Adhesion strength of individual human bone marrow cells to fibronectin. Integrin beta1-mediated adhesion

The purpose of this work was to study the adhesion strength of individual bone marrow cells, using a micropipette aspiration technique. The adhesion strength of the primary human bone marrow cells to fibronectin-coated substrate, by blocking the beta1 integrin with and without antibodies, was also determined. Human bone marrow stromal cells of the second passage were seeded at a density of 500 cells/cm2 on two different substrates: plastic culture dish (PCD) and PCD coated with fibronectin. In short adhesion times (15-180 min) the cells attached without spreading and remained almost spherical. A negative pressure of about 3500 Pa was applied, through the micropipette, on individual bone marrow cells and the detach process was recorded. The tip of the micropipette was bent at an 130 degrees angle to the corpus of the pipette and it was manipulated to be on the upper side of the cell and vertically to the bottom of the plate. It was observed from the experiments that the cells exhibited smaller adhesion strength at early adhesion times (30-85 min). After 85 min the adhesion strength increased abruptly and remained relatively constant for the adhesion period from 85 to 180 min for all substrates. Monoclonal antibodies against integrin subunit beta1 were used for integrin blocking experiments. The data suggested that the attachment of osteoblasts to a plastic culture dish without fibronectin coating occurred earlier than to the one coated with fibronectin PCD. In longer adhesion time the coating with fibronectin increased the adhesion strength at 107%. Blocking of integrin beta1 with monoclonal antibody resulted in decrease of the adhesion strength at 49%.