Studying On Composites Made Of β Tricalcium Phosphate(TCP) Anti-tb Microspheres Drug-loaded Releasing In Vitro And In Vivo

Objective1.Independently, development for continuously controlled-release microspheres with big size(234μm) loaded two couplet anti-tb drugs in a bone tuberculosis lesions area.2.Finding of a favourble method to detect the concentration of two kinds of anti-tb drugs respectively in high performance liquid.3.The best option of Isoniazid(INH), Rifampicin(RFP) and PLGA(poly lactic acid/glycolic acid copolymer), which obtained from standard screening in the quality and coating rate, and detecting the concentration of drug released in vitro on each point time by HPLC. That summarizes releaseing characteristics of microspheres made of the PLGA, Isoniazid and rifampin in vitro.4.Preparing, icrospheres stent made of calcium phosphate(β-TCP) and composive PLGA-INH-RFP, AND observing characterization of stents, AND to study the adhesion and microspheres.5.Implantating the compoud stents into the experimental animals, and detecting the plasma concentration in venous plasma and osteogenesis. Methods1.Preparating microspheres containing drugs through double emulsion solvent evaporation(W/O/W) and studying microspheres characterization.2.Detection method: Hanbon C18 preparative column(4.6mm×250mm,5μm), mobile phase is methanol 0.04 mol.L-1 and disodium hydrogen phosphate(75:25, PH adjusted with phosphoric acid to 4.5), the rate of the flow is 1ml/m, 264 nm wavelength, and 10 u L injection volume.3.Studying the characteristics of microspheres, the determination of drug loadings, encapsulation efficiency and sustained releasing in vitro experiment.4.Composites are made of βTCP and microspheres that is in the proportion of 30%, 40%, and 50% of the mass of the microspheres respectively, using lyophilization to make drug-loaded stents, measuring material porosity and compressive strength, and observing their appearance in the electron microscope.5.Studying on drug metabolism in vivo, the drug-loaded scaffolds are implanted in healthy rabbits in bilateral femoral condyle, measuring venous plasma concentration of the drug respectively, after1, 4, 8, and 12 weeks. Researching in βTCP-PLGA drug-loaded microspheres scaffold metabolism and time of minimum inhibitory concentration in vivo. Observing bone formation in the bone densitometer, radiological, pathological. Results1.Independently developed microspheres loaded PLGA-INH-RFP, whose surface is finishing and color is red and orange uniformly, 90% of the microspheres’ diameter are 234mm.2.Contents of INH and RFP are detected by high performance liquid, establishing the standard curve and regression equation accurately, the retention time of two kinds of anti-TB drugs are 1.6 m and 3.6m, and the internal standard methyl testosterone is 7m. Peak area of INH, RFP and the internal standard ratio is the X-axis, and Y-axis is the concentration of the drug, establishing the standard curve and linear analysis. Linear regression equations are that INH:y=0.6062x+0.2696 R2=0.9993, RPF:y=0.5574 x +0.4678 R2 =0.9994.3.The best ratio combination of INH: RFP: PLGA mass is 20:15:60, measuring INH drug-loaded(%) is 7.8±0.13 and encapsulation efficiency(%) is 13.71±0.89, while RFP’s is 9.87±0.23 and 31.7±0.71. Experiments showed that microspheres releasing process is relatively stable, INH was 10.31% in the burst of microspheres, to 24 days acumulated up to 91.84%, but RFP’s is 7.13 % and 78.35% in vitro.4.the compund scaffold made of microspheres that is in the proportion of 30%, 40%, and 50% whose porosity respectively is 41.3%±1.01, 42.6%±0.87, and 44.5%±1.22 and compressive is 1.23±0.03 MPa, 0.96±0.04 MPa and 0.58±0.02 MPa. Observing Drug-loaded microspheres embedded in βTCP, whose particle size is substantially between 150-200mm, showing a sphere or spheroid in SEM.5.Implanting βTCP/PLGA-INH-RFP scaffold into the experimental animals, having measured blood drug inhibitory concentration for 12 weeks continuously, which can be maintained for 12 weeks. Imaging observation showed that,after eight weeks, observing increased shadow of bone mineral density in the femoral condyle defects of the experimental group, and generated entirely in the bone defect to 12 weeks. Conclusion:The microsphere with large site of PLGAF loaded INN-RFP, whose surface is smooth, and appearance is uniform. In vitro release process, that releases smoothly without burst release and antibacterial drug concentration maintains for 24 days. In vivo drug releasing process, βTCP-PLAG microspheres scaffold loaded drugs released in the bone defect slowly, maintaining an effective plasma concentration. At the same time, as the degradation of βTCP-PLAG microspheres scaffold loaded drugs, which has good osteogenesis in bone defects. Thus, that provides a theoretical basis for further clinical study of bone and joint tuberculosis treatment.