3D打印载PaMZ/BMP-2的nHA抗结核人工骨体外缓释及抗结核性能研究

Study on in vitro sustained release and anti-tuberculosis performance of 3D printed PaMZ/BMP-2-loaded nHA artificial bone

中文关键词:  脊柱结核  人工骨  PaMZ  缓释  抗结核

英文关键词:Spinal tuberculosis  Artificial bone  PaMZ  Sustained release  Anti-tuberculosis

中文摘要:

  【摘要】 目的：观察3D打印的载普托马尼（Pretomanid，Pa）、莫西沙星（Moxifloxacin，M）、吡嗪酰胺（Pyrazinamide，Z）抗结核药物组合（PaMZ）和骨形态发生蛋白-2（bone morphogenetic prtoein-2，BMP-2）的纳米羟基磷灰石（nano-Hydroxyapatite，nHA）抗结核人工骨在体外的缓释情况及抗结核性能。方法：取3D打印的载PaMZ/BMP-2的nHA抗结核人工骨和空白人工骨各5份，分别浸泡于磷酸盐缓冲液（phosphate buffer saline，PBS）中，采用高效液相色谱法于1d、3d、5d、7d、14d、28d、42d、56d、70d和84d共10个时间点检测二者浸提液中药物浓度。用84d的浸提液制备改良罗氏培养基，实验分为3组，实验组（A组）为含载PaMZ人工骨浸提液培养基，阳性对照组（B组）为含利福平培养基，阴性对照组（C组）为含空白人工骨浸提液培养基，分别培养结核分枝杆菌H37Rv菌株和临床分离的利福平耐药结核菌株，每组20个样本，观察培养阳性率。取载PaMZ人工骨和空白人工骨各50mg分别装入2种结核菌液的培养瓶内作为实验组和对照组，不加材料的含菌试管为空白组，于0d、1d、3d、5d、7d、10d和14d共7个时间点取菌悬液培养4周，根据菌落计数绘制时间杀菌曲线。结果：载PaMZ人工骨可有效缓释84d，Pa在84d时浓度为43.92±1.37μg/ml，累积释放率为（71.00±0.71）%；M在84d时浓度为180.22±12.36μg/ml，累积释放率为（96.01±1.54）%；Z在84d时浓度为472.05±65.39μg/ml，累积释放率为（96.39±2.22）%。H37Rv菌株培养阳性率：A组5%，B组0，C组100%，C组与A、B组比较有统计学差异（P＜0.05）；临床分离的利福平耐药结核菌株培养阳性率：A组5%，B组95%，C组100%，A组与B、C组比较有统计学差异（P＜0.05）。时间杀菌曲线显示实验组2种菌株在各时间点的菌落计数均位于对照组和空白组下方，实验组3d开始表现出明显的抑菌性能，10d后未培养出结核杆菌。结论：3D打印载PaMZ/BMP-2的nHA抗结核人工骨在体外可稳定缓释新型抗结核药物组合PaMZ达84d，在84d内可有效抑制敏感型和利福平耐药结核菌的生长，具有良好的抗结核性能。

英文摘要:

  【Abstract】 Objectives: To observe the in vitro sustained release and anti-tuberculosis performance of 3D printed nano-Hydroxyapatite(nHA) anti-tuberculosis artificial bone loaded with PaMZ a novel triple anti-tuberculosis drug combination of Pretomanid(Pa), Moxifloxacin(M), Pyrazinamide(Z) and bone morphogenetic prtein 2 (BMP-2). Methods: 5 copies of the 3D printed PaMZ/BMP-2-loaded nHA artificial bone and blank artificial bone were immersed in phosphate buffer saline(PBS) simulated body fluid, respectively. And drug concentrations of the extracts at 10 time points of 1d, 3d, 5d, 7d, 14d, 28d, 42d, 56d, 70d and 84d were detected by high performance liquid chromatography. The modified Roche medium was prepared with the extract at 84d. The experiment was divided into 3 groups: the experimental group(group A) of the medium containing the PaMZ-loaded artificial bone extract, the positive control group(group B) of the medium containing rifampicin, and the negative control group(group C) of the medium containing blank artificial bone extract. The H37Rv strain and the clinically isolated rifampicin-resistant tuberculosis strain were cultured respectively, with 20 samples in each group, and the positive rate of culture was observed. 50mg of PaMZ-loaded artificial bone and 50mg of blank artificial bone were respectively put into culture flasks containing 2 kinds of tuberculosis liquid as experimental group and control group, and the tuberculosis-containing test tube without material were the blank group. The bacterial suspension was taken at 0d, 1d, 3d, 5d, 7d, 10d, 14d time points to culture for 4 weeks, and the time-kill curve was drawn according to the colony count. Results: The PaMZ-loaded artificial bone effectively sustained-released for 84 days. The concentration of Pa at 84 days was 43.92±1.37μg/ml, and the cumulative release rate was (71.00±0.71)%; the concentration of M at 84 days was 180.22±12.36μg/ml, and the cumulative release rate was (96.01±1.54)%; the concentration of Z at 84 days was 472.05±65.39μg/ml, and the cumulative release rate was (96.39±2.22)%. The positive rate of H37Rv strain culture: 5% in group A, 0 in group B, 100% in group C, and group C was statistically significant compared with groups A and B(P<0.05); The positive rate of rifampicin-resistant tuberculosis strains isolated from clinical: 5% in group A, 95% in group B, and 100% in group C, and group A was statistically different compared with groups B and C(P<0.05). The time-kill curve showed that the colony count of two kinds of Mycobacterium tuberculosis in experimental group at each time point was lower than that of control group and blank group. Experimental group showed obvious bacteriostatic performance from the 3rd day, and no Mycobacterium tuberculosis was cultured after 10 days. Conclusions: 3D printed PaMZ/BMP-2-loaded nHA artificial bone provides stable sustained release of the novel anti-tuberculosis drugs combination PaMZ for 84 days in vitro, which can effectively inhibit the growth and multiplication of Mycobacterium tuberculosis including sensitive Mycobacterium tuberculosis and rifampicin-resistant Mycobacterium tuberculosis within 84 days, so it has good anti-tubercular properties.