The Effects On RAW264.7 With Expression Of Rank By CPC/PLGA At The Early Stage Of Setting

OBJECTIVETo evaluate the degradation behavior of CPC/PLGA at the early stage of setting, in which the effect on proliferation and osteoclast-related factors RANK of RAW264.7, clarify the mechanism which PLGA can promote the degradation of CPC.METHODS1、The changes of calcium and phosphorus ions at the early stage of CPC and CPC/PLGA setting and the effects on the proliferation of RAW264.6Groups:Control group(RAW264.7 cell). CPC group (CPC+RAW264.7 cell). CPC/PLGA group (CPC/PLGA+RAW264.7 cell). CPC and CPC/PLGA were cocultured with RAW264.7 depending the groups. The proliferation of RAW264.7 was tested with MTT method at 1、3、5、7、9d respectively, making growth curve and calculating relative growth rates to study the effect on proliferation of RAW264.7 by CPC/PLGA composites.In the study of media characterization of CPC/PLGA composites, the groups were divided into cell-free and cell group, both groups included control group, CPC group and CPC/PLGA group. CPC or CPC/PLGA powder were spatulated together with liquid at ratio of 2:1, filled into the molds, which were put into plates, immersing with 5ml DMEM at 37℃,5%CO2 for 7 days, refreshing the media every day. The media was collected to test PH value and concentration of calcium and phosphorus with ICP-OES at 1、3、5、7d to clarify the changes of PH value and contain of calcium and phosphorus ions.2 、The effects on osteoclast-related factor RANK of RAW264.7 by CPC/PLGA composite at the early stage of settingThe groups were divided into control group(RAW264.7) 、 CPC group(CPC+RAW264.7)、CPC/PLGA group(CPC/PLGA+RAW264.7). After co-cultured with CPC or CPC/PLGA composites for 7 days, RAW264.7 were collected. The RNA of RAW264.7 were isolated for RT-PCR to test the expression of RANK. Through studying the effects on expression of RANK of RAW264.7 by CPC/PLGA composites at the early stage of setting, clarify the mechanism which PLGA can promote the degradation of CPC.RESULTS1 、The changes of calcium and phosphorus ions at the early stage of CPC and CPC/PLGA setting and the effects on the proliferation of RAW264.6According to the results of MTT, the proliferation of RAW264.7 cells got to the logarithmic growth phase on the third day, on the seventh day reaching growth peak. The relative proliferation rate of CPC and CPC/PLGA group were 79.43% and 66.67%, respectively. And then, the relative proliferation rate of these two groups increased gradually. CPC group reached the highest value 97.83% at the seventh day, while CPC/PLGA group reached the highest value 94.03% at the ninth day.In the study of cell-free group, the result of PH test shows that the PH of control group> PH of CPC/PLGA group> PH of CPC group. In seven day’s observation, the PH of media showed an undulate situation, without decreasing or increasing trends. In the study of cell group, each group showed a decreasing trend with the culture time. The results of changes of calcium ion in cell-group co-cultured with CPC or CPC/PLGA indicated that the concentration of calcium ion of control group was significantly higher than CPC group at 1d、3d、5d、7d (P<0.05). The phosphorus ion concentration in the cell-group co-cultured with CPC or CPC/PLGA indicated that the phosphorus ion concentration of control group is significantly lower than that of CPC group at 1d、3d、5d、7d (P<0.05). The phosphorus ion concentration of CPC group are significantly higher than that of control group (P<0.05), which the highest phosphorus ion concentration is (0.49±0.08) mmol/L.2、 The effects on osteoclast-related factor RANK of RAW264.7 by CPC/PLGA composite at the early stage of settingThe result of RT-PCR shows that there is no significant difference in expression of RANK between control group and CPC group at each time point. But at 6h and 5d, the expression of RANK in CPC/PLGA group are significantly higher than control group (P<0.05), which the highest value reached 95.35±29.43, showing at 6h. The result of Immunofluorescence shows that at 6h、3d、5d、7d, the mean fluorescence intensity values of RANK in CPC group and CPC/PLGA group are higher than control group (P<0.05)CONCLUSIONS1、 There is no significantly negative effects on proliferation of RAW264.7 in the early stage of CPC/PLGA setting. The PH value and concentration of calcium and phosphorus ion show different behavior between cell-free group and cell group, indicating that RAW264.7 may effect the behavior of PH value. The concentration of calcium and phosphorus ion at the early stage of CPC and CPC/PLGA setting show different behavior, indicating that RAW264.7 effect the concentration of calcium and phosphorus ion in different ways.2、CPC/PLGA can upregulate the expression of RANK in RAW264.7.