We investigated the effect of prolonged rhBMP-2 treatment on telomerase activity, replicative capacity and senescence of nucleus pulposus cells (NPCs) during long term culture. We obtained intervertebral disc (IVD) tissues with grade III degeneration from four patients. NPCs were isolated and passaged serially in three groups: control group, low-dose rhBMP-2 group and high-dose rhBMP-2 group until the cells reached the end of their replicative lifespan. Cumulative population doubling level (CPDL), telomerase activity and senescence markers, senescence-associated β-galactosidase (SA-β-gal), p53, p21, and p16, were assessed. The replicative capacity of NPCs in the high-dose rhBMP-2 group was decreased significantly compared to the control and low-dose rhBMP-2 groups. Mean telomerase activity was significantly greater in the high-dose rhBMP-2 group compared to the control and low-dose rhBMP-2 groups. The percentage of SA-β-gal-positive NPCs increased more rapidly in the high-dose rhBMP-2 group with passaging compared to the control and low-dose rhBMP-2 groups. The expression of p53, p21, and p16 in both low and high dose rhBMP-2 groups increased in all passages compared to the control group. We found that prolonged high-dose rhBMP-2 treatment increased telomerase activity of human NPCs, but decreased replicative capacity and lifespan in long term culture. We also found that excessive growth stimulation by prolonged high-dose rhBMP-2 treatment can promote NPCs senescence and result in growth arrest.
Keywords: bone morphogenetic protein-2; human; intervertebral disc; nucleus pulposus; replication; rhBMP-2; senescence; telomerase activity.