Research paper
Dynamic mechanical analysis of collagen fibrils at the nanoscale

https://doi.org/10.1016/j.jmbbm.2011.08.020Get rights and content

Abstract

Low frequency (0.1–2 Hz) dynamic mechanical analysis on individual type I collagen fibrils has been carried out using atomic force microscopy (AFM). Both the elastic (static) and viscous (dynamic) responses are correlated to the characteristic axial banding, gap and overlap regions. The elastic modulus (∼5 GPa) on the overlap region, where the density of tropocollagen is highest, is 160% that of the gap region. The amount of dissipation on each region is frequency dependent, with the gap region dissipating most energy at the lowest frequencies (0.1 Hz) and crossing over with the overlap region at ∼0.75 Hz. This may reflect an ability of collagen fibrils to absorb energy over a range of frequencies using more than one mechanism, which is suggested as an evolutionary driver for the mechanical role of type I collagen in connective tissues and organs.

Graphical abstract

Highlights

► High resolution force mapping on individual collagen fibrils. ► Discrete difference in the modulus correlated with periodic banding. ► Dynamic indentation testing at physiologically relevant frequencies (0.1 to 2 Hz). ► Amount of dissipation on the gap/overlap regions is frequency dependent.

Introduction

Collagenous based tissues, such as skin, bone, ligaments and tendons, are essential biomechanical components in mammalian locomotion. Tendon tissues exhibit a particular hierarchical structure going from large fascicle bundles at long length scales down through collagen fibres, collagen fibrils to the fundamental collagen monomer level (tropocollagen). The synonymous periodic banding of ∼67 nm that is present in collagen fibrils was originally modelled by Hodge–Petruska, where the collagen monomers are arranged end-on-end, in a quarter staggered arrangement (Petruska and Hodge, 1964). Adjacent monomers are stabilised by covalent lysyl oxidase cross-linking (Kadler et al., 1996).

Ultimately, it is regularly reported that this two dimensional model leads to ‘overlap’ and ‘gap’ regions (Kadler et al., 1996, Lin and Goh, 2002), giving the fibril its corresponding banding appearance in TEM and AFM, where it is suggested that the gap region has 20% less packing density than the overlap region (Minary-Jolandan and Yu, 2009). It is generally assumed that the overlap region would correspond to a topographic peak in the fibril, whereas the gap region is associated with the trough region. If the gap region was directly correlated to the trough of the fibril and the overlap region to the peak of the fibril, it is reasonable to assume that the gap (trough) and overlap (peak) regions have different mechanical properties associated with the packing densities of the tropocollagen monomers. It has been reported that an intermediate hierarchy exists between collagen fibrils and tropocollagen where the collagen monomers (∼1.5 nm diameter) from bovine cornea are organised into 4 nm diameter ‘microfibrils’ that are tilted by 15° (Holmes et al., 2001).

Several works have carried out quasi-static nano-indentation on a range of collagen fibrils from bovine tendon (Grant et al., 2008), rat tail (Wenger et al., 2007) and sea cucumber (Heim et al., 2006). The mechanical properties of reconstituted collagen fibrils have been shown to be mechanically sensitive to their hydrated state (van der Rijt et al., 2006, Grant et al., 2008, Yang et al., 2008) and can be manipulated by their aqueous environment (Grant et al., 2009). Although, mature human collagen fibrils and fascicles, with a greater proportion of cross-links, are reported to be insensitive to changes to the salinity and pH of the testing media (Svensson et al., 2010a). Nano-dissection of collagen fibrils using a sharp AFM probe has revealed a sub-structural periodic banding (Wen and Goh, 2004, Wenger et al., 2008). This technique was used to show that the collagen fibril ‘shell’ and ‘core’ of rat tail tendon, as revealed upon scraping, had no differences in the mechanical (stiffness, modulus, hardness) or adhesive properties (Wenger et al., 2008). However, in contrast, collagen fibrils derived from calf skin had exhibited differences in the measured adhesive force between its ‘shell’ and ‘core’ regions (Strasser et al., 2007).

Individual collagen fibrils have also been mechanically tested via AFM tensile testing (van der Rijt et al., 2006, Svensson et al., 2010b), three point bending (Yang et al., 2008) and also shown to undergo stress relaxation via testing with a MEMS device (Shen et al., 2011). Despite this, the mechanical properties of collagen at the sub-fibrillar level are not fully understood, although, it has been reported that the sub-structure of tendon collagen fibrils form a rope-like structure (Bozec et al., 2007). In depth computational modelling work (Buehler, 2008) and X-ray diffraction (Gupta et al., 2006, Gupta et al., 2010) have been carried out to link the mechanical properties of collagen at the various hierarchical levels (molecules, fibrils fibres).

AFM has the capability to separate the localised mechanical responses, at the nano-scale, of an ocular tissue to its two-component parts (fibrils and extra-cellular matrix) (Grant et al., 2011). A dynamic indentation technique has been carried out on articular cartilage using a range of probe geometries (Han et al., 2011). However, this study samples the elastic (static) and visco-elastic (dynamic) properties of the periodic banding of individual collagen fibrils from rat tail tendon using AFM.

Section snippets

Sample preparation

Collagen fibrils from rat tail tendons are carefully dissociated using tweezers before transferring to a cleaned silicon wafer glued using a 2 part epoxy adhesive (Araldite, Aero Research Limited, UK) to a glass microscope slide. After rinsing with ultra-pure water and removing any excess liquid using a gentle stream of nitrogen, the collagen fibrils were then allowed to dry in air in a covered petri dish (Revenko et al., 1994) and used within 4 h.

AFM nano-mechanics

Force volume (FV) imaging (60×60 pixels) on

Results and discussion

Fig. 1(a) shows an AFM height scan (700 nm×700 nm) of a collagen fibril, having a peak to trough height difference of 4.1 ± 0.3 nm, an axial banding period of 67.4 ± 1.8 nm. Fig. 1(b) shows a corresponding high resolution force map on the collagen fibril shown in Fig. 1(a). The force map clearly shows that for identical loading for all pixels, the periodic banding has a variation in the recorded indentation depth. The modulus for each of the bands was calculated by separating the force curves

Conclusions

Periodic banding nano-mechanics on collagen fibrils show distinctively different elastic and visco-elastic properties. These properties can be rationalised using the common model of a 2D staggered array of tropocollagen molecules to describe fibril periodic banding. High resolution static nanoindentation reveals that the periodic banding has discrete mechanical properties, with the peaks having a larger modulus in comparison with the troughs of a collagen fibril. Furthermore, dynamic

Acknowledgement

We gratefully acknowledge funding from the Biotechnology and Biological Sciences Research Council (BB/D011191/01).

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