Analysis of osteocyte differentiation. A) The picture shows a representative image of an osteon formation following osteocyte differentiation of MSCs. Image taken on an upright inverted microscope with a 20× objective. The graph represents the expression follow up of osteopontin (B) and osterix (C) during osteocyte differentiation of MSCs treated with OS or HS. mRNA levels were normalized with respect to GAPDH, which was chosen as an internal control. Each experiment was repeated at least three times. The histogram shows the mRNA expression levels. They are expressed as arbitrary units (*P <0.05). D) The picture shows Alizarin red staining of MSCs treated with OS or HS and then induced to differentiate into osteocytes. Control: cells not induced to differentiate. The Alizarin red staining intensity for each cell culture dish was acquired with a CCD camera and analyzed with Quantity One 1-D analysis software (Bio-Rad). We calculated the sum of the fluorescent pixel values of stained cells and then determined the average fluorescent pixel intensity. HS, healthy weight sera; MSCs, mesenchymal stem cells; OS, overweight sera.