| Objective To observe the 3D printed nano-Hydroxyapatite containing triple antituberculosis drugs PA-824,moxifloxacin,pyrazinamide(Pa Mz)and human morphogenetic protein 2(BMP-2),NHA)The effect of anti-tuberculosis artificial bone on the osteogenic differentiation and cell adhesion of rabbit bone marrow mesenchyml stem cells(BMSCs)in vitro to evaluate the effect of bone marrow mesenchymal stem cells and anti-tuberculosis artificial bone compatibility.Methods A 3D composite scaffold loaded with triple anti-tuberculosis drugs PA-824,moxifloxacin,and pyrazinamide was prepared by 3D printing;the human morphogenetic protein-2 sustained-release microspheres were attached to the scaffold by a scraping method to prepare a pseudo-conjugation Artificial bone with osteogenesis and antibacterial properties.Use whole bone marrow adherent culture method to obtain and purify rabbit bone marrow mesenchymal stem cells,and use flow cytometry,osteogenic and adipogenic staining for cell identification;combine bone marrow mesenchymal stem cells with each group of artificial bones 1,3 After 7,14,and 21 days,the cell proliferation of each group was observed by CCK-8 method at each time point to evaluate the cytotoxicity of the material;the degree of calcium nodule deposition and alkaline phosphatase expression activity were used to evaluate bone marrow mesenchymal stem cells Osteogenic differentiation effect,RT-PCR and Western blot detection of osteogenic related genes and protein expression levels;scanning electron microscope to observe the adhesion characteristics of bone marrow mesenchymal stem cells on the surface of composite artificial bone.Results A composite scaffold containing PA-824,moxifloxacin,pyrazinamide,and polylactic acid-glycolic acid copolymer was successfully prepared in the early stage,and the human morphogenetic protein-2 sustained-release microspheres were attached to its surface.The scaffold was milky white in quality.The surface is hard and rough.Scanning electron microscopy observed that the surface of human morphogenetic protein-2 microspheres are well attached,the scaffold has a complete spatial structure,and the gaps are connected;rabbit bone marrow mesenchymal stem cells are isolated and purified,and the morphology is short spindleshaped fibers on the 7th day.Spindle-like,whirlpool-like growth,in good condition;flow cytometry results show positive expression of CD29 and CD44,negative expression of CD45 and CD90,indicating that the cells are bone marrow mesenchymal stem cells;cells are cultured by adipogenic induction and differentiation,visible in 7 days Lipid droplet formation,oil red O staining positive,osteogenic differentiation of cultured cells,calcium nodule formation can be seen in 21 days,Alizarin red staining is positive,further proving that the cells are bone marrow mesenchymal stem cells;cytotoxicity test results show that each The proliferation status of bone marrow mesenchymal stem cells in the experimental group and the positive control group was better than that of the blank control group(P<0.05),but there was no difference in cell proliferation between the experimental group and the positive control group in the first 14 days(P>0.05),it was confirmed that PaMZ/BMP-2/nHA composite artificial bone was non-toxic;the alkaline phosphatase activity results of the three groups showed that the blank control group had lower alkaline phosphatase activity than the experimental group and positive control group at each time point.There is a significant statistical difference(P<0.05),but the difference between the experimental group and the positive control group is not significant(P>0.05).Alizarin red staining results show that PaMZ/BMP-2/nHA artificial bone and BMP-2 can promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts,and there are calcium nodules deposition;RT-PCR and Western blot detection found that,Runx-2 and type I collagen were significantly expressed in the experimental group and the positive control group.The results further confirmed the ability of PaMZ/BMP-2/nHA artificial bone to promote bone differentiation;21 days scanning electron microscopy observed that the cells adhered tightly to the scaffold,and It grows in the shape of eight claws or long fusiform.Conclusion The nHA anti-tuberculosis artificial bone loaded with PaMZ/BMP-2 has good biocompatibility in vitro,can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells,and is expected to become an ideal bone tissue engineering material. |
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