【作者】 李小康；

【导师】 郭征； 张智勇；

【作者基本信息】 第四军医大学 ， 外科学， 2013， 博士

【摘要】 感染仍然是骨科植入物的严重并发症之一。植入体内的金属异物是导致此类感染发生的危险因素，所引发的一系列体内反应包括巨噬细胞功能的减弱，局部免疫系统的钝化等都为病原体的增殖创造了便利的条件。此外，植入物表面吸附的蛋白还能促进细菌黏附到植入物表面引发感染。过去我们大多关注对环境和个人污染的清除及围手术期全身抗生素的应用，而新的策略则是针对该类感染发病的特殊机制通过植入物表面修饰改性来降低感染的风险。全身用药时，可能会因患处血液循环受损和周围瘢痕组织的影响导致病灶处的抗生素浓度无法达到有效水平，从而影响金属材料植入后感染防治效果，还会带来全身的毒副作用。研究证实，细菌一旦被隔离在无血运的骨组织中，抗生素的全身治疗很难在局部达到满意的药物浓度。而且，一旦病原体附着于骨或其它生物材料表面，就会产生代谢和表型的改变，使病原体能够对抗生素产生抵抗和逃脱免疫监督，局部较低的抗生素浓度反而有可能会成为细菌耐药性选择的动力。为了改善骨感染的治疗效果，抗生素的局部给药方法得到研究与开发。其中，抗生素缓释系统以其具有的药物持续性和可调控性释放特点而受到青睐。本项目组在前期低弹β钛合金和药物控释研究的基础上，对钛合金表面进行规则微形态改性，然后进行多巴胺涂层修饰并以其为媒介将庆大霉素明胶微球缓释系统成功构建于钛合金表面，通过前期的表面表征分析，药物释放实验和体外抑菌实验等证实该金属表面抗生素缓释系统具有微球结合牢固，药物释放持续可控，体外抑菌作用明显等优点。本研究主要在前期工作的基础上继续探索该金属表面抗感染缓释系统的体内外生物相容性及动物感染状况下的材料植入对已发感染的控制效果。目的在前期低弹β钛合金表面抗生素缓释系统成功构建的基础上，进一步通过体外细胞实验和荧光染色研究多巴胺涂层修饰对成骨细胞黏附的影响及其生物相容性，通过小鼠肌袋植入实验观察复合材料的体内生物相容性，建立兔胫骨骨髓炎模型，并最后通过兔胫骨骨髓炎模型的体内植入，研究钛合金表面抗生素缓释系统对于感染的控制作用，从而为抗感染金属植入物的研制和开发奠定理论和实验基础。方法1.钛合金表面多巴胺修饰的细胞相容性检查：为探索多聚多巴胺涂层的生物相容性，将家兔来源的骨髓基质干细胞接种于多聚多巴胺涂层修饰后的钛合金表面，然后分别采用MTT定量计算和激光共聚焦显微镜定性观察的手段，分析材料表面细胞的粘附、增殖情况，并与未涂层材料作对比，观察涂层修饰后材料生物相容性的变化。然后钙黄绿素-AM (Calcein-AM)和碘化丙啶(PI)溶液，分别对活细胞和死细胞进行荧光染色，观察细胞生长存活情况。钙黄绿素可显示胞浆，PI显示死细胞的细胞核。采用激光共聚焦显微镜对活细胞生长形态进行观察。胞浆结构完整的绿色代表正常细胞，胞浆碎片状结构不完整、细胞核红染的代表晚期凋亡细胞。2.复合材料的体内生物相容性检测：将30只Balb/c小鼠随机分为3组，A组植入复合抗感染材料，B组植入单纯钛合金材料，C组采取切开缝合处理。术后1、2、3、6周取材，采集外周血用流式细胞仪检测T淋巴细胞亚群分类CD4和CD8的百分比变化情况并进行组织学观察，评估复合抗感染材料在体内的生物相容性。3.骨科金属植入物感染动物模型构建方法：（1）将健康家兔48只随机分为5组，实验组A、B、C、D组每组10只，对照组8只。实验组动物分别在胫骨近端髓腔内注入浓度分别为1.0×10~5、1.0×10~6、1.0×10~7、1.0×10~8CFU/ml的金黄色葡萄球菌悬液1ml，并在胫骨髓腔内置入长4.0cm，直径0.25cm钛合金棒一根；对照组置入相同规格的钛合金棒后注入生理盐水1ml；（2）分别通过大体观察、体温测量、X线检查、细菌学培养、组织学观察、感染率和死亡率评价模型制备效果，筛选建模所需最佳细菌浓度。4.抗感染金属植入物对家兔感染模型感染控制作用评价：所有新西兰大白兔称量体重、测量体温后随机编号，均匀地分为2个实验组（A组和B组），兔左侧胫骨为实验侧，用来注入菌液建立感染后行材料植入。A组为实验组，植入复合抗生素的金属材料，B组为对照组，植入未复合抗生素的金属材料，取材时间点为术后2周、4周和8周。所有动物行胫骨侧位X线检测，各时间点取材后进行大体观察，细菌培养和HE染色组织学观察。结果1.钛合金表面多巴胺修饰的细胞相容性检查： MTT法测定两组材料各时间点的吸光值后，采用Wilcoxon秩和检验进行统计分析，分析结果显示，第1天两组之间材料表面细胞数量差异无统计学意义。第3，5，7，9天，未涂层组材料表面数量略高于涂层组，组间差异有统计学意义（(Pa<0.01,Pb<0.05)，表明多聚多巴胺涂层对材料表面的细胞增殖有负面影响，但影响程度较轻。两组材料上钙黄绿素绿染的细胞证实，活细胞生长状况良好，涂层组凋亡细胞多于未涂层组，实验结果与MTT测定结果相符。2.复合材料的体内生物相容性检测：流式细胞测定结果的方差分析显示，术后第一周，涂层材料组的CD4／CD8比值同切开缝合组相比无统计学差异，而未涂层材料组的CD4／CD8比值显著低于其他两组（P＜0.01），第二周和第三周的测定结果与第一周结果类似。第六周时，三组之间的CD4／CD8比值均无显著差异。组织学观察结果显示，1、2、3、6W时，未涂层组与涂层组相比，纤维组织增生明显,炎细胞浸润较多。涂层组随时间增长，纤维增生和炎细胞浸润大大减少，未涂层组6W时依然可见明显的纤维组织增生和炎症细胞浸润。3.感染动物模型制备结果：模型动物在最初两周局部软组织出现肿胀，体温明显升高随后降到正常。术后4周影像学显示实验组动物患肢出现骨膜反应、骨坏死及髓腔纤维化等表现，细菌培养显示实验组中感染程度随接种细菌浓度增高而加剧，C、D两组感染率为100%，接种1.0×10~7(CFU)/ml浓度细菌的C组具有最高的成活率为80%，D组具有最高的死亡率为70%。术后4周病理学观察到骨纤维化、骨坏死等典型感染表现。4.抗感染金属植入物对家兔感染模型的感染控制作用评价：对实验动物的术后X线随访发现，2周时实验组(A组)与对照组（B组）均有骨膜反应表现；4周和8周时对照组的骨膜反应加重同时有骨质溶解破坏和死骨出现。细菌培养结果显示，实验组骨组织和植入物培养结果一致，培养板上未培养出菌落，表明实验组复合材料在家兔感染模型上发挥了明显的杀菌、抑菌作用。而对照组的培养板上几乎长满了白色菌落，表明对照组骨组织和植入物表面存在大量细菌，两组差异明显。组织学观察结果显示实验组胫骨组织学切片镜下，髓腔脂肪细胞排列规则，结构完整，造血细胞充满脂肪细胞间隙，胫骨皮质层结构完整，排列紧密，细胞结构完整，可见明显的细胞核存在。对照组为复合空白微球材料组，动物胫骨髓腔组织病理学可见髓腔梗死及纤维化，浆细胞和单核细胞积聚髓腔，正常的空泡状脂肪细胞结构消失而被大量脓液占据，髓腔侧及皮质骨内可见坏死骨片，并且随时间延长，对照组坏死骨片范围扩大。结论1.钛合金表面多聚多巴胺涂层修饰在体外实验中会对细胞黏附造成轻微的负面影响，钙黄绿素染色发现多巴胺涂层无明显的生物毒性。明胶微球的复合使材料在小鼠体内表现出良好的生物相容性。2.成功构建出骨科金属植入物并发感染的动物模型，该模型制备方法简单且成活率高，为植入物抗感染研究提供了良好的动物模型。3.钛合金表面抗生素缓释系统在兔骨髓炎模型中呈现出良好的感染治疗效果，同时结合低弹钛合金良好的力学相容性和表面改性修饰良好的生物相容性，有望集合骨整合和杀菌两方面的优势，为骨科金属植入物相关感染(IAOMI)的防治提供有效的解决方案。更多还原

【Abstract】 Infection is still one of the serious complications of orthopedic implants. Implantedmetallic foreign body are the risk factors leading to these infections, caused by a series ofin vivo reactions include the weakening of the macrophage function and passivation of thelocal immune system, which provide convenient conditions for the proliferation ofpathogens. In addition, adsorption of protein on the surface can promote bacterial adhesionto the implant to cause infection. Sterilization of the environment and personalcontamination is our most concerned in the past. However, the new strategy aim directly atpathogenesis of such infections, which reduce infection risk by surface modification of themetal implants.Systemic administration of antibiotics have the possible disadvantage of not beingsufficiently effective due to impaired blood circulation and scar tissue around the implants,also, bringing systemic toxic side effects. The study confirmed that once the bacteria isisolated in avascular bone tissue, systemic treatment of antibiotics is difficult to achievesatisfactory drug concentration locally. Moreover, once the pathogen attached to the boneor other biological material surface, occurrence of metabolic and phenotypic changes willlead to the drug resistance to the antibiotices and escaping of immune surveillance. On the contrary, the low concentration of antibiotics may provide the power to the strains of drugresistance selection. In order to improve the therapeutic effect of bone infection, local drugdelivery method have been widely investigated and developed. Controlled release systemfor antibiotics has been favored due to its continuity and adjustable release characteristics.On the basis of the research on low elastic beta titanium alloy and controlled drugrelease, we use a surface-adherent polydopamine film onto a micro-grooved surface,which will enable the gentamicin gelatine microsphere to combine to the micro-groove onthe material surface effectually, then forming an antibiotic system on the materialsurface. Through characterization, drug release and in vitro antibacterial experiments, weconfirmed that metal surface with the antibiotic drug delivery system had advantages suchas solid combination, continued and controlled drug release, obvious inhibitory effect invitro. In the present study, on the basis of preliminary groundwork, biocompatibility andanti-infection effect of the antibiotic release system will be explored in vitro and in vivo.ObjectiveOn the basis of pre-established antibiotic drug delivery system on the surface of thelow elasticity β-titanium alloy, the biocompatibility of the poly-DOPA coating