NTA by NanoSight LM10 (Malvern Instruments Ltd, Malvern, UK) assessed vesicle size and concentration within the media samples46 (link). Calcific SMC-derived EVs collected from conditioned media by ultracentrifugation at 100,000 × g were resuspended in 1 ml calcifying media and divided into five 200 μl portions. One portion was collected for initial time point analyses. Aggregation was detected by NTA in the remaining portions incubated at 37 °C for 1, 3, 5, or 7 days. NTA determines nanoparticle number and size distributions from the Brownian motions of objects illuminated by laser light47 (link). The samples were diluted 1:7 in PBS prior to injection into the NanoSight chamber. Samples were continuously injected via a syringe pump (Malvern), and five NTA videos were collected for 1 min each. The camera gain was set at a constant value of 9, and the threshold value for vesicle detection was set to 2. The resultant size and concentration output data from each video were averaged to generate the final distribution for each sample.