Toluidin blue

To assess bone resorption activity, monocytes were seeded on bovine cortical bone slices (ImmunoDiagnostic Systems) and differentiated into MF or OC. Following complete cell removal by several washes with water, bone slices were stained with toluidin blue (Sigma-Aldrich) to detect resorption pits under a light microscope (Leica DMIRB, Leica Microsystems). Surface of bone degradation areas were quantified manually with ImageJ software. Cross-linked C-telopeptide collagen I (CTX) concentrations were measured using betaCrosslaps assay (ImmunoDiagnostic System) in the culture medium of OC grown on bone slices [25 (link)].

MSC differentiation protocols were applied as described in detail previously [35 (link)]. For adipogenic differentiation we used adipogenic induction and maintenance medium [35 (link)], alternately, twice a week for 21 days, before cells were fixed with 50% ice-cold ethanol and stained with 0.2% Oil Red O solution (Sigma, Germany) for lipid visualization. For chondrogenic differentiation, pellet cultures were performed and maintained for 21 days in serum-free chondrocyte induction medium refreshed three times a week (with freshly added TGF-β₃). Pellets were fixed in formalin and embedded in 3% agarose for paraffin block preparation. Slices were prepared and stained with 1% Toluidin Blue (Sigma, Germany) for proteogyclan visualization. For osteogenic differentiation, we applied osteogenic induction medium for 21 days, refreshed three times per week. Finally cells were fixed with 70% ice-cold ethanol and stained with 40 mM Alizarin Red (Sigma, Germany) solution for calcium deposit visualization. Staining was photographed with a Cool Snap™ HQ2 digital camera (Photometrics, USA) on an Axiophot 2 microscope (Carl Zeiss, Germany).

To assess bone resorption activity, macrophages were seeded on bovine cortical bone slices (Boneslices, Inc.) and differentiated into osteoclasts. The dimensions of bone slices are (i) Diameter: 6mm; (ii) Thickness: 0.4mm. Resuspended OC precursors were seeded in 96-well plates on 0.4 mm thick bovine cortical bone slices at a density of 1 × 10⁵ viable cells per bone slice. Following complete cell removal by several washes with water, bone slices were stained with toluidin blue (Sigma-Aldrich) to detect resorption pits under a light microscope (ECLIPSE, TS100, Nikon). The surface of bone degradation areas was quantified manually with ImageJ software (version 1.47).