Cyber shot 3

Morphological properties as well as the mitotic- and binucleate index were monitored and analyzed after seeding pulp fibroblasts in a cell density of 1x 10⁵ cells/ml on glass cover slips in 24 well plates (greiner bio-one, Frickenhausen, Germany). After cell adherence the culture media were substituted with the above specified Biodentine^® suspensions or fresh culture medium as control. For binucleate induction cells were incubated with 1μg/ml cytochalasin B (Applichem, Darmstadt, Germany). Cell morphology was photodocumented every 2h for 24h with the incubator microscope unit IncuCyte (EssenBioScience, Hertfordshire, UK). After 24h cell were fixed with 4.5% formalin (Carl Roth, Karlsruhe, Germany) for 10min and washed with PBS⁺⁺. Thereafter cell nuclei were counterstained with DAPI (Roche, Mannheim, Germany). Photographs were taken with a digital camera (Sony Cyber Shot 3.3, Sony, Cologne, Germany) connected to a Zeiss Axioskop (Zeiss, Oberkochen, Germany). The mitotic index and binucleate index were determined evaluating the number of cells showing mitotic phases (pro-metaphase to telophase) or binucleates (S1 Fig). Per condition and culture 1000 to 1089 cells were blindly analyzed.