Colin A. Grant

Tuning the Elastic Modulus of Hydrated Collagen Fibrils

Systematic variation of solution conditions reveals that the elastic modulus (E) of individual collagen fibrils can be varied over a range of 2-200 MPa. Nanoindentation of reconstituted bovine Achilles tendon fibrils by atomic force microscopy (AFM) under different aqueous and ethanol environments was carried out. Titration of monovalent salts up to a concentration of 1 M at pH 7 causes E to increase from 2 to 5 MPa. This stiffening effect is more pronounced at lower pH where, at pH 5, e.g., there is an approximately 7-fold increase in modulus on addition of 1 M KCl. An even larger increase in modulus, up to approximately 200 MPa, can be achieved by using increasing concentrations of ethanol. Taken together, these results indicate that there are a number of intermolecular forces between tropocollagen monomers that govern the elastic response. These include hydration forces and hydrogen bonding, ion pairs, and possibly the hydrophobic effect. Tuning of the relative strengths of these forces allows rational tuning of the elastic modulus of the fibrils.

Effects of hydration on the mechanical response of individual collagen fibrils

Collagen fibrils prepared from bovine Achilles tendon have been mechanically tested through nanoindentation by an atomic force microscope using force volume analysis. In ambient conditions where the fibrils are expected to be dehydrated, the elastic modulus was determined to be 1.9±0.5GPa, while under aqueous fluid, it decreased by three orders of magnitude to 1.2±0.1MPa. In air, fibril fracture occurred along the axis and the crack lengths were quantized to the D-banding periodicity. The apparent hardness of the fibrils was estimated to be in the range of 0.35–0.6GPa.

Poly(vinyl alcohol) Hydrogel as a Biocompatible Viscoelastic Mimetic for Articular Cartilage

The prevalence of suboptimal outcome for surgical interventions in the treatment of full‐thickness articular cartilage damage suggests that there is scope for a materials‐based strategy to deliver a more durable repair. Given that the superficial layer of articular cartilage creates and sustains the tribological function of synovial joints, it is logical that candidate materials should have surface viscoelastic properties that mimic native articular cartilage. The present paper describes force spectroscopy analysis by nano‐indentation to measure the elastic modulus of the surface of a novel poly(vinyl alcohol) hydrogel with therapeutic potential as a joint implant. More than 1 order of magnitude decrease in the elastic modulus was detected after adsorption of a hyaluronic acid layer onto the hydrogel, bringing it very close to previously reported values for articular cartilage. Covalent derivatization of the hydrogel surface with fibronectin facilitated the adhesion and growth of cultured rat tibial condyle chondrocytes as evidenced morphologically and by the observance of metachromatic staining with toluidine blue dye. The present results indicate that hydrogel materials with potential therapeutic benefit for injured and diseased joints can be engineered with surfaces with biomechanical properties similar to those of native tissue and are accepted as such by their constituent cell type.

Tensile properties of human collagen fibrils and fascicles are insensitive to environmental salts.

To carry out realistic in vitro mechanical testing on anatomical tissue, a choice has to be made regarding the buffering environment. Therefore, it is important to understand how the environment may influence the measurement to ensure the highest level of accuracy. The most physiologically relevant loading direction of tendon is along its longitudinal axis. Thus, in this study, we focus on the tensile mechanical properties of two hierarchical levels from human patellar tendon, namely: individual collagen fibrils and fascicles. Investigations on collagen fibrils and fascicles were made at pH 7.4 in solutions of phosphate-buffered saline at three different concentrations as well as two HEPES buffered solutions containing NaCl or NaCl + CaCl2. An atomic force microscope technique was used for tensile testing of individual collagen fibrils. Only a slight increase in relative energy dissipation was observed at the highest phosphate-buffered saline concentration for both the fibrils and fascicles, indicating a stabilizing effect of ionic screening, but changes were much less than reported for radial compression. Due to the small magnitude of the effects, the tensile mechanical properties of collagen fibrils and fascicles from the patellar tendon of mature humans are essentially insensitive to environmental salt concentration and composition at physiological pH.

Dynamic mechanical analysis of collagen fibrils at the nanoscale.

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.

Static and dynamic nanomechanical properties of human skin tissue using atomic force microscopy: Effect of scarring in the upper dermis

Following traumatic injury, skin has the capacity to repair itself through a complex cascade of biochemical change. The dermis, which contains a load-bearing collagenous network structure, is remodelled over a long period of time, affecting its mechanical behaviour. This study examines the nanomechanical and viscoelastic properties of the upper dermis from human skin that includes both healthy intact and scarred tissue. Extensive nanoindentation analysis shows that the dermal scar tissue exhibits stiffer behaviour than the healthy intact skin. The scar skin also shows weaker viscoelastic creep and capability to dissipate energy at physiologically relevant frequencies than the adjacent intact skin. These results are discussed in conjunction with a visual change in the orientation of collagenous fibrils in the scarred dermis compared with normal dermis, as shown by atomic force microscopy imaging.

