Phosphate buffered saline (pbs)

MSCs (2.5 × 10⁵) from old and young individuals were cultured on slides (Sigma-Aldrich, St. Louis, MO, USA) pre-treated with poly-d-lysine (Sigma-Aldrich, St. Louis, MO, USA) in 6-well plates (Corning Inc., New York, NY, USA) for 8 h (day 1). MSC-derived EVs (2 × 10⁷) from young and old individuals were stained with 10 μM 3-3′-diethylthiacarbocyanineiodide (DiI) and added to each culture of MSCs; PBS (MP Biomedicals, Illkrich-Graffenstaden, France) was added as a control. At 2, 3 and 6 days, the cells were washed three times with PBS (MP Biomedicals, Illkrich-Graffenstaden, France) and fixed with 4% (w/v) paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 10 min, and then the slides were mounted using ProLong® Gold antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI; (Thermo Fisher Scientific, Carlsbad, CA, USA). The cells were examined with an Olympus BX61 microscope using a DP71 digital chamber (Olympus, Tokyo, Japan) with software DP-Controller and DP-Manager software. A NeoScope JCM-6000 Plus scanning electron microscope (Nikon-Izasa, Barcelona, Spain) was used to take supplementary images.

Porcine PFJs were obtained from the right hind legs of Large White pigs aged either 4–6 or 12 months old (John Penny & Sons, Leeds, UK), within 24 hrs of slaughter. The two ages were used to give a model of different bone properties such as elastic yield and modulus. Tissue samples were kept hydrated throughout preparation using phosphate buffered saline (PBS; MP Biomedicals LLC, UK) and stored at -20°C. Prior to testing, samples were thawed at room temperature.

CIMVs were prepared as previously described [37 (link)] with some modifications. Briefly, immature bone marrow-derived DCs or tsDCs were transfected with RNA-B16 or RNA-RLS₄₀ precomplexed with cationic liposomes L or ML at N/P ratio 4/1 as described above. Transfected DCs were washed with serum-free media, resuspended in serum-free IMDM supplemented with 10 µg/mL cytochalasin B (AppliChem GmbH, Darmstadt, Germany) and incubated for 30 min under SC. After that, cells were vigorously vortexed for 30 s. CIMVs were collected by sequential centrifugation at 100× g (10 min, 4 °C), 600 g (20 min, 4 °C), and 15,000× g (30 min, 4 °C). The resulting pellet was washed with PBS (MP Biomedicals, Santa Ana, CA, USA) and centrifuged at 15,000× g (30 min, 4 °C). CIMVs were resuspended in 0.1 mL of PBS and stored at −80 °C until use. CIMV concentration was measured on the basis of the total protein concentration with Qubit Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA ) [19 (link)]. CIMV-based vaccines were administered s/c at the crest area in the interscapular region at a dose of 15 µg of CIMVs (total protein counts) per mouse in 0.2 mL of Opti-MEM.