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

English Keywords:Spinal tuberculosis  Artificial bone  PaMZ  Sustained release  Anti-tuberculosis

English Abstract:

  【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.