| Chapter one:The preparation of first-line anti-TB drug of core-shell structure microsphere by coaxial electrostatic spray technology and releasing characteristics of microsphere in vitro.ObjectiveTo explore the preparation conditions of core-shell microspheres, prepare the first-line anti-TB drug of core-shell structure microsphere, and evaluate its release in vitro.Materials and methodsPLGA and PLLA were used as carrier materials in this study. Different concentrations (5%,7.5%,10%and12.5%) of blank core-shell microspheres were prepared by coaxial electrostatic spray system (the rate of inner pump:1.5ml/h, the rate of outer pump:1.5ml/h, voltage:9.5-10.0KV). The internal structure and characterization of the microspheres were observed by confocal microscope and scanning electron microscope to obtain the parameters for preparation of best core-shell microspheres. According to the parameters and physicochemical properties of the first-line tuberculosis drug, all group microspheres were prepared as follows. Group A:blank core-shell microspheres; group B:isoniazid, ethambutol hydrochloride (shell), rifampicin, pyrazinamide (core); group C:isoniazid, ethambutol hydrochloride (core), rifampicin, pyrazinamide (shell). LC-MS/MS was used to detect the concentration of drugs at each time point in vitro.Results(1) The best concentrations of PLGA and PLLA:When the concentration of carrier materials at12.5%, the spinning phenomenon was appeared. The core-shell morphology can be observed by confocal microscopy in groups with concentration of5%,7.5%,10%. The microspheres presented as a cake at the concentration of5%. The surface of microsphere appeared like wrinkle at the concentration the7.5%. The microspheres with concentration of10%presented with smooth surface under electron microscope examination.(2) Diameter of microsphere:The diameter of Group A was18.07±1.174μm, group B was18.09±0.80μm, and group C was18.17±0.903μm.(3) The in vitro release experiment:In group B, rifampicin and ethambutol hydrochloride were released slower than isoniazid and pyrazinamide (P<0.05); In group C, four first-line anti tuberculosis drugs synchronous release.Conclusion(1)The best concentration of carrier materials for core-shell structure microsphere preparation was10%;(2) When water soluble drugs (isoniazid, ethambutol hydrochloride) were wrapped in the core, and lipid soluble drugs (rifampicin, pyrazinamide) were wrapped in the shell, the four drugs can achieve synchronous released. Chapter two:Effect of core-shell microspheres on the biological behavior of BMSCs of SD ratsObjectiveTo evaluat the biological safety of the microspheres by using BMSCs of SD rats in vitro.Materials and methodsBMSCs were isolated from the tibia of male SD rats (250-300g), and the generation of P3cells were identified and divided into control group, blank microspheres group and drug loaded microspheres group. The growth curve, activity, bone induction capacity and migration of BMSCs in different experimental groups were observed.Results(1) Identification of BMSCs:88.5%of the cells were in G0/G1phase of the cell cycle,6.02%were in G2/M phase, and5.53%were in S phase. The CD44was positive expressed in99.34%of BMSCs, and the CD90was positive expressed in98.25%of BMSCs. Both CD34and CD45were negative expressed in majority of BMSCs. Only1.47%of BMSCs expressed CD34and1.56%expressed CD45.(2) BMSCs growth curve, cell activity, bone induction capacity and migration:Cells in each groups showed "S" type growth (P>0.05). There were no significant difference between each groups in OD at3,6,9day by MTT examination (P>0.05). No significant difference presented in osteogenic induction after2weeks (P>0.05).ConclusionThe prepared core-shell structure microspheres with first-line anti tuberculosis drugs had no effect on capacity of growth, osteogenesis and migration of BMSCs cells in vitro, and had a good biocompatibility Chapter three:Study of tissue repair capacity of microspheres and the distribution of anti tuberculosis drugs in bone defects of SD ratsObjectiveTo evaluat tissue repair capacity of microspheres and the distribution of anti tuberculosis drugs by using bone defects of SD rats.Materials and methods2mm length bone cut out form SD rat tibia and was replanted after inactivation by liquid nitrogen. Bone defect of SD rat model was then established and all rats were randomly divided into normal group, sham operation group, blank microspheres group, drug loaded microspheres group. The animal were randomly selected for X-ray, HE staining, B-ALP, TRAP-5b examination at2,4,6weeks to observe bone tissue repair. The side effects of drugs were judged by LFTs and Renal Function Tests. By detecting the concentration of anti-tuberculosis drugs in local bone tissue, muscle and liver kidney, the drugs distribution in animal were judged.Results(1) Bone tissue repair:No bone delayed union or nonunion happened in blank microspheres group, sham operation group and drug loaded microspheres group at each time point by X examination, periosteum hyperplasia appeared in2weeks, new bone appeared in defect parts and to the lateral growth in4weeks, obvious bony connection formed in bone defect site at6week in HE. B-ALP, TRAP-5b in model’s animal is higher than the normal animal (P<0.05), but no significant difference existed in operation groups at each time point (P>0.05).(2) Liver and kidney toxicity:Compared with the normal animal group, liver and kidney function had no obviously increased in sham operation group, blank microspheres, and microspheres group animal (P>0.05).(3) Drug distribution:Four tuberculosis drugs were not detected in the liver, and kidney. But four tuberculosis drugs were detected at each time point in local bone tissue and muscle tissue, and concentration of drugs were higher than that’s MIC (P<0.05).Conclusion(1) Local implantation of core-shell microspheres with good biocompatibility had no effect on bone tissue repair,. |
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