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[目的]制备一种新型抗结核多孔磷酸钙骨水泥缓释载体,研究其体内外性能。[方法]复乳溶剂挥发法制备利福平-聚乳酸-聚羟基乙酸共聚物微球(利福平-PLGA微球),测定载药量,包封率并行体外缓释实验。将微球以10%、20%、30%(W/W)的比例分别与磷酸钙骨水泥(CPCs)复合,制备载有利福平-PLGA微球的多孔磷酸钙骨水泥,测定材料的孔隙率及抗压强度,筛选出合适比例。将材料的浸提液与Wistar大鼠骨髓间充质干细胞(rBMSCs)共培养,以MTT法测定增殖率,成骨能力以试剂盒检测碱性磷酸酶的水平。将利福平-PLGA微球-CPCs(实验组)及利福平-CPCs(对照组)分别制成相同直径的圆柱状试件,分别植入新西兰大白兔的双侧股骨髁中,于术后2、4、8、12周取植入区旁的髁旁肌,高效液相色谱法测定局部组织药物浓度。术后12周取各组骨标本行组织切片观察,评价材料降解情况及骨组织生长情况。[结果]10%、20%、30%利福平-PLGA微球-磷酸钙骨水泥试件的总孔隙率,大孔率和抗压强度分别为(54.76±1.31)%、(13.67±1.62)%、(11.89±0.96)MPa;(63.76±1.35)%、(23.87±1.67)%、(4.8±0.68)MPa;(72.97±1.10)%、(37.87±2.08)%、(1.03±0.65)MPa。rBM-SCs在利福平/PLGA微球/磷酸钙骨水泥复合材料上生长较好,细胞增殖及碱性磷酸酶水平与空白对照组有明显差异(P<0.05),依据生物材料细胞毒性实验琼脂覆盖法测定材料细胞毒性为0-Ⅰ级。利福平缓释效果优于利福平-CPCs组(P<0.05),且利福平-PLGA微球-CPCs能在较长时间内保持在利福平的最低抑菌浓度(MIC)10倍以上。利福平-PLGA微球-CPCs试件植入体内12周时,材料降解明显快于利福平-CPCs试件组,实验组材料植入区的骨长入率为(84.56±1.47)%,明显高于对照组的(10.56±1.34)%(P<0.05)。[结论]利福平-PLGA微球可显著提高磷酸钙骨水泥的孔隙率促进其降解,微球降解形成互相连通的大孔隙(>50μm)显著提高了骨细胞的长入率,从而加速CPCs降解;同时利福平药物能较长时间缓释,在结核病灶局部能长时间维持有效的抗结核药物浓度,有望达到有效降低骨结核术后复发率的目的,可用于结核性骨缺损的修复与骨重建。
[Objective] To prepare a new anti-TB porous calcium phosphate cement sustained-release carrier and study its in vitro and in vivo performance. [Method] Rifampicin - polylactic acid - polyglycolic acid copolymer microspheres (rifampicin - PLGA microspheres) were prepared by double emulsion solvent evaporation method. The in vitro drug release rate and entrapment efficiency were measured in vitro. The microspheres were respectively compounded with calcium phosphate cement (CPCs) in proportions of 10%, 20% and 30% (W / W) to prepare porous calcium phosphate cement containing rifampicin-PLGA microspheres. Rate and compressive strength, select the appropriate ratio. The leaching solution of the material was co-cultured with Wistar rat bone marrow mesenchymal stem cells (rBMSCs), the proliferation rate was determined by MTT method, and the osteogenic capacity was detected by using the kit for alkaline phosphatase. Rifampicin-PLGA microspheres-CPCs (experimental group) and rifampicin-CPCs (control group) were made into cylindrical specimens of the same diameter, were implanted in the bilateral femoral condyle of New Zealand rabbits in surgery At 2, 4, 8 and 12 weeks after implantation, the paravertebral muscles adjacent to the area were taken and the local tissue drug concentrations were determined by high performance liquid chromatography. Twelve weeks after operation, each group of bone specimens were taken for histological observation to evaluate the material degradation and the growth of bone tissue. [Results] The total porosity, macroporosity and compressive strength of PLGA microspheres-calcium phosphate cement specimens were (54.76 ± 1.31)%, (13.67 ± 1.62)%, ), (11.89 ± 0.96) MPa, (63.76 ± 1.35)%, (23.87 ± 1.67)%, (4.8 ± 0.68) MPa, (72.97 ± 1.10)%, (37.87 ± 2.08)%, (1.03 ± 0.65) MPa. rBM-SCs grew well on rifampicin / PLGA microspheres / calcium phosphate cement composites, cell proliferation and alkaline phosphatase levels were significantly different from the blank control group (P <0.05), according to the cytotoxicity of biomaterials Agar plating method to determine the material cytotoxicity of 0-Ⅰ grade. The release of rifampin was superior to that of rifampicin-CPCs group (P <0.05), and the rifampicin-PLGA microspheres-CPCs could maintain the minimum inhibitory concentration (MIC) of rifampicin for a long time Times more. The material degradation of rifampicin-PLGA microspheres-CPCs was significantly faster than that of rifampicin-CPCs at 12 weeks after implantation. The rate of bone ingrowth in the experimental group was (84.56 ± 1.47)%, , Significantly higher than that of the control group (10.56 ± 1.34)% (P <0.05). [Conclusion] The rifampicin-PLGA microspheres can significantly improve the porosity of calcium phosphate cement and promote its degradation. The formation of macropores (> 50μm) which are interconnected by microspheres degrades the growth of osteocytes and accelerates the growth of osteoblasts Degradation; at the same time rifampicin drugs can be sustained for a longer time to release, in the local tuberculosis can maintain a valid long-term concentration of anti-TB drugs, is expected to achieve effective in reducing the recurrence rate of postoperative tuberculosis purposes, can be used for tuberculosis bone defect repair Reconstruction with bone.