| Spinal tuberculosis,an infectious disease that poses a serious risk to human health,often forms specific infectious bone defects due to the destruction of bone substance of the vertebral body and further increases the extent of bone defects after lesion removal,resulting in tuberculous bone defects which must be restored and reconstructed.The lack of materials for restoring these defects and the inadequate concentration of local drugs in the lesions have led to delayed postoperative healing and a high recurrence rate in spinal tuberculosis,while the growing global problem of drug-resistant tuberculosis has made the restoration of tuberculous bone defects even more challenging.How to effectively carry out anti-TB treatment to heal the lesion quickly and how to restore the tuberculous bone defect satisfactorily to reconstruct the stability of the spine is a thorny problem to be solved urgently in spinal surgery.The development and combination of 3D printing technology,local drug delivery system and novel anti-tuberculosis drugs provide a new idea to solve this problem.Objective1.To construct a nano-hydroxyapatite(n HA)anti-tuberculosis artificial bone composite scaffold loaded with a novel triple anti-tuberculosis drug combination(Pa MZ)of Pretomanid(Pa),Moxifloxacin(M),Pyrazinamide(Z)and Bone morphogenetic protein 2(BMP-2)using3D printing technology,and evaluate its mechanical properties and drug sustained-release properties.2.To evaluate the osteogenic and anti-tubercular properties of 3D printed Pa MZ/BMP-2-loaded nano-HA artificial bone by in vitro experiments.3.To establish an animal model to clarify the biosafety and bone defect restoring ability of 3D printed Pa MZ/BMP-2-loaded nano-HA artificial bone in vivo,to clarify its drug release behaviour in vivo,and to indirectly evaluate its anti-tuberculosis properties in vivo.Methods1.The BMP-2/PLGA microspheres were prepared by multiple emulsion solvent evaporation method,and their encapsulation efficiency was determined;the Pa MZ-PLGA solid dispersion solution was prepared according to the optimal ratio.The Pa MZ/BMP-2-loaded nano-HA artificial bone was constructed after adding the Pa MZ-PLGA solid dispersion solution together with BMP-2/PLGA microspheres to hydroxyapatite printing ink of n HA by 3D printing technology.The characterization of 3D printed Pa MZ/BMP-2-loaded nano-HA artificial bone was observed by scanning electron microscope.An electromechanical universal testing machine was adopted to test the compression resistance;the in vitro release behaviour of three anti-TB drugs and BMP-2 were measured by high-performance liquid chromatography and ELISA,respectively.2.The New Zealand rabbit mesenchymal stem cells(BMSCs)were collected and cultured by the whole bone marrow adherent culture method and identified by flow cytometry and multidirectional induced differentiation.After that,the BMSCs were co-cultured with 3D printed Pa MZ/BMP-2-loaded nano-HA artificial bone,and the adhesion ability of the cells on the scaffolds was observed by scanning electron microscopy.The effect of the scaffold on the proliferation of BMSCs was tested by the CCK8 method;the co-cultured cells were subjected to alkaline phosphatase(ALP)activity test and alizarin red staining,and the m RNA and protein expression levels of osteogenic factors RNNX-2 and COL-I by RT-q PCR and Western Blot were tested respectively to evaluate the in vitro osteogenic properties of the artificial bone scaffold.The artificial bone scaffolds were cultured together with sensitive and drug-resistant Mycobacterium tuberculosis respectively,and their early anti-tubercular properties were evaluated by Time-kill assay;the drug-containing medium prepared from the end of the artificial bone drug-release assay was adopted to culture-sensitive and drug-resistant Mycobacterium tuberculosis separately to evaluate their anti-tubercular properties after 84 days of release.3.The New Zealand rabbit spinal bone defect models were built and implanted with 3D printed Pa MZ/BMP-2-loaded nano-HA artificial bone,and a control group was set up to evaluate the biosafety of the artificial bone through general observation of animals,body temperature,weight monitoring,and liver and kidney function tests.The bone defect restoration was observed by spiral CT 3D reconstruction at the 4th,the 8th and the 12th week postoperatively;the in vivo osteogenic properties were evaluated by gross observation of tissues and HE staining at the 8th and the 12th week postoperatively.The SD rat models were built and implanted with 3D printed Pa MZ/BMP-2-loaded nano-HA artificial bone.The drug concentrations in the plasma and muscle tissue around the material at different time points were tested by high-performance liquid chromatography to clarify its in vivo drug release behaviour and distribution,and indirectly evaluate its in vivo anti-tuberculosis performance.Results1.The BMP-2/PLGA microspheres prepared by the multiple emulsion solvent evaporation method were spherical with a porous and slightly rough surface,good dispersion,uniform size and shape,with an average diameter of 75.09±34.28μm and an average encapsulation efficiency of(45.81±2.47)%;the 3D printed Pa MZ/BMP-2-loaded nano-HA artificial bone—n HA/PLGA had a mass ratio of(61/30)%,a porosity of 60%,a pore size of200μm,rough surface and homogeneous structure under electron microscopy,and a compressive strength of 4.82±0.29 MPa in mechanical test;the three anti-TB drugs Pa,M and Z loaded on the artificial bone showed good sustained-release properties in vitro,with cumulative release rates of(71.0±0.71)%,(96.01±1.54)%and(96.39±2.22)%up to 84 days,and peak concentrations of 73.49±2.33μg/m L,331.85±19.56μg/m L and 1556.17±68.43μg/m L,respectively,and BMP-2 could be released continuously in vitro for 28 days with cumulative release rate of(91.02±3.25)%and the peak concentration of 0.13±0.02μg/m L.2.The cultured rabbit BMSCs met the criteria for stem cell identification by flow cytometry and multi-directional induction of differentiation;BMSCs could adhere and proliferate on the surface of the scaffold when co-cultured with 3D printed Pa MZ/BMP-2-loaded nano-HA artificial bone.The proliferation activity of the cells in the experimental group was not statistically different from that of the positive control group after14 days of co-culture by the CCK8 method(P>0.05),which was significantly better than that of the blank control group(P<0.01).The results of the ALP activity assay