. We found that 20 min of OMA actuation resulted in cellular elongation in the direction of the stimulus and enhancement of nuclear YAP1 accumulation, an effector of ERK phosphorylation. Cellular response was dependent on direct conjugation of RGD peptides to the OMA particles. Repeated OMA mechanical stimulation for 5 days led to enhanced myogenesis as quantified using cell alignment, fusion, and sarcomeric myosin expression in myotubes. OMA-mediated myogenesis was sensitive to the geometry of stimulation but not to MEK1/2 inhibition. Finally, we found that OMA stimulation in regions proximal to the nucleus resulted in localization of the transcription activator YAP-1 to the nucleus, further suggesting the role of YAP1 in mechanotransduction in C2C12 cells. These findings demonstrate OMAs as a novel tool for studying the role of spatially localized forces in influencing myogenesis.

Programmable Mechanically Active Hydrogel‐Based Materials

“…The pNIPAM layer of OMAs collapse when exposed to 785 nm NIR light (Figure 12b) and can apply mechanical stimulation to cells when the particle surface is conjugated with cell adhesion ligands (Figure 12c). In these studies, mechanical stimulation with these MAMs enhanced differentiation and alignment of muscle progenitor cells [57] (Figure 12d), fibroblast actin polymerization (Figure 12e), and T cell calcium signaling (Figure 12e). [26] In the study of muscle cells in particular, the spatial resolution afforded by light responsivity as opposed to other MAM mechanisms allowed for the study of differentiation mechanisms on a sub-cellular level.…”

“…The diameter of these OMAs collapses from ≈500 to ≈250 nm upon illumination, and can be used to apply forces to external systems, such as living cells and biomolecules (Figure 4b). [26,57] However, metallic nanoparticles are not the only way to achieve IR responsivity in MAMs. Graphene nanostructures have also been employed, and these provide light-to-heat conversion similar to metals.…”

“…Cezar et al applied the biphasic ferrogel, a hydrogel MFRMAM containing a gradient of iron oxide for programmed magnetic responsiveness, for controlled release of the chemotherapeutic drug mitoxantrone, as well as release of cells for therapeutic purposes. [132] Other groups have employed NIR-driven molecule release as a remotely actuated [57] Copyright 2020, American Chemical Society. b,e,f) Reproduced with permission.…”

“…Scale bar = 5 µm. a,c,d) Reproduced with permission [57]. Copyright 2020, American Chemical Society.…”

Systems for Muscle Cell Differentiation: From Bioengineering to Future Food

“…Ramey-Ward et al applied cyclic strain by means of gold nanorods coated with a light-responsive hydrogel coating, on which myoblasts were attached. The stimulation of integrin receptors that mediate cell-ECM interactions was shown to enhance myogenesis [98]. Considering the potential application of scaffolds for skeletal muscle regeneration, the mechanics of fibrous, porous cylindrical scaffolds under the action of external cyclic strains have been modeled [99].…”