Static and time-dependent mechanical response of organic matrix of bone.

Bone derives its mechanical strength from the complex arrangement of collagen fibrils (type-I primarily) reinforced with hydroxy-apatite (HAp) mineral crystals in extra- and intra-fibrillar compartments. This study demonstrates a novel approach to obtain organic matrix of bone through its demineralization as well as mechanically characterize it at small length scales using static and dynamic indentation techniques. Sample surface preparation protocol used in the present work maintained the surface integrity of demineralized bone samples which resulted sample surface of roughness (RMS) magnitude of approximately 14 nm (averaged over 1 × 1 μm area duly verified by atomic force microscope (AFM)). Elemental composition analysis via energy dispersive X-ray spectroscopy (EDX) (for probed depth upto 2 μm) confirmed the complete removal of HAp mineral from bone samples during their demineralization using EDTA leaving collagen molecule assemblies unaffected as represented by Second Harmonic Generation (SHG) imaging. The modulus magnitudes of organic matrix obtained using from quasistatic as well as dynamic indentations (at constant frequency of 30 Hz) as ∼2.6 GPa and 4.5 GPa respectively, demonstrated the influence of loading rate on the estimated mechanical properties. For indentation depth to surface roughness ratio greater than ∼5:1, interestingly, measured material properties of organic matrix were found to depend on increasing magnitude of indentation depth of up to ∼500 nm value which probed from few collagen fibrils to next level of hierarchy i.e. collagen fibers. These findings are very useful to accurately determine the elastic and visco-elastic response of organic matrices of mineralized tissues for various applications including tissue engineering, bio-mimetics, etc.