【作者】 张泽华；

【导师】 许建中；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 背景和目的:世界上约1/3人口感染过结核杆菌,2007年全球估计有1300万结核病患者,而中国结核病患者约占17%,仅次于印度。日益严峻的结核耐药形势危机社会公共卫生安全,目前学术界、政府机构关注焦点集中于肺结核,骨关节结核耐药所占比例小,没有获得足够的关注。骨关节结核虽然比例小,但基于中国结核病负担重、人口基数大,骨关节结核将越来越常见,应引起骨科医师的重视。自1963年发明利福平用于抗结核治疗以来,大大缩短了结核病疗程,成为联合化疗的基石。但系列研究表明利福平口服生物利用度差,血浆中药物浓度较低。脊柱结核病理特殊,有脓液、干酪组织、死骨、硬化骨等病变,血供差,口服用药难以保证病灶区内足够的药物浓度。提高病灶内药物浓度可能增强其杀菌效果,为维持病灶内有效药物浓度,缓控释制剂成为研究焦点。因此,本实验主要目的包括(1)筛选一种抗结核药物用于脊柱结核局部缓释;(2)冷冻干燥同种异体骨复合纤维蛋白多聚体作为抗结核药物缓释载体,制备一种新型植入型缓释系统;(3)体内外评价植入型抗结核药物缓释系统的特性。内容与方法:(1)骨关节结核病灶结核杆菌培养、药敏试验:纳入72例骨关节结核患者,通过液体培养基及BACT/ALERT 3D系统进行结核分枝杆菌快速培养,培养阳性者接种L-J培养基,按绝对浓度法进行药敏试验,研究分析重庆地区骨关节结核耐药流行情况,以选用耐药率较低的药物;(2)骨关节结核病灶利福平、异烟肼浓度检测:分别建立高效液相法检测利福平、异烟肼条件,检测36例骨关节结核患者病灶内利福平和异烟肼药物浓度,以明确局部用药的必要性;(3)高浓度利福平对耐利福平结核分枝杆菌杀菌效果测定:复苏18株利福平耐药菌株,采用微阵列基因芯片检测rpoB基因突变,研究递增浓度利福平(2,4,8,16,32,64,128,256μg/ml)对耐利福平菌株杀菌效果,并分析rpoB基因突变与利福平耐药度相关性。(4)山羊冷冻干燥同种异体骨支架材料的制备:遵守美国组织库协会(AATB)标准,制备山羊冷冻干燥同种异体髂骨及松质骨,扫描电镜观察微观结构,测量孔隙率及密度;(5)选用纤维蛋白多聚体固定利福平晶体,优化其配方:采用正交试验设计,组成9种纤维蛋白胶组成配方(纤维蛋白原3个浓度,凝血酶3个浓度,氯化钙40mg/ml,氨甲环酸10mg/ml),制备纤维蛋白胶圆饼,甲醇作为利福平溶剂,将利福平晶体分布于纤维蛋白多聚体网络结构内,体外缓释优化纤维蛋白胶配方;(6)负压灌注法制备植入型利福平缓释系统:进一步研制负压灌注系统,依次将配方优化后纤维蛋白多聚体和利福平灌入松质骨孔隙内,制备植入型利福平缓释系统,扫描电镜观察其微观结构,测定载药量、载药率。(7)植入型利福平缓释系统体外缓释参数及抗结核活性:山羊血清作为体外缓释分散介质,37℃避光,定期换液取样,高效液相色谱法检测其缓释特性;结核分支杆菌标准菌株接种液体培养基,体外检测3种植入型利福平缓释系统抗结核能力。(8)利福平对MSCs增殖和成骨分化的影响:分离、培养人骨髓来源间充质干细胞,培养基内加入梯度浓度(4-128ug/ml)利福平,定期(1、3、5、7d)显微镜观察、MTT法检测不同浓度利福平对MSCs增殖的影响;成骨诱导28天,茜素红染色、骨钙素染色、real-time RT-PCR及western blot检测不同浓度利福平对MSCs成骨指标的影响;(9)植入型利福平缓释系统体内缓释研究:建立山羊体内评价模型,将植入型利福平缓释系统分别置入椎间、髂骨内缺损处,髂骨处定期抽液,高效液相色谱法评价植入型利福平缓释系统体内缓释特性,并利用大体观察、X线片、CT三维重建、组织病理学评估植入型利福平缓释系统椎间植骨成骨融合效果。研究结果:(1)骨关节结核病灶结核杆菌培养、药敏试验:72例中培养阳性31例(43.1%),其中人型结核杆菌28例,牛型结核杆菌3例。全部敏感16例,耐药15例,其中耐1种药物8例,耐2种药物5例,耐4种药物1例,耐5种药物1例。耐药种类:异烟肼耐药8例,利福平均耐药2例,链霉素耐药4例,乙胺丁醇耐药2例,力克肺疾耐药2例,左氧氟沙星耐药9例;(2)骨关节结核病灶利福平、异烟肼浓度检测: 6例能维持利福平有效杀菌浓度4.60±0.76 ug/ml,7例检测不到,其余23例患者低于有效杀菌浓度,平均0.78±0.45ug/ml;异烟肼浓度:34例患者结核病灶异烟肼浓度平均0.543±0.132(0.332-0.704)ug/ml,达到有效杀菌浓度。(3)高浓度利福平对耐利福平结核分枝杆菌杀菌效果测定:18株利福平耐药菌株中有17株(94.4%)检测到rpoB基因突变,1例未检测到突变。突变位点位于531 (55.56%), 526 (11.11 %), 516 (16.67 %) and 513 (11.11%);与低水平耐药相比,高水平耐药(250μg/ml )出现531-Ser突变较高。11例MIC超过256μg/ml(超过设计浓度高限,故未检测),其余7株结核杆菌MICs为32-256μg/ml,特别是低水平耐药者多可被较高浓度利福平杀灭、抑制。(4)山羊冷冻干燥同种异体骨支架材料的制备:冷冻干燥同种异体髂骨支架材料呈乳白色,质硬,具有骨组织天然三维网状孔隙,孔隙内洁净,相互连通。密度为0.39±0.03 g/ml ,孔隙率61.23±9.12%。孔径293±23.2 (188～487)um,皮质骨厚度1.59±0.16 (1.09～2.08)mm。(5)选用纤维蛋白多聚体固定利福平晶体,优化其配方:在9组正交实验中, 3、5、8号纤维蛋白胶配方三维网状结构与利福平颗粒均匀交替分布,这3组虽有突释现象,但缓释曲线较平稳,能维持较高的利福平浓度,49天时其浓度分别为6.22、8.23、7.63 ug/ml。(6)负压灌注法制备植入型利福平缓释系统:采用3、5、8号配方制备3组植入型利福平缓释系统,外观呈鲜红色,药物分布均匀,纤维蛋白胶将骨块孔隙封闭。扫描电镜可见纤维蛋白胶将骨块孔隙封闭,纤维蛋白胶将利福平晶体固定于孔隙表面和内部。(7)植入型利福平缓释系统体外缓释参数及抗结核活性: 8号配方仍有突释,3、5号配方突释现象有明显改善,缓释曲线平稳。3号配方可维持有效杀菌浓度42天,49天时检测不到。5号配方至少可维持49天。三组均可有效杀灭结核杆菌标准菌株。(8)利福平对MSCs增殖和成骨分化的影响:0-16 ug/ml利福平与MSCs具有良好的相容性,对其增殖无明显影响;利福平浓度≥32ug/ml明显抑制MSCs增殖;0-8 ug/ml利福平对MSCs成骨诱导分化无明显影响,利福平浓度≥16ug/ml明显抑制MSCsⅠ型胶原、骨桥蛋白、碱性磷酸酶表达;(9)植入型利福平缓释系统体内缓释评价:植入型利福平缓释系统体内缓释时,随时间延长局部成骨环境内利福平浓度逐渐降低,维持利福平有效杀菌浓度超过42天,浓度为13.55ug/ml,49天时利福平浓度为2.23 ug/ml。术后9月实验组与对照组均达到骨性融合。结论:1、结核杆菌快速培养联合绝对浓度法药敏试验准确度高,费用低,可检测常用一线、二线药物敏感性,适用于指导骨关节结核患者个体化化疗方案的制定;骨关节结核利福平耐药率相对较低;2、部分患者骨关节结核病灶中利福平难以维持有效杀菌浓度;3、高浓度利福平抑制MSCs增殖、成骨分化,为植入型利福平缓释系统的参数优化提供了安全指标;4、优化配方制备的植入型利福平缓释系统体外缓释曲线较平稳,能维持有效杀菌浓度至少49d;体外抗结核作用可靠;5、植入型利福平缓释系统可维持体内局部有效杀菌浓度至少42d,无明显全身毒性,不影响最终骨性融合。更多还原

