This incubation step with the implant material was repeated twice with fresh cell culture media. Three eluates were pooled for cell culture experiments. DFCs were seeded onto cell culture plates and cultivated in standard cell culture media. After cell seeding (12 to 24 h), cell culture media were changed, and cells were cultivated in cell culture media with material eluates. After 24 h of cultivation, cells were harvested for flow cytometry analyses or protein isolation for Western blots (see below).

Cell counting kit 8 assay

Numbers of vital cells were evaluated after days 1, 2, 3, and 6. For cell counting, cell cultures were incubated with the cell counting kit 8 (CCK8) ready to use solution according to manufactures instructions (Dojindo, Rockville, MD, USA). The optical density (O.D.) was measured at a wavelength of 450 nm. For the evaluation of the cell adherence (normalized to standard cell culture dishes), cell proliferation (normalized to cell number at day 1 of cell culture) relative cell numbers were calculated.

Flow cytometry analysis

The induction of apoptosis in DFCs and dNC-PCs was evaluated by measuring the Cell Event® Caspase3/7 Green Flow cytometry assay (Life Technologies, Carlsbad, CA, USA). For the Caspase3/7assay, cells were cultivated in eluates as described above. After 24 h, cells were harvested by trypsin-EDTA treatment, washed with PBS, and stained first with Caspase3/7 Green Detection Reagent (25 min, 37°C). After this step 1-mM SYTOX® AADvanced dead cell stain solution was added to the sample (5 min, 37°C). Cell fluorescence was analyzed at 488-nm excitation and applied to standard fluorescence compensation. Emission of fluorescence was measured with 530/30 BP (Caspase3/7 Green Detection Reagent) and with 690/50 BP (SYTOX® AADvanced dead cell stain) filters.