新型抗结核多孔磷酸钙骨水泥缓释载体的制备与性能研究

Study on preparation and properties of novel anti-TB porous calcium phosphate cement

[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 develop a novel anti-TB porous calcium phosphate cements（CPCs） by incorporating the rifampicin-loaded poly（lactic-co-glycolic acid） microspheres（RFP-PLGA Ms） and to evaluated the performance of the composite as a bone substitute in vitro and in vivo.[Methods]RFP-PLGA Ms were prepared using a double-emulsion solvent-extraction [（water-in-oil）-in-water] technique.Drug-carried rate,envelopment rate and the characteristics of drug release in vitro were developed.Porous CPCs were developed using RFP-PLGA Ms with their different weight ratios（10%,20%,30%）.The porosity and the compression strength of the three composites were measured to determine the proper weight ratio of RFP-PLGA Ms.Wistar rat bone marrow stromal stem cells were also cultured with different leaching liquor of three kinds of materials（20%RFP-PLGA-CPCs,RFP-CPCs,CPCs）.Cell viability analysis was performed by MTT assay,and alkaline phosphatase was measured with alkaine phosphatase kit.At 2,4,8 and 12 weeks after implanted in the rabbit＇s femoral condyle,the muscles beside the femoral condyle were obtained and RFP in the muscles were measured by high performance liquid chromatography（HPLC）.At 12 weeks the samples with the materials were obtained,and evaluated by pathological anatomy.[Results]For the 10%,20%,30% of RFP-PLGA-CPCs composites,the total porosity,large porosity and compressive strength were（54.76 ± 1.31）%,（13.67 ± 1.62）% and（11.89 ± 0.96） MPa;（63.76 ± 1.35）%,（23.87 ± 1.67）% and（4.8 ± 0.68） MPa;（72.97 ± 1.10）%,（37.87 ± 2.08）% and（1.03 ± 0.65） MPa.The material cytotoxicity was 0-Ⅰ level by agar overlay test.RFP release in RFP-PLGA-CPCs group was better than in RFP-CPCs group（P0.05）,and the former maintained the RFP level at a minimum inhibitory concentration（MIC） over 10 times for a longer period.At 12 weeks RFP-PLGA-CPCs were implanted,and the degradation of RFP-PLGA-CPCs was significantly faster than in the control group.The former of bone ingrowth rate was（84.56 ± 1.47）%,significantly higher than（10.56 ± 1.34）%（P0.05）.[Conclusion]RFP-PLGA Ms can significantly increase CPC porosity and promote its degradation.RFP can release for a long time and maintain an effective local anti-tuberculosis drug concentration,thus reducing the recurrence rate of bone tuberculosis.It can be used to repair tubercular skeletal defect.