0.22 μm filter

Isolation of EVs from naïve hBMSCs CM and osteogenic CM was performed by ultracentrifugation as described previously [24 (link),26 (link),27 (link)]. In brief, the CM was centrifuged at 300× g (Sigma, Osterode a. Harz, Germany) and 4 °C for 10 min to remove cells, and subjected to centrifugation again at 2000× g to remove dead cells. Afterwards, centrifugation for 30 min at 10,000× g was applied to remove cell debris, then the supernatant was passed through 0.22 μm filters (Sarstedt, Nümbrecht, Germany), and was subjected to ultracentrifugation once at 120,000× g for 70 min at 4 °C. After careful removal of the supernatant, the pellet was resuspended with PBS and centrifuged again at 120,000× g for 70 min at 4 °C. The hereof resulting EV pellets were resuspended in the presence of 25 mM Trehalose (Carl Roth, Karlsruhe, Germany) in PBS. The protein concentration of EVs was measured using BCA (bicinchoninic acid) Protein Assay Kit Pierce (Thermo Scientific, Rockford, IL, USA) according to manufacturer’s instructions.

For isolation of DNA, biofilms were prepared as described above. Supernatants from at least 12 wells per sample were collected and sterilized with 0.22 μm filters (Sarstedt). Sodium chloride was added to 1 ml supernatant to a final concentration of 250 mM. DNA was precipitated with 2.5 volumes of 96% (v/v) ethanol at -20°C o/N and harvested by centrifugation. DNA was washed once with 70% (v/v) ethanol, air-dried, and then dissolved in 50 μl demineralized water. To confirm the source of the isolated DNA, PCR was performed on the following genes: prfA, secA, lmo0849 and lmo1215. The primers used are listed in Supplementary Table S1. Taq polymerase S (Genaxxon BioScience GmbH) was used for amplification and annealing temperatures and extension times were optimized for each amplicon/primer pair. Lm EGD-e chromosomal DNA was used as control. DNA was analyzed by electrophoresis on 0.8% agarose gels in 1x TAE buffer and 1 kb or 50 bp ladders (Fermentas) were used as markers.

Passage 1 SBOs were grown until 80% confluence and the medium was replaced by serum free DMEM. Conditioned medium (60 mL in total) was collected after 24 h of incubation and filtered through a 0.22 μm filter sterilizer. The collected medium was defined as nonsclerotic SBOs or sclerotic SBOs cultured conditioned medium (CM) and was stored at −80 °C before use in the subsequent experiments (Figure 1A). In this study, we used ultracentrifugation method to isolate exosomes. The SBOs CM was centrifuged using a Beckman Coulter Microfuge 18 Centrifuge (Bechman Coulter Life Sciences, Indianapolis, United States) at 300× g at 4 °C for 10 min to remove detached cells and was filtered through 0.22 μm filters (Sarstedt, Numbrecht, Germany) to remove contaminating apoptotic bodies, microvesicles, and cell debris (Figure 1B). Clarified CM was then centrifuged in a Beckman Coulter Optima^TM L-90K Ultracentrifuge at 100,000× g at 4 °C for 90 min with a Type SW41 rotor to pellet exosomes. The exosome-containing pellets were resuspended in ice-cold PBS. A second round of ultracentrifuge using Beckman Coulter OptimaTM MAX-80XP Ultracentrifuge at 100,000× g at 4 °C for 90 min with a Type TLA 110 rotor was carried out, and the final exosome pellets were resuspended in PBS after carefully removing the supernatant [22 (link)] (Figure 1B).