Insights from Nanoscale Microscopy Techniques: Differential Characterization of Osteoblast Matrix Vesicles

Introduction: Matrix vesicles (MVs), a specialized subtype of extracellular vesicles (EVs), play an active role in bone formation by mediating the mineralization process. Transmission electron microscopy (TEM) is widely utilized method for assessing the quality and purity of EVs, providing insight into their microstructure. Atomic force microscopy (AFM) provides high-resolution non-optical imaging capabilities, enabling the examination of the topographic and mechanical properties of EVs. Scanning electron microscopy (SEM) allows the detailed EVs surface characterization, using electron beams to interact with sample atoms. In this study, we employed nanoscale microscopy techniques to characterize osteoblast MVs obtained at different stages of growth, aiming to discern potential differences among them. Methods: MVs were isolated from MC3T3-E1 subclone 14 cell line using differential centrifugation at days 7, 14 and 21. MVs samples were diluted (1:1000) in SCL buffer and fixed with 1% glutaraldehyde (v/v). For TEM imaging, the fixed samples were dripped onto a carbon-coated copper grid, dried, and analyzed using a JEOL-JEM-100 CXII transmission electron microscope. For AFM, fixed MVs were dropped onto freshly-cleaved mica plates, dried at room temperature, and examined using a Shimadzu SPM-9600 Scanning Probe Microscopy operating in tapping mode. For SEM analysis, MVs were fixed with 2% glutaraldehyde and 2% paraformaldehyde (v/v) in 0.1M phosphate buffer (PBS). Subsequently, MVs were washed with PBS, post-fixed in 1% OsO₄ buffer, dried using a critical point drying system, coated with a gold film, and analyzed using a JEOL-SEM-JSM-6610LV microscopy. Results: All microscopy techniques successfully captured high-quality images of MVs. TEM images showed that MVs from day 7 (7d-MVs) were smaller and more homogenous compared to those from days 14 and 21 (14d-MVs and 21-d Mvs, respectively). However, SEM images did not exhibit significant differences among the analyzed groups, possible due to limitations in magnification preventing detailed observation of vesicles structure beyond x10,000. Conversely, AFM demonstrated significant distinctions between 14d-MVs and 21d-MVs compared to 7d-MVs. AFM 3D topographic images revealed that 7d-MVs showed higher average roughness, indicating a more irregular surface, while 14d-MVs and 21d-MVs exhibited smoother and more uniform surfaces. Conclusions: Our findings provide insights on the dynamic nature of MVs and underscore the importance of selecting appropriate microscopy technique. AFM proved to be the most effective technique in our study for assessing topographical and mechanical changes among MVs groups. Future analyses will be crucial to confirm the impact of these changes on the biological role of MVs in mineralization.