Collagen is the most abundant protein in the human or animal body. It is found in ligaments, tendons, skin, cartilage, bones, cornea, sclera (white of the eye), and blood vessels to name but a few. All of these tissues are biomechanically important; therefore, knowing the mechanical properties of collagen at all hierarchical levels or length-scales is of critical importance. Our studies have concentrated on the mechanics of collagen fibrils from bovine Achilles tendon, which is a self-assembly of aligned monomeric tropocollagen molecules.

The mechanical properties of gel-like materials, such as hydrogels, are affected by the addition of reinforcing fibres [1]. The use of collagen fibrils within an agarose matrix for tissue engineering applications has been recently reported [2], showing that the biophysical properties of collagen gels increases with increasing agarose concentration. Therefore, being able to make detailed characterisation and subsequently manipulate the mechanical properties of the reinforcing fibrils has deeper possibilities for fields such as tissue engineering.

The radial compressive modulus of dehydrated collagen fibrils at the nanoscale level has been recorded using atomic force microscopy [3, 4]. Here, the small AFM probe is used to measure the force-indentation response of the collagen fibril. In general, to carry out such AFM based nanoindentation measurements, the cantilevers are calibrated using the standard thermal tuning method [5]. Then the force-indentation curves are fitted to the Hertzian spherical theory (see fig. 4) to obtain the elastic modulus. This method has been successfully employed to measure a wide range of biological samples such as hydrogels [6], cancer cells [7], cartilage [8] and bone [9].

Collagen fibrils contain a high proportion of water, which we observe as at least a two fold increase in the diameter during rehydration experiments [10]. The water content, therefore, plays an important role in the elastic modulus of collagen fibrils [11, 12]. For example, the compressive modulus measured using nanoindentation AFM increases by three orders of magnitude when the fibril is dehydrated. Understanding elastic response of collagen fibrils under quasi-static loading conditions in different aqueous environments, and comparing this with dehydrated conditions, is a basis for probing collagenous biomaterials at all length and time-scales.

The insets of Figure 1a and b shows an AFM scan of an individual collagen fibril in neutral buffer and its corresponding 50x50 pixel force volume map (FV).

Each pixel of the FV is a single force plot at that location with the scale bar showing the indentation depth in the sample for that given force plot. Each force plot in the FV map is taken at constant load and tip velocity. To interpret the FV indentation map, the darkest regions correspond to zero indentation (i.e. on the hard silica substrate), whereas the lighter regions show a measureable surface indentation was made in the fibril. Force plots from the centre of each fibril are extracted and fitted to Eqn shown in fig. 4. 1 to avoid geometrical effects at the edge of the fibril.

The fragility and fracture behaviour of de-hydrated collagen fibrils is shown in Figure 2. Figure 2a shows a typical fibril with the expected D-banding periodicity of 67nm commonly observed by AFM and EM. The level of load at which plastic behaviour is viewed following indentations is much higher in air than in liquid environments, where we have not observed plasticity even at 40% strain [11]. The load at regular positions along the fibril has been increased in series as; 1, 2, 3, 4, 5 and 10µN in a single nanoindentation measurement. Figure 2b shows how the plastic deformation increases as load increases and the asymmetric shape of the indent highlights the anisotropic mechanical response of the fibril due to the aligned tropocollagen molecules. Deformation was only detected when indenting over 1.5µN, however, above this critical load one can manipulate the collagen fibril structure to give an array of indents across the fibril. In fact, if you indent at high enough load during the FV, the residual plastically deformed array is shown in figure 2c in comparison with the non-deformed periodic banding.

Experimentation on the mechanical properties of collagen fibrils has been carried out with varying media conditions to show that the biomechanical properties of these reconstituted fibrils can be tuned [10]. Figure 3a shows that the modulus under neutral pH conditions approximately doubles after titration of 1M NaCl. However, a much more dramatic increase is seen by changing the media to pH 5 and titrating in up to 1M KCl, which increases the modulus 10-fold. Figure 3b shows that even greater effects on the modulus can be achieved when using organic solvents, such as increasing concentration of ethanol/water solutions. At 100% ethanol, the modulus has now increased 100-fold in comparison with that found in the neutral buffer (no salt). The inset of Figure 3b shows a fibril in 100% ethanol, where periodic banding can still be resolved, however, height measurements indicate that the ethanol is dehydrating the fibrils. The temperature of the scanning media is also known to affect the modulus of biological samples. We found that the modulus of a single collagen fibril decreases approximately 15% when increasing the temperature from room temperature to 37°C. This change of modulus is accompanied with a slight (~3.5%) enlargement of the fibril diameter (data not shown).