TOPOGRAPHIC ANALYSIS BY ATOMIC FORCE MICROSCOPY OF OSTEOBLASTIC MATRIX VESICLES OBTAINED IN DIFFERENTS DAY OF GROWTH

Biomineralization is defined as the physicochemical process leading to the deposition of hydroxyapatite (HA) on specific areas of the extracellular matrix. Matrix vesicles (MVs), a special type of extracellular vesicles, actively participate in bone formation by mediating the mineralization process, starting from the initial formation of HA to the subsequent propagation of minerals. These calcifying bioactive vesicles, carry lipids and proteins and are produced by osteoblast and other cells responsible for endochondral and membranous ossification under physiological conditions. Atomic Force Microscopy (AFM) is a high-resolution non-optical imaging technique that enables the topographic and mechanical studies of biomembranes on the nanometer scale. Here, AFM was applied to characterize osteoblast MVs obtained at different time points during biomineralization to investigate whether there are differences among them. MVs were isolated from MC3T3-E1 subclone 14 cell line at different time points (7, 14 and 21 days) using differential centrifugation. AFM micrographs were obtained by Shimadzu SPM-9600 Scanning Probe Microscopy operating in tapping mode. MVs were also characterized by Dynamic Light Scattering, Zeta potential and Nanoparticle Tracking Analysis. AFM 3D topographic images showed that MVs were spherical-like particles with a rough surface. The 14-day and 21-day MVs appeared to be more like each other than the 7-day MVs. In addition, 7d-MVs exhibit a higher average roughness compared to 14-day and 21-day MVs, which indicates that 14-day and 21-day MVs surface were smoother, and more uniform compared to the 7-day MVs, with fewer irregularities. Regarding MVs characterization, our results showed that the 14-day MVs were smaller, with a lower zeta potential, compared to both the 7-day and 21-day MVs. The vesicle concentration was higher on day 14, and the total of protein content was elevated (~50%) in 14-day and 21-day MVs compared to 7-day MVs. In conclusion, our findings provide insights on the dynamic nature of MVs by revealing changes in their topography, viscosity, size, zeta potential, morphology, and protein content. These observations suggest that MVs undergo functional changes during the mineralization process, which can potentially impact their biological roles and interactions within their environment. Understanding the role of MVs role in biomeralization holds promise for the development of novel therapeutic applications.