Effects of extracellular calcium on the proliferation and differentiation of porcine osteoblasts in vitro

We examined the effects of various extracellular calcium concentrations on DNA content, procollagen type I carboxy-terminal propeptide (PICP) release (reflects type I collagen synthesis), and alkaline phosphatase activity of porcine osteoblasts. Osteoblasts seeded in control medium (2.2 mM calcium) were transferred to low (0.5 or 1 mM) calcium medium or to high (3, 5, 7, or 10 mM) calcium medium at different stages of the culture period and for different incubation times. When osteoblasts were transferred to low or high (3 or 5 mM) calcium medium 1 or 2 days after plating and kept in that medium until the end of the culture period, PICP release was inhibited, but DNA content and alkaline phosphatase activity were unchanged, except in 5 mM calcium, which inhibited alkaline phosphatase activity. Short-term culture of subconfluent and near-confluent osteoblasts in 7 or 10 mM calcium for 48 h inhibited DNA content. DNA content returned to normal levels when cells were transferred back to control medium, whereas alkaline phosphatase inhibition induced by 5, 7, or 10 mM calcium was not reversible. Short-term culture in high calcium media did not affect PICP release. Thus, in porcine osteoblasts, low and high extracellular calcium concentrations affect DNA content, PICP release, and the expression of osteoblastic phenotype markers (alkaline phosphatase activity). These effects are dependent on the duration of calcium treatment and the state of differentiation of the osteoblasts.