【作者】 王会学；

【导师】 叶晓健；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 最近几年来的流行病学调查表明,全球结核病的发病率呈增高趋势。根据调查估计,全球现有的结核病人约2000万,我国现在患结核病的人数居世界第二位,约有结核病患者500万左右。在所有结核病患者中,患有骨与关节结核的病人约占1%~ 2%。随着艾滋病患病率的逐年增高和各种免疫抑制剂的使用增多,其患病人数也呈逐年递增趋势。在众多骨与关节结核病的治疗方法中,主要的方法有药物治疗和外科手术治疗两种。很多骨与关节结核的病人,尤其是有明显的骨、关节破坏并导致临床严重症状的病人,最后往往需要行病灶清除等外科手术干预。随着近年来内固定材料的发展,对于较严重的骨与关节结核病人的治疗,目前多倾向于尽早行骨结核病灶清除+植骨重建内固定术。但是,在行骨结核病灶清除后,由于骨质破坏较多,残腔骨缺损严重,往往引起临床上局部残腔重建困难,尤其是脊柱及四肢的较大的骨与关节周围的严重结核病灶。同时,局部残留的结核杆菌也容易导致骨结核复发,这可能导致自体或人工骨移植失败,引起脊柱的后突畸形或脊髓受压以及关节畸形等远期并发症。另外,在行病灶清理术后,病人仍旧需要全身同时使用多种抗结核药物,且需要很长的治疗时间,一般至少6～9个月。抗结核菌的药物的毒副作用较为明显,多种药物、长疗程的治疗,全身用药毒副作用增大,每天都要服药,病人依从性差。有鉴于此,许多研究者或骨科医生开始尝试局部放置抗结核药物。但骨关节结核病灶清除后单纯的局部放置抗结核药物,药物浓度难以控制,持续时间较短,容易被引流到体外,且由于术后体位及重力等原因,难以在残腔内全方位立体释放。因此,合成一种既具有充填重建、诱导成骨作用,又具有持续立体缓释抗结核药物作用的缓释骨替代材料支架复合体,是目前临床上行骨与关节结核病灶清除术的迫切需要。在目前常用来研究的抗结核药物缓释载体中,研究较多的是有机高分子材料。但有极高分子缓释材料机械强度低,化学稳定性差,在体内容易被生物酶降解而发生崩解释放,且其内所结合的药物难以实现零释放。就其制作合成工艺而言,其载药过程是直接将药物加压沉积于载体上,仅使载体和药物进行简单的机械性混合,药物很难均匀地分散在载体基质上。同时,所合成的有机缓释载药材料质地也不均匀,从而直接影响到药物的释放速率和浓度。介孔二氧化硅材料是一种无机材料,孔径在2～50nm之间,具有如下特性:高比表面积,高孔容,高度有序的孔道结构,孔径和孔道结构可控,化学稳定性好,结构稳定;良好的生物兼容性和生物可降解性,孔道内外易于化学改性,可以实现良好的药物控释。由于介孔二氧化硅具有以上这些明显的优点,现已作为载药缓释材料被深入研究和使用。鉴于介孔二氧化硅为无机材料,且是一种很好的缓释药物的载体,并能较方便可靠地和无机材料支架进行有效的复合,故而成为本课题所选的缓释载体材料。由于单一使用抗结核药物容易导致菌抗药性结核杆的出现,故临床使用抗结核药物现在都采用联合用药,其中最常用的效果比较肯定且贯穿整个疗程的的两种药物是INH和RFP,有文献已经证实,将二者共同包封同时使用,药物疗效和安全性良好。对于支架材料的选择,考虑到缓释微球的结合,我们选择多孔β-磷酸三钙生物陶瓷(β-TCP)这一比较成熟的骨替代支架材料。综上所述,本实验拟将介孔二氧化硅缓释微球材料复合到多孔β-TCP组织工程骨支架材料中,并将INH和RFP共同包封其中。而后对其进行检测和缓释性能的验证,以期望这种复合体能够在动物体内的局部立体空间内,全方位、长时间地释放所包封的抗结核药物;同时还可以起到骨缺损的充填和促进成骨,加快重建的作用。