Understanding the effects of mechanical stretching on all levels of collagen structure is of high importance in understanding the mechanical properties and biological function of collagen. Previously atomic force microscopy (AFM) measurements have been used extensively for investigating the effect of mechanical stretching on individual collagen fibrils. These measurements reveal several important details such as lengthening of the D-Band and stiffening of the fibril due to extension, however AFM provides little detail regarding the effect of stretching on the structure of collagen molecules within the fibril. Here we utilize polarization resolved second harmonic generation microscopy (PSHG) in conjunction with AFM measurements to investigate the effects of stretching on collagen molecular structure. PSHG is an optical microscopy technique with high sensitivity to the molecular structure of helical proteins and can be used to determine the average pitch angle of the collagen triple helix. We demonstrate that there is a continuous unwinding of the collagen triple helix with increasing strain, and that the molecular extension is on the order of double the D-band extension. We additionally show significant changes in collagen molecular density as a result of stretching with density peaking at ~3% D-band strain and then dropping off linearly. A comparison of results obtained for fibrils isolated from functionally distinct tendons will also be presented.

Collagens are important structural proteins in the human body playing a key role in specifying mechanical properties of many tissues including tendons, bones and airways. There, collage is mostly found in the form of fibrils with typical diameters around 100 nm and lengths up to several mm. In addition to macro mechanical competence of tissues, collagen fibrils are important for the extracellular matrix (ECM) acting as cell attachment and mechanotransduction. Collagen fibrils exhibit time-dependent material properties and are thought to behave viscoelastic in physiological loading regimes. While experimental techniques for biomechanical characterization of tissues at the macroscale are more or less well established, experiments and data on individual collagen fibrils is scarce. We present experimental data from force-controlled experiments on individual fibrils using dynamic mechanical analysis (nano-DMA) as well as creep tests. Nano-DMA on collagen fibrils from 14 week old wild type mouse tail-tendon in the phase I mechanical regime show loss tangents of up to 0.2 and storage moduli of up to 5 GPa (at 2 µN average force). In addition, loss tangents decreased from the lower (0.1 Hz) to the higher (1 Hz( frequency applied. Creep tests were conducted on similar samples, with half of them cross-linked by incubation with methylglyoxal (MGO). Creep test data from cross-linked and native fibrils were fitted a Burgers material model in Kelvin-Voigt configuration (strain response of fibrils under constant force). Both creep rate of collagen fibrils and residual strain after unloading was reduced by MGO cross-linking. In addition, cross-linked fibrils showed an almost 2-fold increase in tensile modulus. In contrast, cross-linking did not affect transient viscoelastic behavior of collagen fibrils tested. The observed behavior can be explained by cross-linking influencing deformation mechanisms (straightening, uncoiling, sliding) already well below 10% applied strain.

Collagen type I is the most abundant protein in mammals. It is constituted by molecules known as tropocollagens. Tropocollagen molecules are the building blocks to form collagen nanoribbons and microfibrils. Those structures have a periodic structure known as the D-band [1]. This contribution aims to image in real-time the aggregation of single tropocollagens into collagen microfibrils [2]. High-speed AFM (HS-AFM) was applied to imaging the collagen self-assembly with a spatial resolution of 10 nm and time resolution of 0.3 s. The formation of the microfibril is driven by electrostatic forces between charged aminoacid residues therefore a change in pH leads to the binding and unbinding of the microfibril [3]. By tuning the pH of the buffer solution, it was possible to image either the self-assembly of tropocollagens (pH > 7) or the disassembly of the collagen nanoribbons and microfibrils (pH < 7).

Single collagen fibrils were stretched along their entire length by depositing them on a highly stretchable foil of polydimethylsiloxane (PDMS). Kelvin-probe Force Microscopy (KPFM) was then performed on strained fibrils to probe their electromechanical response. Native fibrils and fibrils exposed to glutaraldehyde, which is a typical protein cross-linking agent for cell cultures, were compared. The results show that their surface potential increases towards more positive values for up to 10% strain and then decreases again at even 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 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.