PMC

Effect of dipyridamole on osteoblast differentiation is diminished in A_2BR-knockout mice. Mouse MSCs derived from wild-type mice were cultured with osteogenic medium in the presence or absence of indicated concentrations of 0.5 μM dipyridamole or adenosine receptor agonists or antagonists for 10 d for Alizarin Red staining (A, top panel) or for 4 d for ALP activity assay (B, left panel). MSCs derived from A_2B-knockout mice were cultured with osteogenic medium in the presence or absence of 0.5 μM dipyridamole or indicated concentrations of adenosine receptor agonists or antagonists for 21 d for Alizarin Red staining (A, bottom panel) or for 4 d for ALP activity assay (B, right panel). ALP activity is expressed as nanomoles p-nitrophenol formed per 10⁴ cells; values are means ± sd of 4 independent experiments. **P < 0.01, ***P < 0.001.

Effect of dipyridamole on osteoblast differentiation is diminished in CD39-knockout mice. Mouse MSCs derived from wild-type mice were cultured with osteogenic medium in the presence or absence of 0.5 μM dipyridamole, 1 μM ZM 241385, or 1 μM MRS 1754 for 10 d for Alizarin Red staining (A, top panel) or for 4 d for ALP activity assay (B, left panel). MSCs derived from CD39-knockout mice were cultured with osteogenic medium in the presence or absence of indicated concentrations of 0.5 μM dipyridamole, 1 μM ZM 241385, or 1 μM MRS 1754 for 10 d for Alizarin Red staining (A, bottom panel) or for 4 d for ALP activity assay (B, right panel). ALP activity is expressed as nanomoles p-nitrophenol formed per 10⁴ cells; values are means ± sd of 3 independent experiments. *P < 0.05, **P < 0.01.

Suppression of human osteoclast formation from bone marrow precursors by A₁R-selective antagonist rolofylline or A_2BR agonist Bay 60–6583. Human BMMs derived from patients with MM (A) or healthy control subjects (B) were cultured with hM-CSF and hRANKL (30 ng/ml each), with or without various concentrations of rolofylline or Bay 60–6583 or 1 μM MRS 1754 for 7 d in 48-well plates for TRAP staining (top panels). Numbers of TRAP-positive multinuclear cells containing >3 nuclei (TRAP⁺ MNC) were counted (bottom panels). Values are shown as means ± sd of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. RANKL + M-CSF cells.

Increased mRNA expression of osteocalcin and osterix during osteoblast differentiation by direct A_2B stimulation, as well as treatment with the nucleoside transport inhibitor dipyridamole via A_2BR. Human MSCs derived from healthy bone marrow were cultured with osteogenic medium in the presence or absence of 0.5 μM dipyridamole or indicated concentrations of adenosine receptor agonists or antagonists for 7 d prior to RNA extraction and real-time PCR for osteocalcin (A) and osterix (B). β-Actin served as PCR control. Relative expression was calculated relative to stromal cells without osteogenic medium (fold value 1). Values are shown as means ± sd of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Enhanced osteoblastic differentiation in human MSC cultures by direct A_2B stimulation as well as treatment with the nucleoside transport inhibitor dipyridamole via A_2BR. Human MSCs derived from patients with MM (A) or healthy control subjects (B) were cultured with osteogenic medium in the presence or absence of 0.5 μM dipyridamole or indicated concentrations of adenosine receptor agonists or antagonists for 14 d for ALP activity assay (bottom panels), or for 21 d for Alizarin Red staining (top panels). ALP activity is expressed as nanomoles p-nitrophenol formed per 10⁴ cells; values are means ± sd of 3 independent experiments. *P < 0.05, **P < 0.01.