areinvestigated in vitro by cell cultrue and in vivo by implantation to into mouse musclepouch; To investigate the anti-infection effect of antibiotics controlled release system onthe metal surface, a rabbit tibial osteomyelitis model has been established andimplantation of anti-infection metal implants into infected medullary cavity has beenadopted to study the effect of infection control, to aim at provide a theoretical andexperimental evidence for anti-infection research and development of metal materials.Methods1. Observation of cell biocompatibility of the dopamine modified titanium surface:To explore the biocompatibility of poly-dopamine coating, bone marrow stromal cellsfrom rabbits were seeded in poly-dopamine modified titanium surface, and then the MTTquantitative calculation and laser confocal microscope qualitative observation were usedto analyze cell adhesion, proliferation on the surface and compared with the uncoatedmaterial, observing the the biocompatibility changes after the coating modification. Calcein-AM and propidium iodide (PI) solution has been used to stain the live cells anddead cells respectively, in order to observe the cell growth and survival.2. Biocompatibility testing of composite materials in vivo:30Balb/c mouse wererandomly divided into three groups, group A, group B and group C were seperatelytreated with anti-infective metal implants, pure titanium alloy implants and incision-suturetreatment. After1,2,3,6weeks after operation, collected peripheral blood was tested byflow cytometry to detect changes in percentage of the T-lymphocyte subsets CD4and CD8,histological observation was conducted also, to evaluate the biocompatibility of thecomposite anti-infective material in vivo.3. Establishment an animal model of IAOMI:(1)48healthy rabbits were randomlydivided into five groups, the experimental group A, B, C, D(n=10), and the controlgroup(n=8). In the experimental groups,1ml volume of the serial dilutions of the bacteriaof S. aureus ATCC25923[10~5to10~8colony-forming units (CFU)/ml] were inoculatedinto the medullary cavity of the animals before a titanium cylinder (4.0cm length and0.25cm diameter) was put into. While in the control group, the same volume of saline wasinoculated and then the same size of titanium cylinder was implanted. The efficiency ofthe IAOMI model in rabbit was evaluated by clinical, radiologic, microbiologic andhistological parameters at the4th week after the operation, and then an optimalconcentration of the bacteria inoculated was chosen.4. Evaluation of the anti-infective effects with implantation of modified metalimplants into the rabbit infection model: New Zealand white rabbits were randomlydivided into2groups(A and B) after mesuring the body weight and body temperature. Theleft tibia were chosen as the experimental side, used to establish the infection model formaterial implantation. Group A was experimental group, insertion with the