A periodic mechanical stress system was used, as previously described (21 (link)). The periodic mechanical stress culturing system (Taixing Experimental Instrument Factory, Jiangsu, China), comprised a reciprocating boost pump and a culture chamber, which provided a periodic mechanical stress with a pressure of 0–0.3 MPa and frequency of 0–1 Hz. The cells (1×105 cells/ml) were plated on slides (25×25 mm), and then underwent periodic mechanical stress treatment of 0–0.2 MPa and 0.1 Hz for 6 h (stress group) or were not exposed to stress (control group). The cells were then collected for detection of the expression levels of integrin α1, α5, αV, collagen 2A1 (Col2A1) and aggrecan, the phosphorylation of PLCγ1 at Tyr783 (PLCγ1-Tyr783) and cell migration of the NPs.
In certain experiments, NPs were transfected with either integrin α1 small interfering (si)RNA (siRNA group) or negative control siRNA (NC group), or remained untransfected (control group) prior to the administration of periodic mechanical stress (0–0.2 MPa; 0.1 Hz; 6 h). NPs were also pretreated with U73122 (Gibco; Thermo Fisher Scientific, Inc.), an inhibitor of PLCγ1, in DMSO at a concentration of 10 µM (U73122 group) or with DMSO alone (control group) prior to the administration of periodic mechanical stress. After 6 h of stress, the cells were collected for various assays.
In certain experiments, NPs were transfected with either integrin α1 small interfering (si)RNA (siRNA group) or negative control siRNA (NC group), or remained untransfected (control group) prior to the administration of periodic mechanical stress (0–0.2 MPa; 0.1 Hz; 6 h). NPs were also pretreated with U73122 (Gibco; Thermo Fisher Scientific, Inc.), an inhibitor of PLCγ1, in DMSO at a concentration of 10 µM (U73122 group) or with DMSO alone (control group) prior to the administration of periodic mechanical stress. After 6 h of stress, the cells were collected for various assays.