showed no statistical difference between the experimental group and the positive control group at each time point(P>0.05),but both were significantly better than the blank control group(P<0.01);alizarin red staining showed that the red-stained mineralized material in the experimental group was the most densely distributed,larger and denser;the m RNA expression of RUNX-2 and COL-I in the experimental group and positive control group at each time point was not statistically different by RT-q PCR(P>0.05),but both were significantly better than that of the blank control group(P<0.01);the relative protein expression of RUNX-2 and COL-Ⅰat day 14 and 21 were not statistically different between the experimental group and positive control group by Western Blot(P>0.05),but both were significantly better than that of the blank control group(P<0.01).Time-kill curves showed that the drug-loaded scaffolds had significant bactericidal ability against both the sensitive H37Rv strain and the clinically isolated rifampicin-resistant tuberculosis strains,and the drug-loaded scaffolds began to show significant bactericidal performance at 3th day after implantation and were able to completely inhibit the growth and reproduction of Mycobacterium tuberculosis after 10 days of implantation.The results of the drug-loaded culture medium prepared from the end of the drug-release assay of the drug-loaded artificial bone showed that the scaffold still exhibited the same bactericidal properties against H37Rv-sensitive strains after 84 days of drug release as the traditional anti-tuberculosis drug rifampicin,and also showed significant inhibition of clinically isolated rifampicin-resistant tuberculosis strains.3.All rabbit spinal bone defect models were successfully constructed without incision infection or death,and the body temperature,body weight and liver and kidney function indexes of the animals in the experimental groups were normal,with no statistical differences from those of the control groups(P>0.05).The results of spiral CT reconstruction at the 4th,the 8th and the 12th week postoperatively showed no loosening or detachment of the implants in each group,and by the 12th week,group A with nano-HA artificial bone loaded with Pa MZ/BMP-2,group B with unloaded artificial bone and group C with autologous iliac bone all achieved bony fusion,while group D with no bone implant showed no obvious signs of healing of the bone defect.The CT-Hedberg score showed that there was no statistical difference between group A and group C at the 4th week(P>0.05),but there was a statistical difference between all the other groups(P<0.05).At the 8th week postoperatively,there was no statistical difference between group A and group C and between group B and group C(P>0.05),however,there was still a statistical difference between group A and group B(P<0.05).At the 12th week postoperatively,there was no statistical difference between groups A,B and C compared to each other(P>0.05),but all were statistically different from group D(P<0.05).The observation of the gross specimens at the 8th week after surgery showed that a small amount of exposed artificial bone was found in both groups A and B,but more bone scabs were formed in group A,while the exposed artificial bone was more obvious in group B than in group A.By the 12th week,the artificial bone in group A was completely encapsulated by new bone(NB),but there was still some exposed artificial bone in group B that was not wrapped or replaced by new bone,and the new bone grew in a crawling and wrapping manner along the surface of the artificial bone.HE staining results showed that at the 8th week postoperatively,a large amount of new bone formation was seen in groups A and C,mainly endochondral osteogenesis by differentiation of chondrocytes to osteoblasts,a small amount of new bone formation was also seen around the artificial bone in group B,but no endochondral osteogenesis was demonstrated,and no new bone formation was seen in group D.By the 12th week,the new bone tissue formed in group A were staggered with the scaffold material,and new bone formation could be seen in the gap of the artificial bone,and the artificial bone could also be seen to be replaced by new bone wrapping.In group B,more new bone was formed than before,but most of it was distributed on the surface of the artificial bone.In group C,the occurrence of osteogenesis within the cartilage was still visible,and the fusion of the iliac bone implant interface could be clearly seen;in group D,a small amount of new bone was also formed at the interface of the bone defect under the fibrous tissue wrapping;quantitative analysis of the total trabecular area at the implant-bone defect interface showed no statistical difference between groups A and C at the 8th and the 12h week postoperatively(P>0.05),and a statistical difference between the remaining groups(P<0.05).All SD rat animal models were successfully constructed without incisional infection and death,and the results of local tissue drug concentrations and plasma drug concentrations of Pa,M,and Z in SD rats at different time points detected by high-performance liquid chromatography showed that all three drugs were released well in vivo,and the trend of local tissue drug concentration-time curves was consistent with drug release assay in vitro.The peak local drug release concentrations of Pa,M and Z appeared on day 28,day 14 and day 7after scaffold implantation,respectively.The blood drug concentration was not detected at the initial time point after local implantation of the drug-loaded artificial bone,and the subsequent measured results were much lower than the local tissue drug concentration,and the two had a positive correlation.The R~2 of the regression equations for the three drugs were0.7455,0.8984 and 0.9109,respectively.Conclusion1.The 3D printed Pa MZ/BMP-2-loaded nano-HA artificial bone was successfully constructed in this study.The mechanical properties of that match the compressive strength of human cancellous bone,and it has excellent sustained-release performance which is effective against both sensitive and rifampicin-resistant Mycobacterium tuberculosis.2.The 3D printed Pa MZ/BMP-2-loaded nano-HA artificial bone has no cytotoxicity in vitro and no liver and kidney toxicity in vivo and has excellent osteogenic and anti-tubercular properties,which provides a new idea for clinical restoring the tuberculous bone defects. |
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