Osteoporosis, which is a problem worldwide, particularly in aging societies, causes approximately 9 million fractures every year according to the International Osteoporosis Foundation. The mineralization process is crucial in maintaining the mechanical integrity of bone. Dysfunctional mineralization can alter a variety of bone properties, leading to bone pathological conditions, including osteoporosis. Despite a long study of the mineralization process, the overall process is not fully understood, and very few studies have been performed in hypomineralized model. The project focuses on investigating the mineralization process in both wild-type and a genetic deficiency model to identify any alterations or missing steps that link to their hypomineralized phenotype. The outcome result might help to identify a target for the therapeutic propose. Electron microscopy is the only proven efficiency to provide direct observation with spatial and energy resolution to analyze step by step of mineralization process at nano and sub-cellular scale ranges. Here we performed analytical electron microscopy to investigate the mineralization process, particularly the mechanism that is regulated by cells in actively mineralizing growth plate cartilage.
Firstly, the sample preparation workflow to prepare the growth plate cartilage/ bone is optimized. The protocol demonstrated its efficiency in preserving both ultrastructure and mineral crystallinity, and the cryo fixation is rapid enough to capture dynamic biological events. The study in growth plate cartilage/bone of wild type demonstrated step by step of cell-regulated mineralization. We then carried out the investigation in genetic deficiency mice by which one alkaline phosphatase; PHOSPHO1 has been knocked out. We have identified a couple of irregular mineralizing organelles/ steps compared to the wild type, including irregular secondary ossification.
The last activity was to optimized experimental condition of precession electron diffraction for biomineral. PED is a specialized technique to collect 3D diffraction in TEM. In PED, the electron probe is tilted and rotated around the central axis to produce a series of diffraction patterns. The probe scans across samples with a step size down to 2 nm. This is a powerful tool to study mineral development in bone which contains nano-size minerals i.e. 10-20 nm globular mineral and needle shape mineral with a thickness of 3-4 nm.
