| Tuberculosis(TB) is a very old disease, it is a kind of class B infectious disease in our country. It has got a certain degree of control in the new China. In recent years, the global tuberculosis cases are increasing,Tuberculous arthritis is a common extrapulmonary complication of TB. Because of its difficult treatment and recurrence, it has become a major challenge in medicine. Traditional methods of treatment of bone tuberculosis are combined with systemic chemotherapy and surgical treatment. There are also problems such as low concentration of drug in the treatment of bone tuberculosis, and the toxicity of liver and kidney. Therefore, a suitable drug-loaded scaffold that can postoperatively release an antituberculosis drug efficiently in a lesion area and help repair a bone defect is very important in the clinical treatment of bone tuberculosis. At present, there are several problems in the development of different drug loaded scaffolds, such as single-medicine carriers, excessively fast or excessively slow drug release, degradation of the artificial bone material is unavailable and poor ossification properties. Therefore, it is challenging to study and develop a new antitubercular drug release system with various drug-carrying types and controllable drug release. The choice of drug carrier material and the system design are the key steps in this research work.Ordered mesoporous materials, have a series of advantages, such as high specific surface area and pore volume, the pore structure is regular and orderly, and the pore size is in the nanometer range, easy chemical modification of pore surface and excellent biocompatibility and so on, show great potential in the direction of drug delivery in the field of medicine. MBG can deposit hydroxyapatite better than bioglass made in the traditional way, it is widely studied as a drug carrier with good bioactivity. MBG and polymer composite were prepared together to manufacture composite, which not only can overcome the shortcomings of pure MBG inorganic scaffold, but also improve the biological activity and drug delivery performance of polymer scaffolds.Based on this bioactivity and its drug release function, MBG can be applied into the construction of drug release system.Three-dimensional(3D) plotting can use the accurate accumulation of materials through a computer-aided design model or computed tomography(CT) data under computer assistance to make special biological products tailored to clinical medicine. Different materials and drugs can be adjusted according to the need to adjust the proportion of the material to facilitate printing, many types of molding materials can be used, it has its unique advantages in the preparation of complex spatial structure of the scaffold. By layer by layer printing, layer by layer stacking it can accurately control the structure of the bracket, the biomechanical strength and porosity of the designed scaffold materials can be controlled in a certain extent. Due to a variety of biological materials could be used for printing, high precision of printing, good degree of integration and easy to operate, 3D plotting is widely used in biological tissue engineering and drug controlled release. In recent years, the use of three-dimensional printing technology for the preparation of MBG scaffolds and composite scaffolds have been reported.This study aimed at the needs of the local bone defect repair and in situ chemotherapy after the operation of bone tuberculosis, MBG/ PHBHHx(poly 3- hydroxybutyrate co- 3-hydroxy acid) composite scaffold which loading two kinds of anti-tuberculosis drugs isoniazid and rifampin was prepared by 3D printing technology. In this scaffold, Functionalized different pore size and surface properties of MBG loading high amounts of first-line anti tuberculosis drug isoniazid and rifampicin respectively, in combination with the mesoporous confinement range, surface chemical groups, and gradual degradation of PHBHHx. This achieves the slow release of the drug as long as possible. This study of the drug loaded stent could meet the requirements of combination, high dose and sustained release in the clinical chemotherapy, not only has a very high academic value, but also has important practical application value, is expected to provide a new development of antituberculosis drug loaded composite scaffold and has its innovation.The first part of the experiment: preparation and characterization of drug loaded composite bone scaffold(DLCS)Objective: to preparation of MBG/PHBHHX drug loaded anti-tuberculosis composite bone scaffold(DLCS) through 3D printing technology, then clearly the morphology by means of TEM and SEM, and detect the basic physicochemical properties and biological activity.Method: choose three block copolymer surfactant P123, via the evaporation induced self-assembly method, prepare MBG powder; on the surface of mesoporous channels of MBG were grafted hydrophilic carboxyl group and a hydrophobic methyl to chemically modified, then to load anti-tuberculosis drug isoniazid and rifampicin separately; then blend the drug-loaded MBG powder and PHBHHx to prepare a slurry suitable for printing, slurry was placed in a 3-D printer to prepare a diameter is 6mm, as high as 8mm cylinder drug loaded composite bone scaffold, named for DLCS. At the same time, the control group drug loaded scaffold was prepared by soaking, which was named for commericial cap. The crystal phase composition and morphology of composite bone scaffold was observed by the application of scanning electron microscopy(SEM). Then detect stent compressive strength and pore order rate etc to assess physical and chemical properties. Through the detection of deposition of hydroxyapatite and bone cell adhesion ability and the influence on the activity of alkaline phosphatase(ALP) to evaluate the biological activity of DLCS.Results: we use 3D printing technology successfully prepared MBG/PHBHHx drug loaded composite bone stent, porosity of the scaffold material was 72.55 ± 1.91(%), The average compressive strength of the 20% elements was 3.15± 0.69 MPa and SEM scanning shows that DLCS surface has a lot of pores and ordered pores are