Osteogenic Potential Modulation by miR-29a

We assessed the impacts of miR-29a-AS and pre-miR-29a on the osteogenic potentials of the harvested primary bone marrow mesenchymal cells. Briefly, primary bone marrow mesenchymal cells harvested from femurs were mixed with red blood cell lysis buffer (11814389001; Sigma-Aldrich, St. Louis, MO, USA) to isolate mononuclear cells. Upon incubating mononuclear cells in Dulbecco’s modified Eagle medium with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) overnight, adherent cells were collected and incubated in osteogenic medium (10⁵ cells/well, 24-well plates) (StemPro™ Osteogenesis Differentiation Kit; Thermo Fisher Scientific, Waltham, MA, USA) for 18 days. The extent of mineralization was assessed employing von Kossa Stain Kits (ab150687; Abcam, Cambridge, UK), following instructor’s protocol. Calcium in mass deposits would be stained with black color by the Kit, and the black color-stained area (mm²/filed) of von Kossa-stained mineralized matrices in each ×125 magnification field were measured applying light microscopy (Zeiss Image Analysis System, Oberkochen, Baden-Württemberg, Germany) [19 (link),21 (link)].