Figure 4

Functional and gene associated changes of osteocytic cells (OT) in response to normal (NM) and OA osteoblast derived matrices (OA-M). (A to E) Gene expression profiles of osteocyte markers were determined by qRT-PCR in osteocytic cells cultured either on normal or OA matrices for 48 hours. The results are expressed as relative gene expression after normalization using the 18srRNA and GAPDH housekeeping gene. (F to H) Protein localisation of DMP1, SOST and E11 osteocyte markers was determined by confocal microscopy (Scale bar: 50 μm for higher and 25 μm for lower magnification). (I) Cell adhesion of osteocytic cells cultured on normal and OA matrices was determined by staining with crystal violet. (J) Zymographic analysis shows the expression of MMP2 and MMP9 in osteocyte conditioned media collected after 24 hours of incubation after culturing on normal and OA matrices. (K) The flow cytometric apoptosis analysis of osteocytic cells on normal and OA matrices was determined by Annexin V/PI staining. All images and graphs are representative of experiments performed on osteocytic cells cultured on matrices derived from four different normal and OA patients. Results are shown as mean ± SD, * P ≤0.05 by Student’s t-test.