Acetylsalicylic acid (ASA) and other non-steroidal anti-inflammatory drugs have been shown to potentially inhibit bone healing and bone formation in both animal and clinical studies. Due to the extensive diffusion of ASA-based long-term therapies, the implications of such a side-effect are of interest in all types of bone surgery, including bone grafting procedures and dental implant placement. In this study, we investigate the effect of ASA at therapeutic concentrations on the proliferation and osteogenic differentiation of human bone marrow stromal cells (BMSCs). Primary cultures of BMSCs were isolated and expanded. Their proliferation in response to ASA 50, 100 and 200 μg/ml was evaluated by MTT assay and 3H-thymidine incorporation. Cell cycle machinery was also investigated by FACS and analysis of inhibitors of cyclin-dependent kinases (CDKIs). ASA inhibited BMSC proliferation and DNA synthesis in a dose-dependent manner down to 60% of control (ASA 200 μg/ml) at 72 h. Cell cycle analysis showed a decrease of BMSCs in the S and G2/M phases with a concomitant accumulation in GO/1 in ASA treated cells. The finding was associated to increased levels of some CDKIs, namely p27Kip1 and p21Cip1, whereas ASA did not affected p16Ink4A level at any of the concentrations employed. The matrix mineralization, that represents the major feature of the osteogenic commitment, was assessed by a specific staining procedure (von Kossa) and by calcium content determination. Both the methods demonstrated an extensive reduction (>90%) of extracellular calcification at 200 μg/ml ASA. On the basis of our results, we can hypothesize that the widely reported inhibition of bone healing by ASA might be sustained both by a direct anti-proliferative effect on BMSCs and by an alteration of the extracellular calcification.
TRPC6 regulates cell cycle progression by modulating membrane potential in bone marrow stromal cells