Journal List > J Korean Soc Endocrinol > v.21(3) > 1063861

Oh, Baek, Lee, Oh, Kim, Han, Lee, Son, Kang, and Kang: The Effect of Oxidative Stress on the Proliferation and Differentiation of Human Bone Marrow Stromal Cell-Derived Osteoblasts

Abstract

Background

The objectives of our study were to assess the effects of oxidative stress on the proliferation, differentiation and apoptosis of human bone marrow stromal cell (BMSC)-derived osteoblasts and to explore pathways by which osteoblast cell apoptosis was induced.

Methods

Mononuclear cells including BMSCs were cultured to osteoblastic lineage. Different doses of hydrogen peroxide (H2O2) were added to the culture media. The colony forming units-fibroblastic (CFU-Fs) were stained with crystal violet and alkaline phosphatase (ALP). The MTT assay was done to see the effect of H2O2 on cell viability. The effect of H2O2 on osteocalcin gene expression was determined by RT-PCR. The matrix calcification measurement was performed. FACS analysis was performed to determine the osteoblasts apoptosis. Caspase-3, -8 and 9 activity assay and cytochrome c release were measured.

Results

The size and number of ALP (+) CFU-Fs were also decreased by H2O2 treatment. When compared with the control group, H2O2 significantly decreased the total number of cells of each culture well during MTT assay. H2O2 significantly diminished expression of osteocalcin mRNA. N-acetylcystein (NAC) blocked the diminution of cell viability and the inhibition of osteocalcin mRNA expression by H2O2. H2O2 reduced matrix calcification. FACS analysis revealed H2O2 increased percentage of apoptotic cells. Addition of H2O2 resulted in the increase of caspase-9 and -3 activity but not caspase-8, and release of cytochrome c to the cytosol.

Conclusion

These data suggest that, in primary human BMSCs, oxidative stress inhibits proliferation of stromal cells and inhibits the differentiation to osteoblastic lineage. In addition, oxidative stress induces apoptosis of human BMSC-derived osteoblasts and this may be mediated by mitochondrial pathway of apoptotic signal.

Figures and Tables

Fig. 1
Oxidative stress measurements. Increased accumulation of ROS in human bone marrow derived stromal cells stimulated by H2O2. Even number of cells were loaded with 20 µg/ml DCHF-DA for 20 minutes. The cells were then treated with 0.1 mM, 0.3 mM and 0.5 mM H2O2. DCF fluorescence was monitored over the course of 2 hour. The results are presented as a mean ± SEM, n = 5, each performed in duplicate.
*P < 0.05 compared with their control groups.
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Fig. 2
Effects of H2O2 on CFU-Fs and ALP (+) CFU-Fs formation. CFU-F formation from human bone marrow cells of the same donor. 4×105 mononuclear cells, which were derived from the bone marrow aspirated from the iliac crest, were plated in 10 cm Petri dishes in α-MEM with 20% heat-inactivated FBS, 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid, and grown for 15 days in the control (A) and presence of 0.1 mM (B) or 0.3 mM (C) H2O2. H2O2 was added after the attachment period (4 to 5 days). The colonies were fixed and stained with crystal violet (upper panel) and alkaline phosphatase (lower panel).
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Fig. 3
Effects of H2O2 on CFU-Fs formation. Effects of H2O2 on the number (A), the mean colony area (B) and the total colony area of CFU-Fs (C). The bone marrow was aspirated from the iliac crest of 9 young donors. Mononuclear cells were seeded in 10 cm dishes. 0.1 mM or 0.3 mM H2O2 was added 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.
*P < 0.05 compared with its control group.
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Fig. 4
Effect of H2O2 on cell viability in the secondary culture of human bone marrow derived stromal cells. The cells were treated with the vehicle or either doses (0.1 mM, 0.3 mM, 0.5 mM) of H2O2 or 5 mM N-acetylcysteine (NAC) or 5 mM NAC + 0.5 mM H2O2 for 48 hours. The MTT assay was done as described in the method. The results are presented as a mean ± SEM, n = 8, each performed in quadriplicate.
*P < 0.01 compared with its control group.
P < 0.05 compared with 0.5 mM H2O2 cultures.
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Fig. 5
Effect of H2O2 on osteocalcin gene expression 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 in secondary culture. The cells were then treated for 2 days with various dose of H2O2. 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 and mean data of 3 experiments were shown. The results are presented as a mean ± SEM.
*P < 0.05 compared with control.
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Fig. 6
Effect of H2O2 on matrix calcification. 5×104 mononuclear cells were plated in 24 well plates in α-MEM with 10% heat-inactivated FBS, 10 mM β-glycerophosphate, and 50 µg/mL ascorbic acid in secondary culture. After the attachment period, the stromal cells were treated with H2O2. The calcium content of the matrix was determined and normalized to the protein content. The normalized value obtained from the cells incubated under the control conditions was set at 100%. The results are presented as a mean ± SEM, n = 4, each performed in duplicate.
*P < 0.05 compared with control.
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Fig. 7
Effect of H2O2 on apoptosis of human bone marrow stromal cell-derived osteoblasts. Cells were treated with or without 0.5 mM H2O2 for 4 hours, and apoptosis was measured by flow cytometric analysis after staining with annexin V-FITC and propidium iodide. Apoptotic cells were annexin V-positive and PI-negative (A, control; B, 0.5 mM H2O2).
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Fig. 8
Effects of H2O2 on casase-3, casase-8, and caspase-9 activity. Human bone marrow stromal cell-derived osteoblasts were treated for 16 hours with 0.5 mM H2O2. For each analysis, extracts were prepared from 1×106 cells and incubated with caspase-3 (A), caspase-8 (B), or caspase-9 (C) fluorogenic substrates. Fluorescence was measured as released picomoles of AMC in a spectrofluorometer. Triplicated samples were compared with untreated cell lysates.
*P < 0.05 compared with control.
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Fig. 9
Effects of H2O2 on cytochrome c release into cytoplasm. Mitochondrial and cytosolic fractions were prepared from cells treated with 0.5 mM H2O2 for 5 hours. Western blot analysis was carried out with mouse anti-cytochrome c antibody.
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Table 1
Primers for semiquantitative reverse transcriptation polymerase chain reaction of the osteocalcin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
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