Preparation And Characteristics Of A Composite Antitubercular Drugs Delivery System Implanted Into Local Focus For Osteoarticular Tuberculosis Therapy

In recent ten years, with a high uptrend of TB (tuberculosis) there were about 20 million patients suffering from TB all over the world. And there were 5 million TB patients now in china, ranking the second in the world. The patients who had bone and articular tuberculosis accounting for 1~2% of all TB patients in our country usually had to face up to the therapeutic schemes including an anti-tubercular drugs regime or/and a surgery treatment. A bone TB focus debride was often recommended by the special surgeon and accepted by those patients whose bone and joints had been destroyed badly and normal physiological function had been impaired partially or wholly.With the development of orthopedic inner fixation materials, there was prone to perform a focus debride and bone defect reconstructive surgery as early as the special surgeon could. One question was, however, that it was very difficult to reconstruct the residual cavity for much defect of removed disable bone especially around the spine and big joints after the focus debride. Another question was the recurrence of the local residual Mycobacterium tuberculosis which would lead to the failure of implanted autologous or allograft bone or bone materials. Meanwhile, it was recommended and required for those patients to use anti-tuberculous drugs therapy systemicly at least for 6~9 months after the treatment of an adequate surgical debridement, which would result in bad compliance and a lot of side effects.A proper solution to the difficult problem was maybe to implant some controlled release antitubercular drugs into the residual cave. Those controlled release antitubercular drugs were expected to adhered to scaffolds of bone substitute materials which would bring forth temporary support and bone conduction in the local residual cave. But the pure antitubercular drug powders put into the residual cave could not reach every site of the wall of the cave in multi-orientation and the concentration of these drugs would not be controlled easily and lasting for a long expecting time.In a word, a composite material which would possess the controlled release function of antitubercular drugs and bone defect reconstruction was to be demanded in the operation of the debride of bone TB focus at present in order to shorten or even avoid multi-drugs therapy used systemicly following debridement and reduce lesions to hepatic and renal functions.There were many shortcomings in controlled release materials studied now, such as organic polymer materials, mainly including low mechanical strength, fast release from disintegrated enclosing materials degenerated by enzymes in vivo, bad chemical stability and difficulty in complete release for enclosing drugs.And what's more, the procedure of enclosing and loading drugs for these materials was only to press the drug into the materials directly and complete simple compound of the drug and the material. The release concentration and velocity of these drugs compounded by the method were not satisfactory because the loaded materials was not homogeneous substance and these drugs were difficult to diffuse into the materials.Alternatively, mesoporous silica materials as porous inorganic materials may provide a more advantageous choice for controlled and localized antitubercular drugs delivery, which had been investigated deeply as a drug delivery carrier, thanks to its extensive nanopore structure in mesoporous silica. The mesopore structure of 2～50 nm in diameter may render high specific surface area , high pore canal volum, decorated conveniently with other inorganic materials, good bioactivity and biocompatibility. Compared with solid nanoparticles, the mesoporous silica nansparticles (MSNs) are apparently more suitable drug delivery carriers due to its extensive mesoporous structure.As far as antitubercular drugs are concerned, isoniazid (INH) and rifampicin (RFP) were two of efficacious drugs against TB with the traditional duration of treatments instead of simple antitubercular drugs which often led to the occurrence of resistant Mycobacterium tuberculosis bacteria. The bioceramics scaffold ofβ-TCP with porous structure and proper diameter of hole was suitable to the MSNs and the drug.The purpose of the experiment was to design and fabricate a composite scaffold drug delivery system (CS-DDS) made ofβ-TCP bioceramics scaffold and BG-MSNs coating within the scaffold for simultaneously encapsulating INH and RFP. The composite system was expected to combine the merits of multi-drug loading and very sustained and localized drug co-release for effective osteoarticular treatment, and bioactivity for bone repairing. During the debridement of osteoarticular TB, this CS-DDS would supply both a sufficient filling to the bone defect area and effective anti-tuberculosis drug concentrations for the prolonged post-operative multi-drug chemo- therapy to partly or even completely eradicate the residual tubercle bacillus.The synthesis and characteristic test of the CS-DDS and the observe of the controlled release of enclosing INH and RFP in vitro and vivo will support theoretical and experimental for the expecting use of CS-DDS in the debridement of osteoarticular TB.Part one: Preparation and characterization of a composite scaffold drug delivery system (INH-RFP/BG-MSN/β-TCP)Objective: To synthesize a composite scaffold drug delivery system (INH-RFP/ BG-MSN/β-TCP) experimentally and demonstrate its physical characters. Methods: MSNs were synthesized chemically.