Fig. 3.

Osteogenic differentiation of cultured human anulus fibrosus cells was induced with use of the osteogenic supplements. Once cells had reached 100% confluence, they were induced for up to four weeks and then were stained with alizarin red S or assayed for gene expression. Mineralization in induced cells is revealed by alizarin red S (×40) (A). Real-time reverse transcription-polymerase chain reaction analysis of osteogenesis-specific genes alkaline phosphatase (ALP) (B), runt-related transcription factor 2 (Runx2) (C), and osteocalcin (OC) (D) was performed, with 18S rRNA as an internal control (Con). The expression of all three genes was upregulated following the induction protocol in five samples. Error bars represent the standard deviation. OM = osteogenic media.