Unraveling the mechanism of matrix vesicles biomineralization: a cryo-electron microscopy approach

• Grant number: 19/26059-8
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: December 01, 2021
• End date: November 30, 2022
• Field of knowledge: Physical Sciences and Mathematics - Chemistry - Physical-Chemistry
• Principal Investigator: Ana Paula Ramos
• Grantee: Marcos Antonio Eufrásio Cruz
• Supervisor: Nico Sommerdijk
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Radboud Institute for Molecular Life Sciences (RIMLS), Netherlands
• Associated to the scholarship: 17/20846-2 - Exploring the role of lipid-protein-mineral matrix on bone biomineralization: a biophysical approach using self-assembled films, BP.DR

Abstract

Biomineralization of hard tissues such as bone and dentin involves a complex spatio-temporal sequence of events regulated by bone-forming cells. One big question that still perdures on the understanding of this process is the origin of the mineral phase. It has been claimed since their discovery in the decade of 1960, that matrix vesicles (MVs) are the entities responsible to nucleate and deliver calcium phosphate to the bone growth front. MVs act as a smart nanoreactors displaying elegant enzymatic machinery working in orchestration to control the phosphate/pyrophosphate ratio required to trigger calcium phosphate formation. Calcium phosphate is then nucleated templated by the lipid composition of MVs and further delivered to the extracellular matrix in order to propagate collagen mineralization. Therefore, characterization of mineral formation on native MVs using cryogenic and hydrated conditions is a need to fully picture the pathways by which collagen-apatite building blocks are assembled to yield the final bone hierarchical structure. In this project, we propose the use of state-of-art cryogenic electron microscopy techniques to track mineral formation in MVs and their interactions within the extracellular matrix. MVs will be isolated from in vitro cultures of human bone mesenchymal stem cells (hBMSC) differentiated to osteoblasts under osteogenic stimuli. Mineralization in the MVs will be visualized by cryo-transmission electron microscopy with a temporal resolution to decipher the pathway by which mineral is formed in the MVs. Using type-I collagen self-assembled on TEM grids we will follow mineralization guided by MVs at different times in order to verify the steps that culminate in the infiltration of the precursor phase into the collagen fibrils and its posterior crystallization. In order to monitor the formation of MVs in an environment close to the native bone tissue, we will use a 3D-living in vitro model of hBMSC differentiated towards osteoblast/osteocyte phenotype. Using this model, spatio-temporal resolution will be achieved using advanced microscopic tools including 3D confocal fluorescence microscopy and 3D cryo-FIB/SEM as well as their correlation. Professor Nico Sommerdijk is a world-renowned expert on using cryo-EM approaches to explore biomineralization mechanisms and we expect at the end of this project to enhance the current knowledge on the role played by MVs during bone formation. (AU)