Distinct Morphologies of Bone Apatite Clusters in Endochondral and Intramembranous Ossification

Bone apatite crystals grow in clusters, but the microstructure of these clusters is unknown. This study compares the structural and compositional differences between bone apatite clusters formed in intramembranous (IO) and endochondral ossification (EO). Calvaria (IO) and femurs (EO) are isolated from mice at embryonic days (E) 14.5 to 15.5 and post-natal days (P) 6 to 7, respectively. Results show that the initially formed bone apatite clusters in EO (≅1.2 µm²) are >10 times larger than those in IO (≅0.1 µm²), without significant changes in ion composition. In IO (E14.5 calvarium), early minerals are formed inside matrix vesicles (MVs). In contrast, in EO (P6 femur epiphysis), no MVs are observed, and chondrocyte-derived plasma membrane nanofragments (PMNFs) are the nucleation site for mineralization. Apatite cluster size difference is linked with the different nucleation sites. Moreover, an alkaline pH and slow P supply into a Ca-rich microenvironment are suggested to facilitate apatite cluster growth, as demonstrated in a biomimetic mineralization system. Together, the results reveal for the first time the distinct and exquisite microstructures of bone apatite clusters in IO and EO, and provide insightful inspirations for the design of more efficient materials for bone tissue engineering and repair.