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Heparin has been shown previously to potentiate the osteogenic effect of BMP-2.53 Equibiaxial mechanical stretching force (10% maximum strain, 0.5-Hz frequency) was applied to the cells for 24 or 48 hours, after which the cells were collected for gene expression analysis. As shown in Fig. 1A, B, mechanical stretching resulted in increased OPG gene expression at both time points. The increase in OPG expression after 24 hours was about sixfold, as determined by quantitative PCR (Fig. 1B). To study if OPG protein expression is also increased by mechanical stretching, we compared the concentration of OPG present in the culture medium at the end of stretching regimen to the concentration in the culture medium of unstretched control cells. As shown in Fig. 1C, mechanical loading resulted in a significant increase (about fourfold) of OPG concentration in stretch-conditioned medium. Our results indicate that mechanical stretching increases expression of both the OPG gene and the OPG protein. In contrast, these conditions led to a biphasic change in the expression of the OC and ALP genes. We observed an initial decrease in OC and ALP gene expression at 24 hours and an elevation of expression at 48 hours (Fig. 1D�CF). Expression of OPG, ALP, or OC was not detected in C2C12 cells cultured without osteogenic induction (data not shown). Although OPG was detected in C2C12 cells after 3 days of osteogenic induction, we could not detect RANKL protein by ELISA or RANKL mRNA by RT-PCR (data not shown). Because ALP and OC gene expression is considered to be a hallmark of osteogenesis and OPG is secreted primarily by mature osteoblasts, the finding of an increase in OPG expression and concomitant decrease in OC and ALP expression in C2C12 cells after 24 hours of stretching was intriguing. We therefore examined the gene expression profile of C2C12 cells cultured under mechanical stretching to further elucidate which genes and signaling pathways may be involved. For the microarray analysis, C2C12 cells cultured in BM induction conditions for 3 days were either subjected to mechanical stretching for 24 hour or kept unstretched. The gene expression profiles of these cells were analyzed using Affymetrix mouse exon arrays. Of the more than 20,000 mouse genes represented by probes on the array, expression of 2779 genes was significantly altered (p