Periodic Reporting for period 1 - BoneImaging (Cryo analytical microscopy of cell-mediated mineralization in models of bone disease) Reporting period: 2021-06-01 to 2023-05-31 Summary of the context and overall objectives of the project 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. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 1. Successfully optimized anhydrous HPF-FS workflow for cartilage/bone for electron microscopy study. The application of this anhydrous protocol can avoid changes in mineral crystallinity, which is caused by aqueous in fixatives or FS medium while preserving the cartilage ultrastructure in a near-native state with sufficient staining contrast for observation. With rapid freezing, it is also efficient to capture short live biological events (as shown in the result of this project. This workflow is not limited to cartilage/bone preparation but also can be applied to other pathological calcified tissue and disease mineralization models. 2. The result from the study of mineralization in wild type, we tracked the cell-regulated mineralization process for both originally intracellularly (in mitochondria and vesicles) and in the matrix vesicle (the classic theory where cells produce membrane vesicles that accumulate calcium phosphate extracellularly). The results demonstrated step by step of these 2 mechanisms, observing both mechanisms side by side enables a direct comparison, leading to distinguishable measures (i.e. by sizes and mineral morphologies). This result would contribute to the complete understanding of the cell-regulated mineralization process. 3. This project studied the mineralization mechanism in hypomineralized bone (PHOSPHO 1 knock-out mouse). Two alterations of mineralization are identified; however, they co-exist with normal mechanisms, therefore these alterations required more studies and chemical analysis to confirm and establish the link to the phenotype. Exploitation and Dissemination: The sample preparation workflow is transferred to collaborators and the host institute(Centre for Ultrastructural Imaging) for future studies and/or users. The research results are under preparation for publication and present in an international conference (IMC20). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) 1. The sample preparation workflow in this study is not limited to cartilage/bone preparation but also can be applied to other pathological calcified tissue and disease mineralization models. 2. The analysis of the mineralization process in wild type provides insights into the 2 processes that are regulated by mineralizing cells. The results provide step-by-step information on the origin and transportation of intracellular minerals and calcified matrix vesicles, comparing them side by side. This information would contribute to a complete understanding. We also investigate the cell-regulated mineralization in a hypomineralized bone model (PHOSPHO 1 knock-out mice), and be able to identify altered mineralization. However, these alterations co-exist with the normal mechanisms, thus requiring further analysis. These altered steps could be a guide for tissue engineering to restore the mineralization process. 3. Optimized the workflow for PED in biological samples. We have partially optimized the sample preparation and experimental conditions; however, we have identified a new technical issue with a dynamic effect in the biological sample caused by the electron probe, rendering data invalid. We are now in progress to clarify the cause of this dynamic effect. Once completed, this can be applied as a working protocol, including precautions for PED on biological samples. Overview research summary
