Dynamics of intrafibrillar collagen mineralization revealed by multimodal in situ analysis

The deposition of calcium phosphate mineral within a collagen matrix plays a pivotal role in the formation of bone and teeth, yet understanding its precise mechanism and time-dependence remains a significant challenge. Conventional approaches are often constrained to ex situ studies and as a result the intrinsic dynamics governing collagen mineralization remains unclear. To address this knowledge gap, we developed a custom thermal flow cell to enable in situ characterization using Raman spectroscopy and small/wide-angle X-ray scattering for comparable sample settings. This approach allowed us to monitor the intricate process of collagen matrix mineralization from the initial infiltration of precursor phases to the formation of intermediate phosphate phases, and ultimately to the predominant growth of hydroxyapatite. Our findings reveal a striking expansion of the collagen matrix during initial infiltration, followed by compression in the early stages of mineralization, likely driven by water expulsion, which suggests the development of pre-stress similar to that observed in bone. As mineralization progressed, the matrix expanded once again, correlated with crystal growth. Post-mortem analyses confirmed the presence of intrafibrillar mineralization, with remarkable agreement to bone formation at up to 9 h of mineralization before over-mineralization occurred. Our study further identified a tessellated mineralization pattern within the collagen matrix, a feature also seen in bone, pointing to a highly regulated physico-chemical control of the mineralization dynamics. These insights deepen our understanding of the fundamental processes governing bone mineralization with broad implications for designing advanced biomaterials. STATEMENT OF SIGNIFICANCE: A deep understanding of the mineral growth dynamics within a collagen matrix is key for any attempt to mimic bone growth. Often, static snapshots at different time points are used to study it, missing key aspects, e.g. the mechanism of matrix/mineral interaction and the formation of intermediate phosphate phases. Using a custom-made reaction-cell combined with in situ Raman spectroscopy and X-ray scattering, together with electron-microscopy and X-ray-fluorescence, we reproduced and characterized bone-like intrafibrillar collagen mineralization, revealing an initial collagen expansion during precursor infiltration, followed by contraction, associated with the onset of mineralization including non-hydroxyapatite aggregates. Subsequently, expansion ensued as hydroxyapatite dominated mineral growth. Consequently, our work highlights the importance of transient phosphate phases during bone mineralization and variable stress induced during the process.