Endochondral ossification: insights into the cartilage mineralization processes achieved by an anhydrous freeze substitution protocol
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
Growth plate cartilage (GP) serves as a dynamic site of active mineralization and offers a unique opportunity to investigate the cell-regulated matrix mineralization process. Transmission electron microscopy (TEM) provides a means for the direct observation of these mechanisms, offering the necessary resolution and chemical analysis capabilities. However, as mineral crystallinity is prone to artifacts using aqueous fixation protocols, sample preparation techniques are critical to preserve the mineralized tissue in its native form. We optimized cryofixation by high-pressure freezing followed by freeze substitution in anhydrous acetone containing 0.5% uranyl acetate to prepare murine GP for TEM analysis. This sample preparation workflow maintains cellular and extracellular protein structural integrity with sufficient contrast for observation and without compromising mineral crystallinity. By employing appropriate sample preparation techniques, we were able to observe two parallel mineralization processes driven by chondrocytes: 1) intracellular- and 2) extracellular-originating mineralized vesicles. Both mechanisms are based on sequestering calcium phosphate (CaP) within a membrane-limited structure, albeit originating from different compartments of the chondrocytes. In the intracellular originating pathway, CaP accumulates within mitochondria as globular CaP granules, which are incorporated into intracellular vesicles (500-1000 nm) and transported as granules to the extracellular matrix (ECM). In contrast, membrane budding vesicles with a size of approximately 100-200nm, filled with needle-shaped minerals were observed only in the ECM. Both processes transport CaP to the collagenous matrix via vesicles, they can be differentiated based on the vesicle size and mineral morphologies. Their individual importance to the cartilage mineralization process is yet to be determined.
Date Issued
2025-01-01
Date Acceptance
2024-11-11
Citation
Acta Biomaterialia, 2025, 191, pp.149-157
ISSN
1742-7061
Publisher
Elsevier BV
Start Page
149
End Page
157
Journal / Book Title
Acta Biomaterialia
Volume
191
Copyright Statement
© 2024 The Authors. Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
(http://creativecommons.org/licenses/by/4.0/).
License URL
Identifier
http://dx.doi.org/10.1016/j.actbio.2024.11.015
Publication Status
Published
Date Publish Online
2024-11-12