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The aim of the present in vitro study was to evaluate the influence of different bone substitute materials (BSM) on the viability of human primary osteoblasts (PO), bone marrow mesenchymal cells (BMMC), and nonadherent myelomonocytic cells (U937). Six different bone substitute materials were tested: Bio-Oss Spongiosa (BOS), Tutodent Chips (TC), PepGen P-15 (P-15), Ostim (OM), BioBase (BB), and Cerasorb (CER). Cells were cultivated on comparable volumes of BSM in 96-well plates. Cell culture-treated polystyrol (Nunclon Delta surface; C) served as positive control. After 2 h and 3, 6, 10, and 14 days, viability of cells was evaluated using a standardized ATP viability assay (CellTiter Glo). Nonsurface-dependent effects of the materials were separately tested using nonadherent U937 suspension cells. For statistical analysis, the Mann-Whitney test was used. Results were considered statistically significant at P < 0.05. Cell viability of PO increased significantly on TC, C, and CER followed by BB. No changes were found for P-15 and decreasing viability for BOS and OM. BMMC showed similar results on C, TC, CER, and P-15. Lower viability for BB and no viability could be detected for BOS and OM (Mann-Whitney test, respectively). Nonadherent cells displayed increasing viability in presence of CER, BB, and BOS. No changes were observed for TC and P-15, whereas for OM, no viability was detected after a maximum cultivation period of 3 days. It was concluded that granular hydroxyapatite (HA; TC, BOS, P-15) and alpha- and beta-tricalciumphosphate (CER, BB) support, whereas nanosized HA (OM) limit or even inhibit surface- and nonsurface-related cell viability in the in vitro model used.