通过对合成方法的探索以及对合成材料理化性能的检测、以及其体内外释放抗结核药物特性的检测和对成骨作用的观察,为其最终能应用于临床提供理论和实验支持。实验第一部分:INH-RFP/BG-MSN/β-TCP缓释复合体的制备及表征目的:旨在通过实验室合成INH-RFP/BG-MSN/β-TCP复合缓释材料(CS- DDS),明确其形貌特征。方法:通过化学方法合成MSNs,在300℃条件下将其与β-TCP复合,真空负压吸引法包封INH和RFP,而后涂覆BG,场发射扫描电镜(FE-SEM)观察分析复合材料的晶相组成、微观形貌、化学结构和热稳定性,HPLC检测其载药率。结果:MSNs粒子为介孔材料,主要分布在40nm左右,介孔分布呈有序的柱状排列。介孔的表面积、孔容积及孔径大小分别为958m~2/g, 0.91cm~3/g和2.65nm。β-TCP较大的孔隙结构表面可见MSNs及BG涂布均匀、光滑,复合材料载药率高。结论:通过烧结与真空负压吸引等技术的成功合成了具有包封INH和RFP作用的/BG-MSN/β-TCP复合缓释材料,形貌特征均匀,比表面积良好,可以其可同时装载INH和RFP两种抗结核药物,载药量满意。实验第二部分:INH-RFP/BG-MSN/β-TCP复合缓释材料体内外缓释特性的检测目的:检测INH-RFP/BG-MSN/β-TCP复合缓释材料(CS-DDS)在体外和新西兰兔股骨体内的药物缓释特性,评价复合材料的缓释性能。方法:将一定量的CS-DDS放置到动态更换的SBF液中,取不同的时间点,用HPLC法检测其对应的药物浓度,总结CS-DDS在SBF液中的体外释放特性;将L-929细胞与CS-DDS材料共培养,MTT法检测其对L-929细胞活性的影响;将CS-DDS植入新西兰大白兔的股骨中下段骨缺损及骨髓腔内,分别于第1、3、5、7、14、28、42天取静脉血及肝、肾、脾和局部骨等组织,匀浆离心萃取后取上清液用HPLC检测对应INH和RFP的浓度,总结其体内释药特点;静脉血同时行ALT, AST, BUN及Cr值等肝肾功能指标检测,对照组用载药的β-TCP材料;取实验组第28、42d时的CS-DDS浸泡入SBF中,上清液(空白组用SBF缓冲液)与结核菌一起共同培养4周,观察记录结核菌生长情况。结论:CS-DDS无明显细胞毒性,体外有效释放可达30天,体内有效释放超过42天,其对应时间点的最终检测浓度都在对应药物的最低抑菌浓度(MIC)以上,未见明显肝、肾功能损害。该缓释材料缓释抗结核药物效果肯定,未见明显毒副作用。实验第三部分::INH-RFP/BG-MSN/β-TCP(CS-DDS)缓释复合体对兔桡骨缺损修复的实验研究。目的:评价BG-MSN复合后的β-TCP骨支架材料对兔桡骨骨缺损的修复性能。方法:建立兔桡骨骨缺损的实验动物模型,将定量的BG-MSN/β-TCP材料植入缺损部位,对照组用等量的β-TCP材料。术后16周取材,通过大体观察、影像学观察及组织学观察评价成骨修复情况。结果:肉眼观察BG-MSN/β-TCP充填的桡骨缺损处大量骨痂形成,缺损修复处较牢固,与单纯β-TCP组类似。X线片发现骨缺损处BG-MSN/β-TCP组与单纯β-TCP组成骨都较为明显,缺损处骨修复效果肯定。组织切片显示BG-MSN/β-TCP组骨缺损处有明显血管及成软骨细胞和成骨细胞长入,类似β-TCP组。结论:本实验通过物理的方法将BG-MSN加入复合到β-TCP内,形成的复合材料骨诱导成骨能力肯定,类似于β-TCP,说明其作为抗结核药物的缓释载体,在骨与关节结核行病灶清理术时,可以作为骨充填支架材料,具有良好的充填和诱导成骨能力。更多还原

【Abstract】 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.更多还原