Prenucleation Clusters‐Induced Intrafibrillar Mineralization of Collagen Fibrils via Promotion of Hydroxyapatite Crystallization

Intrafibrillar mineralization (IM) of collagen fibrils is a fundamental process during bone and dentin formation. Elucidating the detailed mechanisms of IM is essential for developing regenerative therapies targeting mineralized tissues. Currently, it is well documented that non-collagenous proteins (NCPs) and their analogues play a critical role in IM by interacting with minerals or associating with collagen fibrils. However, whether NCPs are continuously involved in the IM of collagen fibrils throughout the entire mineralization process, and whether the early-formed minerals can directly induce IM, remains poorly understood. Here, we designed a two-step method to achieve IM, in which the first step (called the premineralization stage): the collagen fibrils are premineralized for 30 min in the presence of pAsp to form prenucleation clusters (PNCs) within fibrils and exclude the association of pAsp and fibrils; the second step: the premineralized collagen fibrils continue mineralization in the artificial saliva in the absence of polyelectrolytes. In this mineralization process, the premineralized formed PNCs could act as nucleation promoters and accelerate the hydroxyapatite (HAP) formation inside collagen fibrils. According to these findings, we propose the PNC-induced intrafibrillar mineralization (PNCiIM) pathway, which is further confirmed by in vitro dentin remineralization. The PNCiIM, to some extent, complements the current mechanisms of IM of collagen fibrils and provides a significant strategy for hard tissue repair.