| OBJECTIVETo evaluate the degradation solution of CPC/PLGA in its effect on proliferation and related factors RANKL and OPG of osteoblast, and clarify the mechanism that PLGA can promote the degradation of CPC and provide theoretical basis.METHOD:Part1.the choose between PLGA,CPC/PLGA1. the change about concentration of lactic acid and glycolic acid of PLGA and CPC/PLGA during the degradation:Step1. Making PLGA degradation solutionTaking 400mg PLGA in 5 mL DMEM medium, at predetermined intervals(15,30,45,60,75d), the supernatant was withdrawn completely. Stored in an incubator at 100% relative humidity and 37?.Detected the lactic acid and glycolic acid by high-performance liquid chromatography (HPLC).Step2. Making CPC/PLGA degradation solutionTaking 400mg CPC/PLGA composite mixing with Na2HPO4(200?l),each composite was soaked in 5 mL DMEM medium, at predetermined intervals (15,30,45,60,75d), the supernatant was withdrawn completely. Placed the mixed composites in cylindrical molds to store in an incubator at 100% relative humidity and 37?.Detected the lactic acid and glycolic acid by high-performance liquid chromatography (HPLC)2. Detected the concentration of lactic acid and glycolic acid by high-performance liquid chromatography (HPLC). Then Select the suitable concentration of cell proliferation by MTT. According to the experimental results of MTT experiment to choose suitable concentration of glycolic acid and lactic acid for the experimental degradation solution to carry out subsequent experiments.Part2. The influence of Calcium phosphate cement/poly(lactic -co-glycolic acid)microspheres on osteoblastsTaking the neonatal rat calvaria bone and obtain primary osteoblasts by two-step digestion method.The basic culture medium was DMEM medium, the rats were divided into four groups: 1.The control group (osteoblasts); 2.The CPC group (CPC + osteoblasts); 3.The PLGA group (PLGA + osteoblasts); 4.The CPC / PLGA group (CPC / PLGA + osteoblasts).Mixed each extract above with osteogenic cell ,and then cultured the four different mixed composites.Cultured for 1,3,5,7,9d in vitro, proliferation of osteoblast was determined by MTT, differentiation of osteoblast was determined by the following assays:Alkaline phosphatase (ALP) assay, alizarin red staining, and Real-time reverse transcriptase-polymerase chain reaction (Real-time RT-PCR).RESULTS1.The concentration of lactic acid and glycolic acid in the PLGA gradation solutionPLGA released (3.996±0.57)mg/L lactic acid and (23.685±0.54)mg/L glycolic acid on 45th day.CPC/PLGA released (2.028 ±0.049)mg/L glycolic acid on 15th day.2. The concentration of lactic acid and glycolic acid in the CPC /PLGAgradation solutionThere was an gradual increasing trend on the concentration of glycolic acid from (2.028 ±0.049)mg/L on the 15th day and it reached at its peak,about(7.320±0.034) mg/L on the 60th day. And then it gradually fell down to 4.256 mg/L on the 75th day. Meanwhile, there was only a minor fluctuation of the concentration of lactic acid.It was about (0.171 ±0.024 ) mg/L on the 15th day,and it declined to (0.14±0.051) mg/L on the 45th day and finally (0.091 ±0.038)mg/L on the 75 th day.MTT results showed that osteoblasts was in the logarithmic growth phase from the third day, and the growth curve flattened from the seventh day in which phase the cell proliferation rate slowed down. Compared with CON group, the CPC group and the CPC / PLGA group showed the proliferative capacity enhancement (P <0.05); Compared with CON group, PLGA group's proliferation declined(P <0.05). Compared with the other three groups, CPC /PLGA group , after 21-day culture, the precipitate of calcium nodules was not obvious. The alkaline phosphatase activity in CPC / PLGA group on 3rd,5th,7th day were higher (P <0.05)than that in the control group, and significantly higher than PLGA group (P <0.01); the alkaline phosphatase activity in PLGA group is lower than the control group (P <0.05). RT-PCR results showed that: the expression of CPC / PLGA group on 3rd,5th,7th day osteocalcin were higher than which in the other three groups (P <0.05); that in the PLGA group were lower than the control group (P <0.05) on 5th,7th day .CPC/PLGA group and PLGA group presented higher RANKL expression than that in control group(P <0.05) both on 1st,3rd,5th,7th day; There was no obvious difference on RANKL expression between that in CPC group and that in CON.Compared with CON group, CPC/PLGA group presented significantly higher RANKL expression (P<0.01) on the 3rd day ;PLGA group presented significantly higher RANKL expression than control group (P <0.01) on the 7th day. CPC group presented higher OPG expression than control group (P <0.05) both on 1st,3rd,5th day,lower than control group (P <0.05) on the 7thd; CPC/PLGA group presented significantly higher OPG expression than control group (P <0.01) on the 7thd.PLGA group presented lower OPG expression than control group (P <0.05) both on 1st,3rd,5th,7th day.CONTROLCLUSIONCPC/PLGA extract can promote the expression of RANKL in the early stage of proliferation of osteoblasts;... |
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