| Tuberculosis is an ancient disease that still influences people's health.Spinal tuberculosis is the most common extra pulmonary tuberculosis,accounting for 50%-60% of bone and joint tuberculosis, accounting for1%-3% of tuberculosis. If spinal tuberculosis is not properly treated, it will cause serious complications such as paraplegia, deformity, etc.. With the advent of the era of chemotherapy, anti-tuberculosis chemotherapy has become the cornerstone of the treatment of spinal tuberculosis, while surgery is an important supplementary means to solve the complications.However, when patients use anti-tuberculosis drugs therapy systemicly, the drug concentration in the center of focal is low, besides it easy to induce acquired drug resistance and to cause adverse effects such as hepatic and renal function damage. Therefore, a proper solution to the difficult problem was to implant some controlled release anti-tuberculosis drug delivery systems(DDSs) into the focal of spinal tuberculosis in order to maintain the local high drug concentration. At present, people all over the world has do many researches on organic polymer materials, many of them has developed kinds of controlled drug release materials with good sustained release performance. While few people has reported the safety and effectiveness of those organic polymer controlled drug release materials used in human. For spine tuberculosis, after anterior radical debridement,titanium(Ti) alloy screw plate / rod system and titanium cage were used to reconstruct the spinal stability, the implants can be directly contact with the focus. Thus, fabricating an anti-tuberculosis controlled drug release coating on titanium alloy, it can simultaneously achieve the purpose of killingMycobacterium tuberculosis and reconstruction of spinal stability, and may become a potential spinal tuberculosis DDSs. Poly dopamine(PDA) could adhere onto the surface of organic and inorganic materials by Michael addition or Schiff Base reaction. 4-arm Poly ethylene glycol(PEG) is neutral pH, biocompatible and biodegradable. Porous starch has good adsorption properties, and is safe, non-toxic and biodegradable. Poly lactic-co-glycolic acid(PLGA) has good drug loading performance, and hydrophobic, long release period. Our study make the materials above,loading with isoniazid(INH), attach to the surface of titanium by casting or sol-gel dip coating method, as a drug delivery system for spinal tuberculosis.1. The preparation and characterization of Ti-PDA-PEG-PLGA-INHUsing PDA, porous starch and 4-arm-PEG, loading with INH,attached to the surface of titanium by casting or sol-gel dip coating method,to fabricate the anti-tuberculosis controlled drug release system. Using proton nuclear resonance spectroscopy(HNMR) to verify the end functional group and scanning electron microscope(SEM) to observe the characterization of the surface.2. In vitro drug release and antimicrobial test of Mycobacterium tuberculosisIn vitro release of INH from the Ti-PDA-PEG-PLGA-INH were carried out at 37 ? in 1 mL of PBS. The concentration of INH in the medium was determined at pre-determined intervals by high performance liquid chromatography(HPLC), then calculated the cumulative drug release rate of INH. Implanting the Ti-PDA-PEG-PLGA-INH onto Roche medium contained Mycobacterium tuberculosis at 37 ? for 4 weeks,observing the antibacterial activity in vitro of the anti-tuberculosis controlled drug release coating.3. In vivo drug release and evaluation of biological effects25 randomly selected from the experimental animals as a group, a total of 3 groups were selected as experimental group, control group and blank group, A 6mm diameter, 10 mm depth bone defect of left femur condyle is made by surgical method of each group, Titanium rods covered with PDA-PEG-PLGA-INH coating, titanium rods covered with PDA-PEG-PLGA and titanium rods are implanted into the bone defects according to the categories. INH concentrations in blood and tissues were examined at each time point postoperatively by HPLC, the hepatic and renal functions were tested by examining the values of some serum enzymes including ALT, AST, BUN and Cr in venous blood specimens at 7?14?28?42?56 days postoperatively. Another12 rabbits were randomly divided into experimental group and control group, the experimental group implanted with titanium tablets and titanium rods coated with PDA-PEG-PLGA-INH in the paraspinous muscle and left femoral condyles respectively; control group implanted with a blank sheet of titanium tablets and titanium rods in the same place. Hematoxylin and Eosin Staining were used to observe the biocompatibility of the composite system in vivo at 28 and 56 days postoperatively.The results showed that Ti-PDA-PEG-PLGA-INH controlled drug release coating uniformly distributed on the surface of plates and rods, with translucent form and smooth surface. In vitro INH release kinetics exhibited a short-burst release during the first 8h, and the cumulative release of the INH was about 65%. On the 9th day, the cumulative release of the INH was about 90%, and then the release tends to be flat, and the drug release behavior in vitro could continue more than 20 d. The antibacterial test in vitro showed that the titanium tablets coated with PDA-PEG-PLGA-INH formed obvious bacterial inhibition zones.In vivo release test showed that the concentration of INH in vein blood, muscle and bone tissue around the composite system was increased steadily postoperatively. On about the 28 th day, the concentration reachedthe max(vein blood 114.39±14.03ug/ml, muscle 983.17±122.87ug/g, bone786.00±47.13ug/g); the INH concentrations in muscle and bone tissue around the composite system were still higher than the minimum inhibitory concentration(MIC) on the 56 th day. This anti-tuberculosis controlled drug release coating and the drug concentration did not cause obvious damage to liver and kidney function, but to some extent, some serum enzymes of liver and kidney will increase. The pathological results indicated that mild inflammatory reaction was seen in the 4th week postoperatively, and the reactive capsule formation with loose connective tissue. In the 8th week postoperatively, there's no obvious inflammation occured, and the reactive capsule became more dencer and thicker.In summary, the study have successfully fabricated the Ti-PDA-PEG-PLGA-INH anti-tuberculosis controlled drug release coating,with the experimental results above, it is proved that the Ti-PDA-PEG-PLGA-INH has reasonable release behavior both in vivo and in vitro, effective antibacterial activity in vitro, without significant long-term lesions to liver and kidney and good tissue biocompatibility,which is a potentially effective drug delivery system for spinal tuberculosis. |
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