Invited:
STUDYING THE STRUCTURE AND FORMATION OF BONE AND BONE-LIKE MATERIALS USING ADVANCED MICROSCOPY AND SPECTROSCOPY

A thorough understanding of the structure and growth dynamics of mineralised tissue is key for the development of treatments of widespread bone diseases such as osteoporosis or arthritis. Bone is a hierarchical bio-composite essentially constituted by collagen and the calcium phosphate mineral hydroxyapatite (HAp) providing both toughness and strength to the endoskeleton in vertebrates. To shed light on the nano-level organisation of human bone we have performed electron tomography using scanning transmission electron microscopy (STEM) in conjunction with two-dimensional transmission electron microscopy (TEM), electron diffraction and STEM studying the mineral morphology and crystallinity. We found that the mineral organisation follows a fractal-like pattern from the nanoscale upwards starting from needle-like twisted nanocrystals which form larger assemblies of platelet shaped rotated units across the organic collagen fibril boundaries, hence a cross-fibrillar mineralisation pattern results. This network of HAp crystals exists in conjunction with the highly cross-linked collagen matrix.
Another challenge regarding the understanding of bone formation is the realisation of in situ characterisation approaches allowing for the direct observation of the mineralisation in the context of collagen fibrils. This process is known to be strongly affected by the presence of non-collagenous organics, such as osteopontin which enhances the rate of matrix mineralisation. For this purpose we have investigated - using Raman microspectroscopy - the process of HAp mineralisation of rat-tail derived collagen applying a bespoke heatable flow cell and the polymer-induced liquid precursor (PILP) technique using osteopontin as calcium and phosphate binding macromolecule. Within a time-frame of a few hours we spectroscopically followed the process of infiltration and mineralisation of the collagen being able to identify the different phases tracking the ν₁-PO₄ Raman band around 962 cm^-1 and associated collagen vibrational modes and could clearly distinguish the different phases of collagen mineralisation. Post-mortem samples were studied using TEM and electron diffraction indicating a morphological mineral pattern very similar to that of bone. Our findings show that advanced electron microscopy and in situ spectroscopy offer exciting opportunities to characterise nano-level and molecular-level spatial as well as temporal structural and compositional variations important for the understanding of bone formation.