【Abstract】 Background and Objective:One-third of the world’s population is infected with the Mycobacterium tuberculosis. There were an estimated 13.7 million prevalent cases in 2007, and China accounts for nearly 17 % tuberculosis burden of the world, it’s second only to India in the number of TB patients. Since the WHO declared tuberculosis a worldwide emergency in 1993, spinal tuberculosis has resurged after decades of decline in incidence, as a common form of extrapulmonary tuberculosis in china. Even extra-pulmonary tuberculosis is not contagious, public attention are most focused on pulmonary tuberculosis. Unfortunately and obviously osteoarticular tuberculosis has risen parallel to that of pulmonary tuberculosis. It attempted to call much more attention, if considering the large population base and as important cause of disability and mortality.Since rifampicin was introduced as antituberculous agent in 1963, it has been the cornerstone of drug regimens for the treatment of tuberculosis, its use has greatly shortened the duration of chemotherapy for the treatment of TB. However, studies have shown probable reasons lead to poor variable bioavailability and lower plasma concentrations of rifampicin, and intralesional concentration of rifampicin and pyrazinamide in osteoarticular tuberculosis were mostly at subtherapeutic level, it has been associated with clinical failure and drug resistance. Low level resistance to rifampicin implies the antituberculous effect would be optimized with higher concentration rifampicin. In order to maintain effective rifampicin concentration, novel drug delivery systems has been investigated for sustained or controlled release purpose .So aims of this study included: To select an antituberculous drug for sustained release by analyzing drug susceptibility testing of Mycobacterium tuberculosis in spinal tuberculosis, detection of intralesional isoniazid and rifampicin concentration using HPLC method, and the killing effect of higher concentration rifampicin on rifampicin-resistant M. tuberculosis isolated from patients with osteoarticular tuberculosis in vitro.Materials and Methods:(1)The specimens (pus or caseous tissue) were collected from 72 patients with osteoarticular tuberculosis during surgery. After processed in a routine mycobacteriology laboratory procedure, the specimens were inoculated into vials of the BACT/ALERT 3D system. Mycobacterium tuberculosis strains were identified with PNB or TCH culture medium growth trial. The drug susceptibility testing was performed using absolute concentration method,which included 11 first and second line drugs. (2)Two simple, precise and accurate HPLC-based assays for the quantification of rifampicin and isoniazid were established. The intralesional effect of rifampicin and isoniazid of 36 patients with osteoarticular tuberculosis were accessed by concentration:MIC ratio.(3)A set of 18 rifampicin-resistant M. tuberculosis isolates were prospectively collected for further study. Detection of rpoB gene mutations associated with rifampicin resistance in Mycobacterium tuberculosis was performed using non-fluorescent low-density DNA microarrays. Following secondary culture, susceptibility to rifmapincin was tested at increase concentration of rifmapincin by degrees (2,4,8,16,32,64,128,256μg/ml),which is attainable for prolonged periods of time with local chemotherapy.