Acetylsalicylic Acid Inhibits Proliferation of Human Bone Marrow Stromal Cells and Matrix Mineralization

Abstract

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 microg/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 mcg/ml) at 72 h. Cell cycle analysis showed a decrease of BMSCs in the S and G2/M phases with a concomitant accumulation in G0/1 in ASA treated cells. The finding was associated to increased levels of some CDKIs, namely p27(Kip1) and p21(Cip1), whereas ASA did not affected p16(Ink4A) 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 (greater than 90 percent) of extracellular calcification at 200 microg/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.