β-TCP were coated with MSNs by bonding together in high sintering temperature 300℃and the MSN/β-TCP had been enclosed with INH and RFP by suction under the vacuum negative pressure condition befor the BG were coated onto the MSN/β-TCP by the immersion method. The composition and morphology, the chemical constructure and the heat stability of CS-DDS were analyzed by using FE-SEM and EDS. The rate of loaded drugs was detected by HPLC at last. Conclusion: The composite scaffold drug delivery system (INH-RFP/BG-MSN /β-TCP) with good physical character and specific surface area was synthesized successfully by sintered and the vacuum negative pressure condition. The CS-DDS could enclose both INH and RFP with a satisfactory loading drugs amount.Part two: Characters of the drug release from a composite scaffold drug delivery system (INH-RFP/BG-MSN/β-TCP) in vitro and in vivo.Objective: To evaluate the controlled release character of CS-DDS by detecting the concentration of INH and RFP with respect to different time in vitro and in vivo. Methods: the controlled release character of CS-DDS in vitro was detected by monitoring the concentration of INH and RFP released from CS-DDS in SBF by HPLC with respect to different time. Cellular safety testing was performed by culturing the L-929 cells and materials according to MTT methods. The release characteristics in vivo was detected on 1,3,5,7,14,28,42d after CS-DDS was implanted into femur defect of rabbits. The release concentrations of the antitubercular drugs with respect to time in tissues or blood after the implantation were monitored in the supernatant from the different tissues'soak homogenate by HPLC. Results: MSNs was proved as a mesoporous with diameter of about 40 nm and an order columnar arrange. The superficial area of a MSN particle was 958 m2/g and its pore capacity was 0.91 cm3/g and the diameter was 2.65 nm. The compound material with big pores coated by MSNs and BG uniformly and smoothly could enclosing much heavy antitubercular drugs. 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 different time points. As the control group,β-TCP loaded the antitubercular drugs were also implanted and monitored similarly. Correspondingly, anti-tubercle bacillus tests with the implanted CS-DDS on 28d and 42d were conducted and the control group was used the implantedβ-TCP on 7d and 14d. All materials taken out of rabbit bones were soaked into SBF for regular time and the corresponding supernatant from the SBF had been cultured with Mycobacterium tuberculosis for four weeks at 37℃and its growth condition was recorded. Conclusion: with no apparent cellar poisonous, the antitubercular drugs with an effective concentration above the MIC of the corresponding drugs had being released from the CS-DDS for 30 days in vitro and 42 days in vivo of rabbits. The CS-DDS without significant long-term lesions to liver and kidney and no evident side-effects was proved satisfactorily in the character of its controlled release.Part three: Study on the repairing of bone radius defect of rabbits with the BG-MSN/β-TCP composite materials.Objective: To evaluate the capability of reconstruct the rabbit bone radius defect with the BG-MSN/β-TCP composite materials. Method: Establishment of experimental the rabbits bone radius defect model of the rabbit was performed and proper amount scaffolds of BG-MSN/β-TCP composite materials were implanted into the defect of the rabbit model andβ-TCP were done into controlled rabbit model groups instead of the BG- MSN/β-TCP. The corresponding specimen of radius obtained after 16 weeks of the post-operation was performed tests by watching outcomes, imaging findings and histopathological results to estimate the bone defect reconstructure of the composite materials. Results: a large amount of callus formed in the defect of reconstructive region were observed in the BG-MSN/β-TCP group similar with theβ-TCP group. The comparative affirmative outcome on repairing bone defect was got by X rays tests in the MSN-BG/β-TCP group and theβ-TCP group, while the failure of repairing the bone defect was obtained in the empty group. Histopathological test on the issue slice of bone reconstructure in the BG-MSN/β-TCP group and theβ-TCP group revealed that many osteoblasts growed and much mineral deposited inside of the materials. Conclusion:β-TCP scaffolds coated with MSN and BG were proved successfully to be able to reconstruct bone defect in rabbit radius defect similar with porousβ-TCP scaffolds. The composite system combined the merits of multi-drug loading and very sustained and localized drug co-release for effective osteoarticular treatment was proved to have a better bioactivity for bone defect repairing.