Mgcl2 solution

Total RNA was extracted using the Trizol™ method following the manufacturer's protocol (31 (link), 32 (link)). Briefly, Trizol™ reagent (Thermo Fisher Scientific) was admixed to either whole cell suspension, APs, MVs or EXs (9 part Trizol: 1 part cells/vesicles). The Trizol-vesicle solution was triturated or vortexed to ensure vesicle lysis prior to addition of MgCl₂ solution (Sigma) and chloroform. Each mixture was vortexed vigorously, incubated at room temperature (RT) for and centrifuged prior to transfer of the aqueous phase to fresh 2 mL micro tubes. Then, isopropanol was added and the samples were repeatedly inverted and incubated at RT. The tubes were then incubated at −20°C for 1 h (or overnight). Following incubation, the samples were centrifuged at 12,000 × g for 10 min at 4°C to collect RNA pellet. The RNA pellets were washed in RNAse-free 75% ethanol twice prior to removal of the supernatants and allowing the RNA pellets to air dry. Finally, the RNA was resuspended in 10–20 μL RNase-free water (Invitrogen; depending on the size of the RNA pellets).
Vesicle-derived RNA was quantified and evaluated for quality (as described in the Supplementary Material). RNA samples of sufficient quality were initially subjected to qRT-PCR to confirm the presence of miRNAs prior to subjecting RNA to deep sequencing using the Illumina® NextSeq500 system (below).

Total RNA was isolated using QIAzol Lysis Reagent (QIAGEN GmbH, Düsseldorf, Germany) and miRNeasy Mini Kit (QIAGEN) with on-column DNase digestion (RNase-Free DNase Set, QIAGEN) as described in the manufacturer’s protocol.
Reverse transcription was performed for 45 min at 42°C and 5 min at 99°C utilizing 2.5 U MuLV Reverse Transcriptase (Thermo Fisher Scientific), 1 U Protector RNase inhibitor (Sigma-Aldrich), 0.125 μM Oligo(dT)₁₅ primer (Promega), 1 mM MgCl₂ solution (Sigma-Aldrich), 0.4 mM dNTP mix (Promega), 1×PCR Buffer without MgCl₂ (Sigma-Aldrich), and 100 ng RNA template with a final reaction volume of 20 μL.
Quantitative PCR was conducted using the Rotor-Gene Q cycler and Rotor-Gene SYBR Green PCR Kit (Qiagen) according to the manufacturer’s protocol with β-actin as reference gene. Applied primer pairs (Supplementary Table 1) were used in a final concentration of 0.4 μM. Cycling conditions involved 5 min at 95°C followed by 45 cycles of 5 s at 95°C, and 10 s at 59°C.
RNA isolation and RT-qPCR were carried out using three separate cell culture passages, each measured twice (biological n=3, technical n=2).

All oligonucleotides were purchased from Integrated DNA Technologies (IDT, Skokie, IL, USA). T4 ligase was obtained from Promega (Madison, WI, USA). T7 RNA polymerase and ribonucleotide triphosphates (rNTPs) were purchased from New England BioLabs (NEB, Ipswich, MA, USA). Zeba^TM spin desalting columns were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cyanine 5-UTP was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Mica and Lacey Formvar/carbon-coated copper grids (01883-F) were obtained from Ted Pella (Redding, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin (P/S), fetal bovine serum (FBS), and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Gibco (Waltham, MA, USA). The Stemfect^TM RNA Transfection Kit was purchased from Stemgent (Houston, TX, USA). CelLytic M and MgCl₂ solution were purchased from Sigma-Aldrich (Saint Louis, MI, USA). A Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). Tris-HCl buffer was purchased from Invitrogen (Waltham, MA, USA).

BSA and fatty-acid-free BSA (FF-BSA) (both Sigma Aldrich) were separately dissolved at several concentrations in a 1 mmol/l MgCl₂ solution (Merck Millipore, Darmstadt, Germany) or a physiological buffer, both set at pH 7.5 by adding NaOH. The physiological buffer contained the following (all from Merck Millipore unless stated otherwise): 27 mmol/l NaHCO₃, 112 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgCl₂, 1 mmol/l Na₂HPO₄ (VWR International, Radnor, PA, USA) and 2.5 mmol/l CaCl₂ dissolved in Milli-Q (Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands).

For the cement setting experiments at the beginning of the study (Figure S1, Supporting Information), different magnesium oxides (MgO 22, MgO 27, MgO 291, MgO 2923) from the company Magnesia GmbH (Lüneburg, Germany) were mixed with MgCl₂-solution (4 mol L⁻¹, Merck KGaA, Darmstadt, Germany) in the respective powder-liquid-ratio (PLR). For the following experiments, only MgO 2923 was used for cement preparation since it exhibited the most rapid setting.