Abstract： Objective To investigate the mechanism underlying the response of osteoblast cell line MC3T3-E1 cultured on poly-(lactic-co-glycolic acid) and carboxylated multiwall (PLGA-MWCNTs) nanocomposite films.Methods After the MC3T3-E1 cells were cultured on PLGA-MWCNTs,poly-(lacticco-glycolic acid) film (PLGA) and tissue culture polystyrene (TCPS) for 72 h,RT-PCR was performed to determine the expression of bone-specific genes,including the alkaline phosphatase (ALP),bone sialoprotein (BSP) and osteocalcin (OCN).Western blotting,and immunofluorescence analysis was used to examine the cell viability and the expression of focal adhesion kinase (FAK).The expression of p-ERK1/2,pJNK and p-p38 MAPK proteins in each group of MC3T3-E1 cells was determined by Western blotting.Then,the three groups of MC3T3-E1 cells were added with 30 mmol/L PD98059 (a specific ERK1/2 inhibitor).After culture for 72 h,stained with MTT,Live/Dead and Texas Red-labeled phalloidin,the cells were observed for cell viability and production of actin fiber under confocal laser scanning microscope.Results The mRNA levels of ALP,BSP,and OCN in MC3T3-E1 cells on PLGA-MWCNTs films were higher compared with those on PLGA or TCPS (all P＜0.05),whereas these levels did not differ significantly between cells on PLGA and on TCPS (P＞0.05).The staining pattern of pFAK for MC3T3-T1 cells cultured on PLGA-MWCNTs films were more evenly distributed when compared with those on PLGA or TCPS,and the expression level of pFAK in cells on PLGA-MWCNTs films (2.3±0.3) was much higher than that in cells on PLGA (1.4±0.2) or TCPS (1.0±0.2) (all P＜0.05).The expression level of p-ERK 1/2 in MC3T3-E1 cells on PLGA-MWCNTs films (3.3±0.2) were nearly two times higher than that in the cells on PLGA (1.3±0.2) or TCPS (1.0±0.2) (all P＜0.05).There was no statistical difference in expression of p-JNK or p-p38 MAPK among groups (all P＞0.05).After culture with the ERK1/2 inhibitor PD98059 for 72 h,the cell viability and actin fiber production were reduced in all groups (all P＜0.05).The MC3T3-T1 cells on PLGA-MWCNTs films showed significantly reduced viability and actin fiber production compared with the cells on PLGA or TCPS.Live/dead staining revealed that the cells on PLGA-MWCNTs films were more readily to be red-stained,and these cells appeared less viable compared with those not added with PD98059 (0.33±0.02 vs 0.57±0.03,P＜0.05).Conclusion PLGA-MWCNTs films can promote cell differentiation and actin fiber formation in MC3T3-E1 cells by inducing the expression of bone-specific genes and activating the FAK and ERK 1/2.