Chinese Journal of Tissue Engineering Research ›› 2021, Vol. 25 ›› Issue (28): 4447-4453.doi: 10.12307/2021.056

Previous Articles     Next Articles

Three-dimensional printed artificial bone loaded with anti-tuberculosis drugs and bone morphogenetic protein 2 sustained-release microspheres can promote osteogenesis of bone marrow mesenchymal stem cells

Liu Changhao1, Zheng Jianping2, Shi Jiandang3, Zhu Xi3, Zhou Zhanwen1, Zhang Xu1   

  1. 1School of Clinical Medicine, Ningxia Medical University, Yinchuan 750000, Ningxia Hui Autonomous Region, China; 2Department of Orthopedic Trauma, 3Department of Spinal Orthopedics, General Hospital of Ningxia Medical University, Yinchuan 750000, Ningxia Hui Autonomous Region, China
  • Received:2020-09-08 Revised:2020-09-09 Accepted:2020-10-09 Online:2021-10-08 Published:2021-05-15
  • Contact: Shi Jiandang, Doctoral supervisor, Chief physician, Department of Spinal Orthopedics, General Hospital of Ningxia Medical University, Yinchuan 750000, Ningxia Hui Autonomous Region, China
  • About author:Liu Changhao, Master candidate, School of Clinical Medicine, Ningxia Medical University, Yinchuan 750000, Ningxia Hui Autonomous Region, China Zheng Jianping, Doctoral candidate, Attending physician, Department of Orthopedic Trauma, General Hospital of Ningxia Medical University, Yinchuan 750000, Ningxia Hui Autonomous Region, China
  • Supported by:
    the National Natural Science Foundation of China, No. 8176090169 (to SJD)

PDF

102

Abstract:

BACKGROUND: Insufficient local effective drug concentration after debridement of spinal tuberculosis lesions and repair of residual bone defects are the current problems in the treatment of spinal tuberculosis. New anti-tuberculosis drugs combined with bone morphogenetic protein 2 (BMP-2) have become a new idea to solve this problem, which can effectively promote bone defect repair.

OBJECTIVE: To observe the effect of PA-824, moxifloxacin and pyrazinamide (PaMZ)/BMP-2/nano-hydroxyapatite (nHA) anti-tuberculosis artificial bone on osteogenic differentiation and adhesion of rabbit bone marrow mesenchymal stem cells.
METHODS: Rabbit bone marrow mesenchymal stem cells were extracted and purified. The nHA was used to fabricate the three-dimensional printed artificial bone containing anti-tuberculosis drugs PA-824, moxifloxacin, pyrazinamide and BMP sustained-release microspheres (PaMZ/BMP-2/nHA), and co-cultured with the third generation of bone marrow mesenchymal stem cells as the experimental group. BMP-2 was co-cultured with bone marrow mesenchymal stem cells as a positive control group. Bone marrow mesenchymal stem cells were cultured separately as blank control group. The proliferation, osteogenesis and cell adhesion effects of rabbit bone marrow mesenchymal stem cells were respectively tested at 1, 3, 7, 14, and 21 days of culture to evaluate the influence of the artificial bone on the osteogenic differentiation of rabbit bone marrow mesenchymal stem cells. The cell proliferation and cytotoxicity were detected by CCK-8 assay. The degree of calcium nodule deposition and alkaline phosphatase expression activity were measured to evaluate the influence of osteogenic differentiation. Fluorescence quantitative PCR and western blot assay were used to detect the expression levels of osteogenicgenes and proteins, respectively. The adhesion characteristics of cells were observed by scanning electron microscopy. 
RESULTS AND CONCLUSION: (1) The newly synthesized anti-tuberculosis artificial bone showed good spatial microstructure, which was conducive to cell attachment. (2) CCK-8 assay showed that the cells in each group proliferated well. Among them, the experimental group and the positive control group showed a proliferation state for 14 days, and the blank control group became stable after 7 days, indicating that the artificial bone had good biocompatibility. (3) The alkaline phosphatase activity appeared a trend of increasing first and then decreasing, reaching a peak at 14 days. At 7 and 14 days, the alkaline phosphatase activity of the positive control group was higher than that of the experimental group (P < 0.05), but the activity of the experimental group was higher than that in the positive control group at 21 days. The change was not obvious in the blank control group at each time point. (4) Alizarin red staining experiments revealed that calcium nodules were formed in the experimental group and the positive control group, and the differences in the number and size of calcium nodules between the experimental group and the positive control group were not obvious. The expression levels of bone-related genes and proteins (Runx2 and collagen I) were higher in the two groups than those in the blank control group (P < 0.05). (5) Under the scanning electron microscope, it was observed that the cells adhered tightly to the scaffold and showed “Eight Claws” or long fusiform growth. (6) It is concluded that the newly synthesized anti-tuberculosis artificial bone has a unique spatial structure and good biocompatibility, which can promote the proliferation and osteogenesis of bone marrow mesenchymal stem cells, and is an ideal bone tissue engineering material.

Key words: PA-824, moxifloxacin, pyrazinamide, bone morphogenetic protein 2, nano-hydroxyapatite, anti-tuberculosis artificial bone, mesenchymal stem cells, osteogenic differentiation

CLC Number: