Axiovision imaging software 4

Osteogenic differentiation was initiated by changing the growth medium to the Mesenchymal Stem Cell Osteogenic Differentiation Medium (C-28013, Promocell, Heidelberg, Germany) in ASC, BMSC, and POB of Group 1 (control). Alizarin Red staining was performed as described previously to confirm osteogenic differentiation in the control and test groups [8 (link)]. For this purpose, alizarin fluorescence was visualized by Axio Scope.A1 fluorescence microscope (Carl Zeiss, Jena, Germany) using AxioVision Imaging Software 4.8.2.0 (Carl Zeiss, Jena, Germany). Osteogenic marker expression was determined by RT-PCR using Primer against the genes of osteopontin (OPN) and bone morphogenetic protein 2 (BMP-2) [22 (link)].

For the long-term experiments, Bio-Oss blocks were perfused in bioreactor type 2_enc+ with 0.4 × 10⁶ cells/ml for 60 min using the Ibidi pump system (ibidi GmbH). An oscillating perfusion rate of 0.5 ml/min was chosen. After perfusion, scaffolds were incubated in a roller culture flask for 14 days and the cell culture medium was changed every three days. All experiments were performed in triplicates. The initial osteogenic differentiation was analyzed by Alizarin red staining according to manufacturer's protocol (Osteogenesis assay kit, #ECM815, Millipore, Burlington, MA, USA). Briefly, cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, washed again, and incubated with 0.1% Alizarin red for 30 min. Finally, cells were washed until red stained structures were clearly visible. Visualization and imaging were carried out with the Axioscope A1 microscope using AxioVision Imaging Software 4.8.2.0 (both from Carl Zeiss Microscopy GmbH).