Mineralisation of collagen studied by in situ X-ray and Raman spectroscopy

Bone is a fascinating biocomposite material combining hardness and toughness through the hierarchical and nano-level organisation of a hard mineral (hydroxyapatite) and a soft protein (collagen type I). The mechanism behind the mineralisation of the collagen matrix, which is constituted of fibrils, is still a central problem in the field of biomineralisation research. Current understanding is that the mineral is formed via an amorphous precursor phase that infiltrates the collagen fibrils and subsequently crystallises. However, the mechanisms controlling infiltration and crystal growth remain unclear. To identify the processes involved in the precursor transport and phase transformation across varying length scales, an in vitro model system was used, the polymer-induced liquid-precursor (PILP) process, by which collagen mineralisation can be achieved both intra- and extra-fibrillar [1][2]. The in vitro model system employs a process-directing polymer e.g. osteopontin a biogenic non-collagenous protein (NCP) in native bone formation. Using a bespoke in-situ heated liquid cell to maintain physiological temperatures, for use in a Raman spectrometer and on I22 beamline, collagen mineralisation was successfully achieved and observations were made in real time of the transition from an amorphous precursor phase through to the formation of hydroxyapatite (HAP) crystals. nano- X-ray fluorescence (n-XRF) was performed on I14 beamline which showed the distribution of Ca and P across the fibre revealing there was a higher degree of mineralisation on the edge of the fibre than in the centre of the fibre. In addition, transmission electron microscopy (TEM) was used to examine the mineralised collagen fibre, which revealed HAP crystals both on the surface and embedded within the collagen fibre. Our findings allow for the quantitative characterisation of the kinetics of precursor infiltration and crystallisation across different length scales.