Figure 8
Histological analysis of scaffolds perfused with oscillating perfusion mode. Scaffold blocks were perfused in a bioreactor type2_enc+_osc with 0.5 × 106 cells/ml, followed by 14-day incubation in a roller culture. (A) Scaffolds were stained with Giemsa's Azure-Eosin-Methylene Blue Solution and Toluidine Blue-Methylene Blue-Pyronine solution showing multi-layered stem cell accumulations extending over the surface and the pores of the bone graft substitute. (B) Autofluorescence image of the scaffold block representing background fluorescence visualized by fluorescence microscopy equipped with FITC filter set. Immunohistochemical staining of osteogenic differentiation markers (green) Collagen I (C, D), Osteopontin (E) and Bone Sialoprotein (F) counterstained with Hoechst (blue) to visualize cell nuclei by confocal LSM. White arrows represent newly expressed marker proteins by ASC. Collagen I is expressed in all stem cell layers as well as Osteopontin and Bone Sialoprotein, indicating differentiation of ASC into the osteogenic lineage (LSM 780, Carl Zeiss Microscopy GmbH). (G) Expression analysis of three osteogenic marker genes (i) osteopontin (OPN), also known as bone sialoprotein I, (ii) bone morphogenetic protein 2 (BMP-2), and (iii) the distal-less homeobox 5 gene (DLX-5). Notably, transcript levels of all three osteogenic differentiation genes do not differ significantly between the positive control (chemically differentiated ASCs) and the perfused cells within the scaffold.
