3D cryogenic electron microscopy for nanoscale imaging of bone matrix vesicles

Matrix vesicles (MVs) play a critical role in initiating mineralization during bone formation. However, imaging MVs without compromising their structure has been challenging, and their exact composition and mechanisms remain poorly visualized. In this study, we utilized advanced 3D cryogenic electron microscopy to visualize the mineralization of MVs in their native hydrated state. To analyze the distribution and morphology of MVs in vivo, embryonic chicken bone was high-pressure frozen, preserving it in a vitrified hydrated state. Correlative light and electron microscopy were employed to precisely target regions of mineralizing cartilage and cryogenic focused ion beam scanning electron microscopy (cryoFIB/SEM) allowed the imaging of a large volume of the tissue with nanometric resolution. We show that both mineral-free and mineral-coated vesicles coexist with initial mineral deposits in cartilage. By isolating MVs from the tissue, we characterized their content and their capacity to produce mineral in vitro. Our results show that isolated MVs can drive mineral deposition through their phosphatase activity. Cryogenic electron tomography revealed that mineralization on MVs proceeds from their surface. Our in vitro observations closely mirror the pattern of in vivo mineral deposition, demonstrating that the initiation of cartilage mineralization results from the action of MVs, which locally manipulate supersaturation by converting inhibitors and producing phosphate for mineral deposition. These findings have significant implications for understanding mineral deposition during early stages of bone development and pathological soft tissue mineralization.