Nanofibrils on PDMS: A new approach to investigate the mechanics and electrostatics of collagen

Session

Nanoscale Probing of Physical Properties via AFM & SPM

Authors

Dr Emilie Gachon (1, 2), Dr Patrick Mesquida (1)

Affiliations

1. King's College London
2. CEA Cadarache

Keywords

Atomic Force Microscopy, Kelvin-probe Force Microscopy, collagen, fibrils, surface charge, buckling, elastic modulus

Abstract text

Collagen is a fibrillary protein that provides strength, mechanical stability and shape to connective tissue in vertebrates. It is the basic component of the extracellular matrix and forms a meshwork to which cells need to attach. Type-1-collagen fibrils, the most common ones, have a striated pattern, called D-banding, made up of peaks (overlap regions) and valleys (gap regions) distributed every 67 nm along the length of the fibril. There is evidence that the mechanical properties and the electrical surface charge of fibrils influences cell behaviour in the matrix and, possibly, stem cell differentiation. Understanding these properties is not only important from a fundamental, biophysical or –medical perspective but will also be of great value in the targeted design of collagen-based scaffold materials in tissue engineering. 

In this study, single collagen fibrils were stretched along their entire length by depositing them on a highly stretchable foil of polydimethylsiloxane (PDMS). Kelvin-probe Force Microscopy (KFM) was then performed on strained and unstrained fibrils to probe their piezoelectric response. These measurements were performed on fibrils dissected from adult rodent tail tendon. Measurements were done on fibrils in the native state and on fibrils exposed to glutaraldehyde, which is a typical protein cross-linking agent for cell cultures. The results show that the surface potentials of the gap and overlap, both, increase towards more positive values for up to 10% strain and then decrease again for higher strains. We interpret this phenomenon as breaking of cross-links, which exposes positive charges at the surface of collagen fibrils. This trend correlates with the stiffness of collagen fibrils, where fibrils strain-stiffen for strains up to roughly 15%, and then strain-soften for greater strains. The change in charge described here could affect the interaction of collagen with cell-adhesion proteins and the calcification of fibrils, thereby ultimately affecting collagen-cell interactions and cell behaviour. 

Using the same experimental approach, individual collagen fibrils were deposited on a pre-strained PDMS foil. By releasing the PDMS foil from its initial strain, the attached collagen fibrils spontaneously buckled. AFM imaging was then used to determine the shapes of individual, buckled fibrils. The data obtained then allows calculation of the fibrils’ tensile moduli using the well-known column-buckling theory from mechanical engineering without the need for force measurements. Comparison of our calculated moduli with data obtained by AFM nanoindentation and more sophisticated techniques show that our results are in good agreement. The great advantage of our approach, however, is that it is much easier to use and can be implemented by any lab to quickly determine the mechanical properties of a large number of fibrils without requiring specially built instrumentation.