Harnessing μ-X-Ray Fluorescence Spectroscopy as a Tool to Assess Extracellular Vesicle-Induced Biomineralization

Bone cell-derived extracellular vesicles (EVs) have been increasingly investigated as novel acellular strategies for bone regeneration due to their pro-regenerative potency. The evaluation of such bone repair enhancement strategies commonly lies in the assessment of cell-mediated mineral deposition, associated with destructive and nonhigh-throughput methods. Herein, a robust methodology is presented to assess the osteogenic potential of an EV therapy using μ-X-ray fluorescence spectroscopy (μ-XRF). Mineralizing osteoblast-derived EVs (MO-EVs) are isolated from conditioned media via ultracentrifugation and comprehensively characterized. Their pro-osteogenic potency is validated via alkaline phosphatase activity, alizarin red, and picrosirius red staining for the evaluation of calcium and matrix deposition, respectively. μ-XRF is first employed to quantify calcium and phosphorous levels as markers of minerals generating 2D elemental maps of the cultures. The in-depth downstream analysis of the elemental maps reveals that MO-EVs modulate mineralization in a time- and concentration-dependent manner as MO-EV concentration from 5 μg mL⁻¹ significantly increases mineral coverage and increases calcium/phosphate levels in mineralized phases. Together, these results demonstrate the potential of μ-XRF, allowing the examination of elemental levels, mineral coverage, and chemical phases in a single process and thus, offering a new platform for the therapeutic screening of osteogenic technologies with a resolution accommodating biological workflows.