FIGURE 4.

Senescent osteocyte-secreted factors aggravate osteocyte differentiation and mineralization induced by LPS. A) Differentiated Ocy454 cells were exposed to the various stimuli shown in the figure and described in the Methods, and the expression of recognized osteogenic markers was measured by RT-qPCR. Data are represented as mean ± SEM (n = 6). B) A subset of cells from panel A was used to assess bone mineralization capacity using ARS staining. C) A parallel experiment was performed using primary alveolar osteocytic cells. They were cultured under the same conditions for 15 days, and mineralized nodule formation evaluated using ARS staining. Note that in both experiments decreased mineralization produced by LPS is notably aggravated by the addition of SCM, which is reversed by SB202190. Representative images for both cell models were obtained using a digital camera. D) ARS elution was performed on the Ocy454 cells (n = 6) and normalized ARS absorbance is displayed. Significant changes are indicated by: * compared with control, †LPS compared with LPS+SCM, and ‡ for LPS+SCM compared with LPS+SCM+SB202190. Various levels of significance are based on the number of each respective symbol (one symbol P < 0.05; two symbols P < 0.01; three symbols P < 0.001)