Viscoelastic time responses of polymeric cell substrates measured continuously from 0.1–5000 Hz in liquid by photothermal AFM nanorheology

The mechanical properties of the polymeric substrate or matrix where a cell grows affect cell behavior. Most studies have focused on relating elastic properties of polymeric substrates, which are time-independent, to cell behaviors. However, polymeric substrates and biological systems exhibit a time-dependent, often viscoelastic, mechanical response. While less is known about how time responses dictate cell behavior, cells are likely sensitive to substrate time responses rather than elasticity alone. However, testing this hypothesis is complex due to the lack of nanoscale tools. To overcome this limitation, photothermal actuation-atomic force microscopy nanoscale dynamic mechanical analysis (PT-AFM nDMA), a novel AFM technique that measures sample viscoelasticity over a broad and continuous frequency range, was applied to measure the viscoelastic properties of cell culture substrates made of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) and collagen I (pureCol) in liquid at frequencies ranging continuously from 0.1 Hz-5000 Hz. PT-AFM nDMA to date has not been performed in liquid, but successfully and accurately characterized substrate viscoelasticity and synergized with measurements obtained using a more established AFM technique, bimodal imaging. The results of this study demonstrate that PT-AFM nDMA can be performed in liquid environments, especially relevant to biological samples. Additionally, comparing PT-AFM nDMA measurements of the poly(HEMA) and pureCol substrates in this study to cell behaviors described in the scientific literature on similar substrates suggests that longer substrate time responses at low measurement frequencies promote cell attachment, proliferation, and migration, while shorter substrate time responses promote ECM remodeling and differentiation.