| Tuberculosis is a global public health problem that is seriously harmful to public health. Globally, China is the world's second largest country suffered from tuberculosis. The incidence of TB is higher among all the infectious diseases in China. The proportion of extrapulmonary tubereulosis accounts for 10%-15% of the TB, It primarily affects bones and joints, though other organs may be involved. The most commonly used treatment for bone tuberculosis is through the surgery to remove tuberculosis nidus, while he bone defect can be leaved. At present, the domestic medical market doesn't have medical implant materials which can repair the bone defects injured by tuberculosis perfectly. In recent years, there are many new materials used to restore the common bone defects, after all these new materials can not completely used to repair bone defects injured by tuberculosis. For example, some implant materials partly can repair the bone defects but without anti-TB effect or the materials degradation and ossification can't match well. In addition, TB caused bone defects is slower than the common bone defects in restoration. The research on repairing tuberculous caused bone deficiencies is still at the initial stage. Many scholars have created a lot of new materials,Inevitably, These materials have some disadvantages like anti-TB drug components is single, the drug release is not stable, excessive drug concentration can inhibit osteogenesis and the concentration is too small can't effectively kill tuberculosis bacilli etc. Therefore, we have designed a new stent materials to solve the problem of bone defects caused by tuberculosis which not only can repair bone defects, reconstruct and induce bone formation, but also can release new anti-TB drugs slowly, continuously and three-dimensionally.In this study, according to bone tissue engineering principles, the anti-TB compound bone tissue engineering materials which both have the effect of bone repairing and anti-TB was made by loading the anti-TB drugs release microspheres in bone tissue engineering scaffolds made by 3D printing technology using ?- tricalcium phosphate as basic materials. Then the experiments were did in vivo and vitro to evaluate its function.1. Preparation, characteristics and mechanical properties of ?- tricalcium phosphate scaffold loaded anti-TB drug release was made by 3D printing technologyThe composite materials was made by 3D printing technology. Through environmental scanning electron microscope we found that the material in the support area is the same size of microscopic pore structure,distributed evenly and micro pore connected to each other, the diameter is 2?m ~ 8?m. After testing and calculation, the total porosity of three-dimensional porous ?- tricalcium phosphate scaffold is(61.76 ± 2.53) %, macro pore accounted for 42.5% and micro pore accounted for 19.26%, RFP / PLGA microspheres were dark red and INH / PLGA microspheres were white. There are still some PLGA microspheres failed to encapsulate exist around RFP / PLGA microspheres and RFP / PLGA microspheres. Under the scanning electron microscope, micro ball is spherical and on its surface full of 0.5 ?m-1.5?m size holes that are RFP and INH release channel. The macro pore in drug-loaded microspheres material area is 400?m, the diameters of RFP / PLGA microspheres and RFP/PLGA microspheres between 200?m ~ 300?m, solid microspheres are RFP/PLGA microspheres and hollow microspheres are INH / PLGA microspheres. Microspheres are evenly distributed on the ?-TCP scaffold, the overall porosity of microspheres loaded RFP / PLGA and INH / PLGA total porosity is(30.59 ± 1.33)%. By X ray diffraction analysis we found that the ?- TCP scaffolds ithere is no phase change before and after sintering. Take sintered ?-TCP scaffold used as compressive strength test, the compressive capacity of ?-TCP scaffold with 400?m pores is the largest, the maximum compressive strength is(3.31 ± 0.64) MPa, the mechanical properties achieve the compressive strength of cancellous bone(2~12 MPa).2. The drug release and degradation of porous ?-TCP scaffold loaded anti-tuberculosis drug release microspheres in vitroThrough release and degradation experiments in vitro to testify the anti-tuberculosis, osteogenesis and degradation effects of this new composite materials, And to provide technical support for the in vivo experiment. The results showed that the drug loading ratio of RFP / PLGA and INH / PLGA microspheres are(26.0 ± 1.2)% and(28.0 ± 1.5)%, The quality of each of the porous ?-TCP scaffold is(401.0±13.2)mg, the quality of ?-TCP loaded 50% RFP/PLGA microspheres and 50% INH / PLGA microspheres increase about(79.0 ± 7.6)mg, the quality of drugs is(21.33 ± 2.34)mg(total quality of RFP and INH). The drug release time of this materials is about 90 d. In vitro degradation process, the quality of materials gradually reduce over time and at 15 weeks the quality tends to 0. At the same time the PH value of the material gradually reduce and then increased slightly. The compressive strength of porous ?-TCP scaffold loaded RFP / PLGA microspheres and INH / PLGA microspheres decreased, At first the compressive strength is 3.31 Mpa, after four weeks is 3.11 Mpa, at 8th week is 3.04 Mpa, at 12 th week is 2.98 Mpa, after 13 weeks the compressive strength number doesn't measured any more.3. The cytotoxicity assay of porous ?-TCP scaffold loaded anti-tuberculosis drug release microspheres In vitroThe biocompatibility and osteogenic performance of this new materials were evaluated in this study. Composite materials and mouse bone marrow mesenchymal stem cells were co-cultured, we observed that cells grow vigorously. The control group and the experimental group had no significant differences in cellular morphology at 24, 48, 72 h. Metallurgical microscope was used to observe the microscopic structure of the porous ?-TCP scaffold and co-cultured cells, cells in the porous ?-TCP scaffold like distribution was surrounded, cells grown on the surface and pores of the porous material, some have entered the inner frame. Scanning electron microscopy observed ?- tricalcium phosphate porosity have rat bone marrow mesenchymal stem cells, cells were spindle and irregular in shape, and surface adhesion, the surface of adherent cells, dense growth, growing active and stretch the pseudopod attach material surface.4. The experiments of porous ?-TCP scaffold loaded anti-tuberculosis microsphere in vivoWe conducted a series of experiments of three-dimensional porous ?-TCP scaffold loaded RFP / PLGA and INH / PLGA release microspheres in vivo and find this new materials in one hand have a very well slow release characteristics, in the other hand can repair bone defects and promote bone formation. The rabbits were select randomly by X-ray, The results showed that the bone defect in rabbit models of drug group and control group are basically identical in vivo and vitro. Conventional CT was used to study its degradation in vivo, the results is the same as in vitro. Micro-CT was used to study new bone formation, the results show that after 3 weeks, 6 weeks, 9 weeks, 12 weeks, 15 weeks the new bone volume was no significant difference between drug group and non-drug group(P >0.05); after 3 weeks, 6 weeks, 9 weeks, 12 weeks, 15 weeks the new bone volume was significant difference between drug group and control group(P <0.05); there is significant difference between non-drug group and control group(P <0.05); By comparing we found that the control group and the blank group after 3 weeks, 6 weeks, 9 weeks, 12 weeks, 15 weeks have significant differences in the new bone volume(P <0.05).The results showed that?- tricalcium phosphate bone tissue engineering scaffold loaded anti-TB drug release composite material has a strong anti-TB, osteogenesis effects and good histocompatibility, achieve the expect experimental design requirements. It will have dual role in anti-TB and bone repairing for Clinical applications in the future. |
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