(4)Procurement, processing, sterilization and conservation of the iliac crest allograft scaffold were all performed according to the guidelines of the American Association of Tissue Banks. Its physical characteristics were observed including colour, texture and porosities, the diameter of pores and microstructure were tested with scanning electron microscope.Its porosity and density were tested with liquid displacement technique.(5)Orthogonal experiment design was used in the optimization of a fibrin scaffold for sustained release of rifampicin. The scanning electron microscopy analysis and in vitro study of sustained release of fibrin disc containing rifampicin crystals were carried out to optimize the avaible fibrinogen and thrombin concentration.(6) A new negative pressure impregnation system was designed and manufactured. In the light of previous results, preparation of implantable fibrin polymer/bone allograft composite systems for rifampicin delivery were unerwent using negative pressure impregnation technique using the No.3,5,8 fibrin compositions . Fibrin glue and rifampicin methanol solution were impregnated into the pore of bone allograft under negative pressure respectively. (7) The sustained release of rifampicin from implantable fibrin polymer/bone allograft composite systems was carried out in vitro ,the chemostat filled with goat serum as the release medium was placed in a humidified 37oC /5% CO2 incubator. The HPLC-based assay was used to determined the concentration of rifampincin for optimization the No.3,5 and 8 fibrin compositions .(8) The toxic effects of rifmapicin on the proliferation and osteogenic differentiation of MSCs were assessed. by MTT-measurements, extracellular matrix calcication , immunohistochemistry staining, real-time RT-PCR, and western blot. (9) For in vivo study, the No.5 implantable fibrin polymer/bone allograft composite systems was implanted in the goat’s os ilium and lumbar interbody. The local rifampicin concentration was determined by an assay of effusion drained from implanted sites.Results:(1) Thirty one of the total fifty specimens were culture positive(43.1%). Twenty eight specimens (90.32%) were identified as M. tuberculosis infection and three (9.68%) as M. bovis. The drug susceptibility testing outcomes showed a resistance level of 11.27% for isoniazid, 2.78% for rifampicin and ethambutol, 5.56% for streptomycin, 2.78% for dipasic and 12.68% for levofloxacin.(2)The mean intralesional rifampicin concentration of 23 of 36 patients was 0.78±0.45ug/ml, which was mostly at subtherapeutic level,6 of which was 4.60±0.76 ug/ml(exceeding 10 fold MIC),the other seven were undetectable. The mean intralesional isoniazid concentration of 34 of 36 patients was 0.543±0.132(0.332-0.704) ug/ml, which was achieving 10 fold MIC.(3)Mutations in the RRDR of the rpoB gene were identified in 17 (94.4%) of the 18 rifampin resistant isolates. 