Fig. 4

Osteoclasts differentiated from Teflon-coated bag-derived macrophages remain viable and show increased TRAP-positivity and bone resorption. A Flow cytometry: Gating strategy used to identify osteoclasts differentiated from Teflon bag-derived macrophages. Cell viability was assessed by staining for 7-AAD and Annexin V-FITC. A 1:1 mixture of untreated and heat-incubated (10 min at 95 °C) osteoclasts was used as positive control. Shown is one representative scatter plot out of n > 3 donors. B Monocytes (2.0 × 10⁵) differentiated to osteoclasts in the presence of high concentrations of M-CSF and RANKL in cell culture plates or in Teflon bags were stained for TRAP activity (scale bar: 10 µm) and visualized by microscopy. Black arrows indicate TRAP-positive and cells with ≥ 3 nuclei. The corresponding quantification of TRAP-positive cells is shown on the right. C Bone mineral resorption of osteoclasts on synthetic inorganic bone matrix is shown in representative bright-field images and corresponding quantification on the right. D Bone resorptive activity of osteoclasts on bovine cortical bone slices was visualized by toluidine blue staining. E Representative electron microscopy images of osteoclasts from two distinct donors seeded on bone slices. nu = nucleus, er = endoplasmic reticulum, bs = bone slice, bm = bone matrix (partly degraded), v = vacuole, asterisk = not degraded collagen fibrils; scale bar: 1 µm. F Quantification of the pit area formed by osteoclasts differentiated on bone slices. G The release of type I degradation products into the supernatant of osteoclasts cultured on bone slices was measured by ELISA