Dental cells were cultivated in standard cell culture media until passage 6. Cell adherence and cell proliferation/growth were measured for the estimation of cell viability on tested rigid and soft materials. In Figure 1, the cell adherence of dNC-PCs on bone substitute materials was better than that of DFCs. However, both dental cells types adhered very well on silicone. Unluckily, dental cells did not adhere on PA; only single cells survived for longer than 48 h (Figure 1B). For the evaluation of cell proliferation, relative cell numbers on implant materials were measured (Figure 2). While cell proliferation of dNC-PCs was moderate (Figure 2A), relative cell numbers of DFCs increased dramatically on bone substitute materials (Figure 2B). However, these results proved the viability of dNC-PCs and DFCs on SB and AP. Interestingly, dental cells formed large spheroid cell clusters on silicone, but cells lost their adherence to this material (Figure 2C), so numbers of silicone adherent cells decreased until day 6 of cell culture (data not shown).

The induction of apoptosis and/or cell death was estimated by flow cytometry and western-blot analyses (Figure 3). While eluates of SB, PA, and silicone did neither induce cell death nor apoptosis, AP induced both cell death and apoptosis in DFCs and dNC-PCs. Both dental cell types expressed the pro-apoptotic marker BAX and the anti-apoptosis marker BCL2 under standard cell culture conditions (Figure 3B). However, BCL2 was not expressed on AP. Interestingly, BCL2 was also not expressed in DFCs after cultivation on SB. The low expression of BCL2 in DFCs may explain the low cell adherence on SB and AP (Figure 1).

We measured the normalized ALP activity in dNC-PCs and DFCs after cultivation on tested materials (Figure 4). While ALP activities in dental cells on bone substitutes were increased or comparable to that of differentiated cells in standard cell culture dishes (control), the specific ALP activity was decreased on silicone (Figure 4B). A PCR array analysis showed that AP induced the expression of osteogenic differentiation markers (Figure 5A). Moreover, differentiated cells formed thick connective tissue like matrices on bone substitute materials (Figure 5B). These results reminded on the differentiation of osteogenic progenitor cells.