T6066

Decellularized bone scaffolds were obtained from human trabecular femoral head specimens (permission number: 187/18, University of Wuerzburg ethics committee), as previously described in [34 ]. Briefly, freshly thawed samples were precisely cut in 3 mm thick slides using an electric diamond band saw (300, Exakt; D64, Walter Messner GmbH, Oststeinbek, Germany) to ensure homogeneous penetration of washing solutions through the complete sample volume. Blood and residual fat material were firstly removed by several cycles of washing in water and a chloroform (288306, Sigma-Aldrich, Steinheim, Germany) and methanol (8388.6, Carl-Roth, Karlsruhe, Germany) mix solution. Further decalcification of bone slices was achieved by incubation for several days in 2.5% ethylenediaminetetraacetic acid (EDTA, E5134, Sigma-Aldrich) in 10 mM Tris-base (T6066, Sigma-Aldrich), from where cylindrical constructs were shaped using a 10 mm biopsy punch. Complete decellularization of bone samples was achieved by enzymatic treatment with 100 Units/mL DNase (DN25, Sigma-Aldrich) and finalized with lyophilization (Martin Christ, Alpha 1–2 LDplus, Osterode am Harz, Germany) for 4 days under a vacuum pressure of 1 mbar. Processed bone scaffolds were stored at −20 °C, and sterilization with 70% ethanol was always performed freshly the day before cell seeding.

Cell lysis and extraction of the cytoplasmic and nuclear fractions was performed using the NE-PER™ Nuclear and Cytoplasmic Extraction kit (Thermo Scientific, 78835) according to the manufacturer’s instructions. The resulting fractions were then mixed with 5× Laemmli buffer (62.5 mM Tris–HCl, pH 6.8 (Sigma, T6066), 2% sodium dodecyl sulfate (Sigma, L4509), 10% glycerol (Sigma, G5516), 5% beta-mercaptoethanol (Sigma, 63689), 0.02% bromophenol blue (Bio Rad, 1610404)) and separated by SDS-polyacrylamide gel electrophoresis.

Phosphate buffered saline (PBS) (Sigma P4417-199TAB), Tris (50 mM, Sigma T6066) buffered saline (150 mM, Fisher Scientific S/3161/65) with Tween-20 (0.01%, Sigma P1379) (TBST) were prepared and used for the study. Microtiter ELISA plates were procured from Costar 9018, Thermo Fisher Scientific whereas the plate reader and RDS-2500 reader were purchased from FLUOstar Omega Microplate Reader (BMG Labtech) and (DETEKT, USA) respectively. Anti-species alkaline phosphatase (A5187 and SAB3700286) was procured from Sigma, USA. The substrate pNPP was procured from BioPanda, USA. The paired antibodies used in the study are summarized in Table 5.Table 5

Paired antibody and recombinant protein standard used for the study.

	Antibody	Recombinant protein standard
UMOD	MAB5144; AF5144 (R&D Systems, USA)	H00007369-Q01 (Novus Biologicals, USA)
OPN	MAB1433; MAB14332R; AF1433 (R&D Systems, USA)	1433-OP (R&D Systems, USA)
IL-9	MAB2091; AF209; MAB209 (R&D Systems, USA)	209-ILB010 (R&D Systems, USA)

Formula of melanin bleaching solution was modified based on previous literature.^{48 (link)} Bleaching solution A: 1% H₂O₂ (v/v) (Sigma-Aldrich, H1009), 15% urea (w/v) (Sigma-Aldrich, U5378) in 0.05 M Tris (Sigma-Aldrich, T6066), pH 10.0. Bleaching solution B: 3% H₂O₂ (v/v), 15% urea (w/v) in 0.05 M Tris, pH 10.0.

200 μl of EVs were digested with 100 μl of lysis buffer [(50 mM NaCl (S5886, Sigma Aldrich), 5 mM EDTA (E9889, Sigma Aldrich), 5 mM Tris, pH 8.0 (T6066, Sigma Aldrich), 1% Sodium Dodecyl Sulfate (SDS, L3771, Sigma Aldrich), 20 mM Dithiothreitol (DTT, 43816, Sigma Aldrich) and 0.5 mg/ml of Proteinase K (P4850, Sigma Aldrich)] O/N at 56 °C. Then, DNA extraction was performed using the standard phenol: chloroform: isoamyl alcohol procedure. DNA concentration was determined using Qubit™ dsDNA High Sensitivity assay kit in a Qubit™ 3.0 fluorometer (Thermo Fisher Scientific) according to the manufacturer’s proceedings.