Determination of Collagen Stiffness via AFM

A commercial atomic force microscope (MFP-3D-BIO AFM, Asylum Research) was used for the determination of the elastic moduli (i.e., stiffness) as previously described (Staunton et al., 2016 (link); Wu et al., 2018 (link)). Briefly, spheroconical probes (LRCH, Team Nanotech) with nominal spring constants of 0.2 N ⋅ m⁻¹, a half cone angle of 18.8°, tip length of >10 µm, and a spherical radius of 700 nm were used to collect force–indentation curves in four to seven 4 × 5 grids of 90 × 90-µm areas at 37°C in PBS buffer with an indenter vertical speed of 2 µm ⋅ s⁻¹. Trigger forces of 20–30 nN resulted in indentation depths ≥10 µm. The elastic moduli were obtained by fitting the initial 10 µm of indentation of each force–indentation curve to a nonadhesive elastic contact model for spheroconical probes (Staunton et al., 2016 (link)). Collagen was assumed to be incompressible, with a Poisson ratio of 0.5 (Lacroix et al., 2018 ). The spring constants of the cantilevers were determined by the thermal noise method before the experiment (Butt and Manfred, 1995 ).