The effect of simvastatin on the proliferation and differentiation of human bone marrow stromal cells

Abstract

Statins have been postulated to affect the bone metabolism. Recent experimental and epidemiologic studies have suggested that statins may also have bone protective effects. This study assessed the effects of simvastatin on the proliferation and differentiation of human bone marrow stromal cells (BMSCs) in an ex vivo culture. The bone marrow was obtained from healthy donors. Mononuclear cells were isolated and cultured to osteoblastic lineage. In the primary culture, 10(-6) M simvastatin diminished the mean size of the colony forming units-fibroblastic (CFU-Fs) and enhanced matrix calcification. At near confluence, the cells were sub-cultured. Thereafter, the alkaline phosphatase (ALP) activities of each group were measured by the time course of the secondary culture. Simvastatin increased the ALP activity in a dose dependent manner, and this stimulatory effect was more evident during the early period of culture. A 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay was performed during the secondary culture in order to estimate the effect of simvastatin on the proliferation of human BMSCs. When compared to the control group, simvastatin significantly decreased the proliferation of cells of each culture well. 10(-6) M of simvastatin also significantly enhanced the osteocalcin mRNA expression level. This study shows that simvastatin has a stimulatory effect on bone formation through osteoblastic differentiation, and has an inhibitory effect on the proliferative potential of human BMSCs.

Fig. 1

CFU-F production from human bone marrow cells of the same donor. 4×10⁶ nucleated cells, which were derived from the iliac crest, were plated in 10-cm Petri dishes in α-MEM with 20% FCS heat-inactivated, 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid, and grown for 15 days in the absence (A) or presence of 10^-8 M (B) and 10^-6 M (C) simvastatin. Simvastatin was added after the attachment period (4 to 5 days). The colonies were fixed and stained with crystal violet.

Fig. 2

Effect of simvastatin on the number (A) and the size (B) of CFU-Fs. The bone marrow was aspirated from the iliac crest of 9 young donors. Mononuclear cells were seeded in 10-cm dishes. Simvastatin was added at the appropriate concentrations after the attachment period. After 15 days, the cultures were fixed, stained, and the number and size of the CFU-Fs were determined. The results are shown as a mean±SEM. ^*statistically different from control cultures, p<0.05.

Fig. 3

Effect of simvastatin on matrix calcification. 8×10⁵ nucleated cells were plated in 6 well plates in α-MEM with 20% FCS heat-inactivated, 10 mM β-glycerophosphate, and 50 µg/mL ascorbic acid. After the attachment period, the stromal cells were treated with simvastatin. The calcium content of the matrix was determined as described in the text and normalized to the protein content. The normalized value obtained from the cells incubated under the control conditions was set at 100%, corresponding to 3.26±0.31 µg calcium/µg protein. The results are presented as a mean±SEM, n=11, each performed in duplicate. ^*statistically different from control cultures, p<0.05.

Fig. 4

Effect of simvastatin on the ALP activity in the secondary culture of human marrow stromal cells. After confluence, 1×10⁵ stromal cells per well were subcultured in α-MEM with 20% FCS heat-inactivated, 10 mM β-glycerophosphate, 50 µg/mL ascorbic acid in 6 well plates. The cells were treated with either the vehicle or 10^-8 or 10^-6 M simvastatin for the indicated time period. The ALP activity was measured as described in the text and normalized to the protein content. The results are shown as a mean±SEM. n=14, each performed in duplicate. ^*statistically different from control cultures, p<0.05. ^†, p<0.01.

Fig. 5

Effect of simvastatin on cell number in the secondary culture of human marrow stromal cells. The cells were cultured as described in Fig. 4 and treated with the vehicle or either doses (10^-8 or 10^-6 M) of simvastatin for the indicated time period. The MTT assay was done as described in the text. The results are presented as a mean±SEM, n=22, each performed in duplicate. ^†statistically different from control cultures, p<0.01.

Fig. 6

Effect of simvastatin on osteocalcin gene expression as determined by RT-PCR. The cells were seeded in 6 well culture plates at a density of 1×10⁵ cells per well and grown for 12 days. The cells were then treated for 2 days with the vehicle control or either doses (10^-8 or 10^-6 M) of simvastatin. 1 µg of the total RNA from each culture was reverse transcribed and subjected to PCR for osteocalcin analysis. GAPDH mRNA expression was also examined by RT PCR as an internal reference. Representative PCR band (A) and mean data (B) of 22 experiments. The results are presented as a mean±SEM, ^*statistically different from control cultures, p<0.05.