Multi-scale mechanical characterization of highly swollen photo-activated collagen hydrogels

Biological hydrogels have been increasingly sought after as wound dressings or scaffolds for regenerative medicine, owing to their inherent biofunctionality in biological environments. Especially in moist wound healing, the ideal material should absorb large amounts of wound exudate while remaining mechanically competent in situ. Despite their large hydration, however, current biological hydrogels still leave much to be desired in terms of mechanical properties in physiological conditions. To address this challenge, a multi-scale approach is presented for the synthetic design of cyto-compatible collagen hydrogels with tunable mechanical properties (from the nano- up to the macro-scale), uniquely high swelling ratios and retained (more than 70%) triple helical features. Type I collagen was covalently functionalized with three different monomers, i.e. 4-vinylbenzyl chloride, glycidyl methacrylate and methacrylic anhydride, respectively. Backbone rigidity, hydrogen-bonding capability and degree of functionalization (F: 16 ± 12–91 ± 7 mol%) of introduced moieties governed the structure–property relationships in resulting collagen networks, so that the swelling ratio (SR: 707 ± 51–1996 ± 182 wt%), bulk compressive modulus (Ec: 30 ± 7–168 ± 40 kPa) and atomic force microscopy elastic modulus (EAFM: 16 ± 2–387 ± 66 kPa) were readily adjusted. Because of their remarkably high swelling and mechanical properties, these tunable collagen hydrogels may be further exploited for the design of advanced dressings for chronic wound care.

Temperature dependent stiffness and visco-elastic behaviour of lipid coated microbubbles using atomic force microscopy

The compression stiffness of a phospholipid microbubble was determined using force-spectroscopy as a function of temperature. The stiffness was found to decrease by approximately a factor of three from ∼0.08 N m−1, at 10 °C, down to ∼0.03 N m−1 at 37 °C. This temperature dependence indicates that the surface tension of lipid coating is the dominant contribution to the microbubble stiffness. The time-dependent material properties, e.g. creep, increased non-linearly with temperature, showing a factor of two increase in creep-displacement, from ∼24 nm, at 10 °C, to 50 nm, at 37 °C. The standard linear solid model was used to extract the visco-elastic parameters and their determination at different temperatures allowed the first determination of the activation energy for creep, for a microbubble, to be determined.

Force spectroscopy of streptavidin conjugated lipid coated microbubbles

AbstractForce microscopy has been used to investigate the mechanical properties of phospholipid coated microbubbles and to quantify their stiffness. The mechanical properties were investigated using tipless cantilevers to compress microbubbles attached to a gold surface under aqueous conditions. The phospholipid microbubbles were produced by microfluidic flow focusing and were found to have stiffness of 25 mN m–1. The attachment of a streptavidin coating increased the microbubble stiffness by a factor of 30 to ∼750 mN m–1. Further, estimation of the frequency response based on values of stiffness obtained by force spectroscopy seems reasonable in comparison with those of an uncoated bubble and a polyethylene glycol coated Bracco SonoVue BR14 bubble, suggesting that the present approach may provide useful information for the development of novel microbubble coatings.

Nano-scale temperature dependent visco-elastic properties of polyethylene terephthalate (PET) using atomic force microscope (AFM).

Visco-elastic behaviour at the nano-level of a commonly used polymer (PET) is characterised using atomic force microscopy (AFM) at a range of temperatures. The modulus, indentation creep and relaxation time of the PET film (thickness=100 μm) is highly sensitive to temperature over an experimental temperature range of 22-175°C. The analysis showed a 40-fold increase in the amount of indentation creep on raising the temperature from 22°C to 100°C, with the most rapid rise occurring above the glass-to-rubber transition temperature (T(g)=77.1°C). At higher temperatures, close to the crystallisation temperature (T(c)=134.7°C), the indentation creep reduced to levels similar to those at temperatures below T(g). The calculated relaxation time showed a similar temperature dependence, rising from 0.6s below T(g) to 1.2s between T(g) and T(c) and falling back to 0.6s above T(c). Whereas, the recorded modulus of the thick polymer film decreases above T(g), subsequently increasing near T(c). These visco-elastic parameters are obtained via mechanical modelling of the creep curves and are correlated to the thermal phase changes that occur in PET, as revealed by differential scanning calorimetry (DSC).