| Objective To investigate the effects of polymethyl methacrylate/nano-hydroxyapatite/collagen/magnesium-calcium alloy(PMMA/n HAC/Mg-Ca)composite materials on the osteogenic differentiation related proteins and genes RUNX2,OSX,ALP and OCN in MC3T3-E1 cells.Methods According to different materials,it was divided into PMMA/n HAC/Mg-Ca composite material(group A),PMMA/n HAC(group B),Mg-Ca alloy(group C)and blank control group(group D).Put the three groups of materials A,B and C into the α-MEM medium containing 10% FBS to pre-incubation one day,then MC3T3-E1 was directly inoculated on the three groups of material surface A,B and C,group D The cells were cultured with α-MEM medium containing 10%FBS.The cells in each group were cultured for 24 hours.After the culture was completed,the cell morphology was observed under the microscope;Culture mouse pre-osteoblasts MC3T3-E1 one day,three days,five days,the cell proliferation activity of the three groups was detected by the CCK-8 method;The cells on the surface of the composite material were observed by scanning electron microscope Adhesion morphology;Alkaline phosphatase activity was detected in four groups;The secretion levels of osteocalcin(OCN)and type I collagen in MC3T3-E1 cells were measured by enzyme-linked immunosorbent assay(ELISA);RT-PCR and Western blot were used to detect the expression levels of osteogenic differentiation related proteins and genes RUNX2,OSX,ALP and OCN.Results1.The CCK-8 test results showed that the cell proliferation activity of the four groups A,B,C and D increased with time;The cell proliferation activity of group A were higher than those of groups B,C and D on 1d,3d and 5d(P<0.05).2.The results of scanning electron microscopy showed that the cells were distributed on the surface of the materials in groups A,B and C,and the cells adhered and grew well on the surface of the materials in groups A and B,and the cells stretched obviously.The surface cells of group C material are round,and the pseudopodia extension is not obvious,and the number of cells adhering to the surface of materials in group C is significantly less than that in groups A and B.3.ALP detection results showed that ALP levels in group A,group B,group C and group D were gradually increasing with the increase of culture time;at the same time,there was no significant difference between group B and group C(P > 0.05),but it was higher than group D(P < 0.05),and ALP levels in group A were significantly higher than those in group B,group C and group D after 3,5and 7 days of culture(P < 0.05).4.ELISA analysis showed that the secretion levels of OCN and Col I in MC3T3-E1 cells cultured in groups A,B,C and D increased gradually;There was no significant difference between group B and group C(P > 0.05),but they were higher than group D(P < 0.05);The secretion levels of OCN and Col I in group A were higher than those in other groups on the 8th,10 th and 14 th day(P< 0.05).5.The results of RT-PCR showed that the m RNA expression level of bone differentiation genes RUNX2 and OSX in group B and C was not significant(P >0.05),but they were higher than those in group D(P < 0.05),with statistical significance;The m RNA expression levels of RUNX2 and OSX in group A were significantly higher than those in the above three groups(P < 0.05).6.Western blot analysis showed that there was no significant difference in the protein expression levels of RUNX2 and OSX in group B and group C(P >0.05),but they were higher than those in the blank control group(P < 0.05);The protein expression levels of RUNX2,OSX,ALP and OCN in group A were higher than those in group B,group C and group D(P < 0.05).Conclusion This experiment confirmed that PMMA/n HAC/Mg-Ca composite has good biocompatibility and bone differentiation ability.PMMA/n HAC/Mg-Ca composite can promote the adhesion and proliferation of MC3T3-E1 cells and the expression of osteogenic differentiation related proteins and genes RUNX2,OSX,ALP and OCN.It can be concluded that PMMA/n HAC/Mg-Ca composite has a positive effect on the osteogenic differentiation of MC3T3-E1 cells. |
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