uniformly distributed, the sequence hole rate is larger, it was closely arranged between layers and the structure was stable. DLCS has a better ability to deposit hydroxyapatite, and hBMSC cells adhered well on the scaffold surface. ALP activity test showed that the difference between DLCS group and control group was not significant(p>0.05), compared with the blank group, ALP activity was low(p<0.05). This is related to the effect of drug release from the drug-loaded scaffold, but with the increase in the number of days, the activity of ALP in the experimental group and the control group appeared significantly increased, indicating the DLCS still has good osteogenic properties.Conclusion: through 3D printing technology can be successfully prepared MBG/PHBHHx drugs-loaded anti-tuberculosis composite bone scaffold. The stent has good biological activity, stable structure, good loading of two antituberculosis drugs, and has certain compressive strength, can provide a new theoretical support for the study of drug loaded artificial bone scaffold.The second part of the experiment: the drug release characteristics of DLCS in vitro and in vivo and its effect on liver and renal functionObjective: aim to test the drug release rhythm and characteristics of DLCS in vitro simulated body fluid(SBF) and in vivo of New Zealand white rabbits. To evaluate DLCS in drug release performance, and to detect the effects on liver and kidney function of animal body at different time periods.Methods: 0.1g of the DLCS and control group(commercial Cap) were taken into the 20 ml SBF solution respectively, then placed in a constant temperature oscillator of 37 centi-degree and 100r/min. Sampling and replacing the same volume SBF solution at intervals. Using HPLC to detect and calculate the concentration of INH and RFP in each medium, and summarize the in vitro drug release characteristics. DLCS and control group each 0.1g implanted into New Zealand white rabbits femoral inferior segment bone defect, the blood and materials were taken from the surrounding tissue at the corresponding time interval after the operation, then calculate the corresponding INH and RFP concentration by HPLC and summarize the in vivo release characteristics of the drug. The liver and kidney function of the blank group, the experimental group and the control group were detected and evaluated, and the effects of materials on liver and renal function in animals was evaluated.Results: in vitro experiments showed that the release of INH and RFP in the control group was close to 100% in the first 2w, While the experimental group INH and RFP close to the 80% release in 6w and 4w respectively, and until 10 w is not yet fully released. Animal experiments show that the local concentration of DLCS in the 12 w group is still above the minimum inhibitory concentration(MIC:INH0.05 g/ml, g/ml RFP0.5), while the control group local concentration of 4w has been reduced to the minimum inhibitory concentration. The drug concentration in the blood was significantly lower than that of the surrounding tissue, most of which was below 5ug/ml. Liver and renal function tests results show that in the experimental group the alanine aminotransferase(ALT) and aspartic acid amino shift enzyme(AST) appeared a transient increase postoperative day 7 and day 14. The urea nitrogen(BUN) and creatinine(CR) value was not significantly increased during the whole experiment.Conclusion: DLCS loading satisfied, Compared with the control group, the drug release rhythm in vitro and in vivo was significantly slower than that in the control group, release performance is stable, the effective inhibitory time can reach more than 12 w in local tissue in vivo, Can meet the needs of the local chemotherapy after operation of bone tuberculosis. The drug releasing process of DLCS has little effect on the liver and kidney function of New Zealand rabbits, and only had a transient effect on AST and ALT.The third part of the experiment: the biological safety and the ability of osteogenesis of the DLCSObjective: to evaluate the biological safety of DLCS, in order to confirm its compliance with the application standards of biological materials and the osteogenic properties, DLCS were tested in vivo, in order to prove its ability to repair bone defects.Methods: the cytotoxicity of DLCS was detected by CCK-8 method, and the acute systemic toxicity test, primary skin irritation test, intracutaneous stimulation test and sensitization test was performed in animal body. we also set up a control group for comparison, to evaluate the biocompatibility and safety of experimental materials. The same as the second part of the experiment, a rabbit model of femoral bone defect was established, experimental group, control group and blank group for each of two groups, 3 rabbits in each group. 6w and 12 w were executed in batches after surgery, after general observation, The regional organization of bone defect underwent Micro-CT scanning and hard tissue biopsy, to study the ability of repairing bone defect with DLCS.Results: CCK-8 assay showed that the survival rate of cells in each time period was above 80%, according to the evaluation criteria for the cytotoxicity was the 1 stages, which was in accordance with the standards of the use of biological materials. Animal acute systemic toxicity test, primary skin stimulation test, intracutaneous stimulation test and sensitization test all showed that DLCS toxicity is low, it has good biological compatibility with the body, and it has good biological safety performance. In the repairing process of femoral bone defect in rabbits, the experimental group and the control group were similar, compared with the blank group has obvious advantages.Conclusion: DLCS has no obvious cytotoxicity, it has good biocompatibility and safety, and the cytotoxicity of the cells is in accordance with the standard of biological materials. DLCS has a strong ability to induce osteogenesis, which can be used in the treatment of bone defect after bone and joint tuberculosis. It can promote the healing of bone defect and has strong clinical application value. |
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