Cells positive for Caspase3/7 Green Detection Reagent were identified as apoptotic cells, while dead cells were positive for SYTOX® AADvanced dead cell stain. However, vital cells were negatively stained for both staining solutions.

Western blotting

For protein isolation, cells were treated with lysis buffer (250 μl phosphatase, 100 mM Na3VO4, 137 mM NaCl, 200 mM Tris, 480 mM NaF, 1% NP-40, 10% Glycerol) on ice for 2 min. A protease-inhibitor (1 Protease Inhibitor Cocktail tablet from Roche) was included to minimize protein degradation. Cell lysates were placed on ice for 10 min. Protein samples were separated by SDS-polyacrylamide gel electrophoresis in pre-casted 12% Tris-glycine gels (Invitrogen, Waltham, MA, USA) and blotted to nitrocellulose membranes. Membranes were blocked with skimmed milk for 1 h and incubated with primary antibodies that were specific for proteins BAX (pro apoptotic protein), BCL2 (anti apoptotic protein), and β-Actin (housekeeper protein). Washed membranes were then incubated with a horseradish peroxidase-labeled secondary antibody. The detection of the secondary antibody was performed via chemiluminescence and X-ray films (GE Healthcare, Pewaukee, WI, USA).

Osteogenic differentiation

DFCs were cultivated until sub-confluence (>80%) in standard cell culture medium before the differentiation starts with the osteogenic differentiation medium (ODM) comprised DMEM (PAA) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 μmol/L ascorbic acid 2-phosphate, 10 mmol/L KH₂PO₄, 1 × 10⁻⁸ mol/L dexamethasone sodium phosphate (Sigma-Aldrich, St. Louis, MO, USA), HEPES (20 mmol/L) and 100 μg/ml penicillin/streptomycin. The differentiation was evaluated by qRT-PCR and ALP activity detection.

ALP activity detection

Cells were washed with PBS buffer and lysed by shock freezing (−80°C). Diluted 1:1 in 1 × PBS, 100 mM p-nitrophenyl phosphate (Sigma) was added to each sample. After incubation at 37°C for 60 min, the reaction was stopped by adding 300 μL of 0.3 M NaOH and the liberated p-nitrophenol was measured at 405 nm. ALP activity values were normalized to total DNA concentration, which were determined by the Quant-iT PicoGreen dsDNA Assay (Invitrogen).

Prime PCR arrays

For the evaluation of osteogenic marker expression, the Biorad PrimePCR array (Development - Hedgehog and PTH signaling pathways in bone and cartilage development) was used, which consists of the most important markers for the osteogenic differentiation. Total RNAs, which were isolated from differentiated dental cells at day 7, were reverse-transcripted with the iScript™ Advanced cDNA Synthesis Kit for RT-qPCR (Biorad) according to the manufacturers protocol. PCRs were made with SsoAdvanced™ Universal SYBR® Green Supermix (Biorad) on the StepOne real-time PCR machine (Life Technologies, Carlsbad, CA, USA). Results were analyzed with the PrimePCR™ Analysis Software (Biorad), and the output is presented as Clustergrams. While red tiles signify a high gene expression, black/gray and green tiles show a middle gene expression and a low gene expression, respectively. Black tiles with a cross designate no gene expression.

Histology

Combinations of SB with dNC-PCs and AP with dental cells yielded from cell cultures after 7 days of osteogenic differentiation were fixed in 4% formaldehyde/0.1 M PBS at 4°C for at least 24 h. Tissues were decalcified with EDTA and subsequently dehydrated in an ascending series of ethanol and embedded in paraffin. Serial sections of 5 μm were cut in different planes for orientation and stained with hematoxylin-eosin (HE).