Distribution of matrix secreted by normal and OA osteoblasts. (A) mRNA levels of bone regulating genes in primary normal and OA subchondral bone osteoblasts (SBOs) at passage one. Values were normalised to 18srRNA and GAPDH mRNA levels (n = 5 subjects). (B) Normal and OA SBOs were cultured in osteogenic differentiation medium. After de-cellularisation matrix mineralisation was determined by Alizarin red. Images were taken at 20X magnification. Total staining density of each well was quantified using ImageJ software (n = 3 subjects). (C to G) Immunoblotting of FN, versican, LN, OPN and COLIII, in normal (NM) and OA osteoblast derived matrices (OA-M). Band density was quantified using Image J software (n = 3 subjects). (H) SEM micrographs and COL1 immunostaining showing the morphology of matrix organisation secreted by normal and OA osteoblasts. Note the poor alignment of OA osteoblast secreted matrix (n = 3 subjects). Scale bar:100 μm. In vivo evidence of disrupted bone matrix in OA patients as determined by the back scatter SEM (BS-SEM) image technique. Scale bar: 200 μm and 100 μm. Results are shown as mean ± SD, * P ≤0.05 (Student t-test).