Il 1β

CD14⁺ monocytes were seeded at a density of 10⁶ monocytes/mL in 24-well plates and cultured for 6 days in RPMI 1640 medium containing 10% FCS, 1% pen-strep and 1% amphotericin B, supplemented with the DC cytokine cocktail (40 ng/mL IL-4 and 100 ng/mL GM-CSF (Sigma-Aldrich, Darmstadt, Germany) for the first 5 days; 40 ng/mL IL-4, 100 ng/mL GM-CSF, 10 ng/mL TNF- α, 10 ng/mL IL-1β, 10 ng/mL IL-6 (Tebu Bio, Offenbach, Germany), and 1 µg/mL PGE2 (Bio Trend, Köln, Germany) for the last 24 h). Four different DC populations were generated, three of them were treated with 5-fold or 10-fold concentrated JPC secretomes (from untreated JPCs (S_CO), and from osteogenically induced JPCs with (S_OB+D) or without (S_OB-D) dexamethasone stimulation for 10 days). The fourth DC population served as a control sample without additional secretome supplementation (control).

The concentration of use for lipopolysaccharide E. coli 026:B6 (LPS) (100 ng/mL), a pathogen-associated molecular pattern (PAMPs), and IL1β (0.1 ng/m) (Tebu-Bio, France) was selected from the literature [22 (link)] to be used, respectively, as a canonical agonist for TLR4 and IL1R complex. The specific TLR4 antagonist CLI-095/TAK-242 (1.00 µM) (InvivoGen, San Diego, CA, USA) was used as a TLR4 inhibitor [22 (link),79 (link)]. We selected low concentrations of BBA (0.10–5.00 µM) (Merck, Darmstadt, Germany) to facilitate clinical transferability. All cell culture reagents were purchased from Sigma-Aldrich (Sant Louis, MO, USA) except otherwise mentioned. PubChem: IL1β CID 159483, CLI-095 CID 9919285, BBA CID 168928.

Cell migration was analyzed by a modified Boyden chamber assay, using a 48-well microchemotaxis chamber (NeuroProbe Inc., Baltimore, MD, USA) with polycarbonate filters with 8 μm pores (NeuroProbe Inc.) between the lower well containing the chemotactic factor and the upper well containing the cells. Recombinant human PDGF-BB (BioLegend, Fell, Germany, 10 ng/ml), rhIGF-1 (Biomol, Hamburg, Germany, 100 ng/ml) and IL-1β (tebu-bio, Offenbach, Germany, 1 ng/ml) were diluted in serum-free DMEM, filled into the lower compartment of the chemotaxis chamber and covered with the chemotaxis filter. The upper wells were loaded with 1 × 10⁴ cells, suspended in serum-free DMEM and incubated for 4 h. Non-migrated cells were removed and migrated cells on the lower side were fixed with 4 % formaldehyde, stained with Giemsa solution (Merck, Darmstadt, Germany) and counted. DMEM in the lower well served as a negative control (basal migration) for each experiment. To distinguish chemotaxis from undirected chemokinesis migration analyses with the growth factors were performed in the presence and absence of a concentration gradient. The CI was determined as the average number of migrated cells in stimulated wells divided by the average number of migrated cells in control wells.