1 Rifs strain exhibited no mutations in this region. The most prevalent mutation sites were in codons 531 (55.56%), 526 (11.11 %), 516 (16.67 %) and 513 (11.11%).High level rifampin resistant strains (250μg/ ml rifampin resistant) had higher mutation frequency at 531-Ser than low level rifampin resistant strains (50μg/ ml rifampin resistant) ( P < 0.05) .Isolates with mutations of rpoB were highly resistant to rifampicin,11 of which with MICs exceeding 256 μg/ml(not determined).The MICs for the remaining 7 resistant isolates were between 32 and 256μg/ml. Especially for low level rifampicin resistant strains, M. tuberculosis growth was inhibited under certain higher concentration. (4) The freeze-dried iliac crest bone allograft was ivory white, clean and porous, presenting sandwich structure with tricortical and cancellous bone. The rifampicin crystal was cemented in the pore by fibrin. Its mean density ,porosity and pore diameter was 0.39±0.03 g/ml, 61.23±9.12% , 293±23.2 um,respectively. (5) The No.3,5,8 fibrin discs showed satisfying microstructure and sustained release property: the rifampicin crystals were surrounded into the three-dimensional fibrin network structures. The release curves were relatively smooth.They could maintain microbial killing effect of rifampicin for at least 49 days. (6) The three group implantable fibrin polymer/bone allograft composite systems for rifampicin were cardinal red, the pore of bone allogrft were filled with fibrin and rifampicin. The rifampicin crystals were surrounded into the three-dimensional fibrin network structures.(7)All the NO.3,5,8 implantable systems for rifampicin could maintain microbial killing effect of rifampicin for at least 42 days,49 day and 49 days,respectively. But release curve of NO.5 was smoother, sudden release could be observed in NO.8.All had effective antituberculous effect in vitro.(8)The rifampicin obviously inhibited MSCs proliferation at concentrations of 32ug/ml; In vitro osteogenesis, rifampicin decreased the ALP,COL 1A1 and OPNexpression in a dose-dependent manner, obviously when the concentration of rifampicin exceeding 16ug/ml. (9) In vivo sustained release in osteogenic microenvironment,the concentration of rifampicin showed time-based decrease, but 13.55ug/ml could be achived in 42 days; Bone repair process was not affected in long term followup.Conclusion :(1)The procedures were accurate and low-cost for first and second line antituberculous drugs susceptibility testing,and could be recommended for refining the optimal individual chemotherapy regimen in spinal tuberculosis. The drug resistance epidemiology of osteoarticular tuberculosis was parallel to that of pulmonary tuberculosis, rifampicin resistance was low.(2) The intralesional rifampicin concentration was mostly at subtherapeutic level; (3) At a high concentration, rifampicin inhibits proliferation and differentiation of human bone marrow mesenchymal stem cells and could compromise the bone-healing process in vitro. (4) The implantable systems with NO. 5 fibrin composition was optimal for rifampicin release and maintain microbial killing effect. (5)The implantable fibrin polymer/bone allograft composite systems for rifampicin could maintain microbial killing effect for at least 42 days in goat model, and result in solid lumbar interbody bone union.更多还原