Strontium Changes Lipid Profile, Release, and Function of Matrix Vesicles Produced by Mineralization-Competent Cells

Abstract

Background: Mineral deposition in the extracellular matrix (ECM) is a highly organized process initiated by matrix vesicles (MVs) released from mineralization-competent cells, such as osteoblasts. In bone pathologies, osteogenic inducers (ions, hormones, nanoparticles) are becoming increasingly vital for the repair of damaged tissue. Among these inductors, strontium ranelate (SR), first suggested for treating osteoporotic patients, stands out. The bioactive strontium ion (Sr²⁺) has a dual mechanism of action in bone homeostasis: it activates osteoblasts, promoting bone formation, and inhibits osteoclasts, limiting bone resorption. Recent research has focused on how Sr²⁺ influences osteoblast function, but its effects on the mineralization process have not been explored. For this study, we hypothesized that Sr²⁺ modulates mineralization-competent cells at two levels: (a) it activates the extracellular signal-regulated kinase 1/2 (Erk1/2) and cAMP response element-binding protein (CREB) osteogenic signaling pathways, increasing mineral towards in the ECM, and (b) it regulates MV release and function. Advanced lipidomic analysis examined how Sr²⁺ affects the MV lipid profile, which is pivotal for MV biogenesis and bone formation. Methods: We performed an MTT assay to assess the cytotoxicity of CaCl₂ and SR. Alizarin Red and Von Kossa staining were used to track mineral deposition towards the ECM. We assessed the phosphorylation states of ERK and CREB by western blotting and the osteogenic-related gene levels by quantitative real-time PCR. Biophysical characterization of 17A11-derived MVs was performed by nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and zeta potential. Mineral deposition and characterization were performed by turbidimetry and Fourier transform infrared spectroscopy (FTIR), respectively. MV activity was studied by alkaline phosphatase activity. We also performed a Western blot analysis to assess MV markers. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) were applied to investigate changes in membrane fluidity and the vesicles’ conformation. We explored the changes in lipid profiles using state-of-the-art lipidomic analysis. Results: Our findings demonstrate that Sr²⁺ activates the Erk1/2 and CREB pathways, leading to a dose-dependent increase in ECM mineralization. Additionally, the viscoelastic properties of MVs from Sr²⁺-stimulated 17IIA11 cells, a preodontoblast progenitor cell line, were altered, as demonstrated by AFM and TEM, which we linked to modifications in their lipid composition, as revealed by the enrichment of ceramide (Cer) and sphingomyelin (SM), both of which play pivotal roles in bone development. Conclusions: Our study demonstrated that Sr²⁺ affects the initiation of the mineralization process by changing the release and lipid composition of MVs, and acts, in part, through Erk1/2 and CREB signaling pathways.