  The mechanical properties of collagen fibrils depend on the amount and the
distribution of water molecules within the fibrils. Here, we use atomic force
microscopy (AFM) to study the effect of hydration on the viscoelastic
properties of reconstituted type I collagen fibrils in air with controlled
relative humidity. With the same AFM tip, we investigate the same area of a
collagen fibril with two different force spectroscopy methods: force-distance
(FD) and amplitude-phase-distance (APD) measurements. This allows us to
separate the contributions of the fibril's viscoelastic response and the
capillary force to the tip-sample interaction. A water bridge forms between the
tip apex and the surface, causing an attractive capillary force, which is the
main contribution to the energy dissipated from the tip to the specimen in
dynamic AFM. The force hysteresis in the FD measurements and the tip
indentation of only 2 nm in the APD measurements show that the hydrated
collagen fibril is a viscoelastic solid. The mechanical properties of the gap
regions and the overlap regions in the fibril's D-band pattern differ only in
the top 2 nm but not in the fibril's bulk. We attribute this to the reduced
number of intermolecular crosslinks in the reconstituted collagen fibril. The
presented methodology allows the mechanical surface properties of hydrated
collagenous tissues and biomaterials to be studied with unprecedented detail on
the nanometer scale.
