Figure 3

Morphological changes of osteocytic cells in response to normal and OA osteoblast-derived matrices. (A) Cells cultured in 2D showed rounded osteocyte morphology when grown on the OA matrices. Alizarin red combined with the haematoxylin staining showed a well aligned osteocytic cell distribution within normal matrix (NM) compared to OA matrix (OA-M). Magnification: 20X. (B) SEM (Scale bar: 100 μm for higher and 20 μm for lower magnification) and (C) confocal images (Scale bar: 50 μm for higher and 25 μm for lower magnification) indicated diversified morphologies of osteocytic cells grown on OA osteoblast derived matrices. Actin filaments are shown in red, microtubules in green and the nuclei in blue. (D) Representative micrographs of vinculin immunofluorescence (green) staining in osteocytic cells grown on normal and OA matrices. Scale bars: 25 μm. (E) Shape factor analysis of the confocal images (phalloidin only) was performed with the Metamorph software. At least 15 cells from three different experiments were analyzed. All images and graphs (A-E) are representative of experiments performed on osteocytic cells that were grown on four different normal and OA patient-derived matrices. Results are shown as mean ± SD. Student’s t test, P <0.05.