antibioticloaded metal material, while group B was the control group inserted with the metalmaterials without the antibiotics. The samples were harvested after the time of2weeks,4weeks and8weeks. All animals were detected by lateral X-ray at the tibia, aslo, thegeneral observation, bacterial culture and histological observation with HE staining atdifferent time points. Results1. Cell biocompatibility on the surface of the titanium alloy after modified bydopamine: The absorbance values for each time point in the two groups were assayed byMTT method, then, the values were statistically analyed by using the Wilcoxon rank sumtest. The results showed that there was no significantly difference for the number ofadhesive cells at the first day between the two groups. The number of the adhesive cells inuncoated group were slightly higher than that in the coated group at days3,5,7,9. Thedifference between the two groups was statistically significant ((P_a<0.01, Pb<0.05),which showed that the poly-dopamine coating have a negative impact about cellproliferation on the surface, but the impact limited to a low extent. Calcein green-stainedcells in the two groups confirmed that living cells grew well, apoptotic cells in the coatedgroup were more than the uncoated group, which was in accordance with the results ofMTT assay.2. Biocompatibility test of composite materials in vivo: The variance analysis of theresults of flow cytometry measurement showed that, after the first week, the CD4/CD8ratio of the coated group had no significant difference with the incision-suture group,while the CD4/CD8ratio of uncoated group was significantly lower than the other twogroups (P <0.01). The measurement results for the second and third weeks were similar tothe first week. At sixth week, CD4/CD8ratio were not significantly different between thethree groups. The histological observations showed that at the time point of1,2,3and6weeks, the uncoated group had a obvious fibrous tissue hyperplasia and moreinflammatory cell infiltration compared with the coated group. Fibrous tissue hyperplasiaand inflammatory cells infiltration were greatly deceased in the coated group with timeincreased. Obvious fibrous tissue hyperplasia and inflammatory cell infiltration still can beseen in the uncoated group at6weeks.3. Establishment of animal infection models: Clinical progression observed by thepostoperative swelling of the local soft tissue, and a significant increase in bodytemperature within the early two weeks and then had a decrease. There were varyingdegrees of periosteal reaction, osteolysis, sequestrum formation in the experimental groups at the4th week after the operations on radiology. Bacteriological culturing showed that theseverity of infection was aggravated in the experimental groups, with the increase of theconcentration of inoculums. The infection rate of group C and D was100%, and there wasthe highest survival rate in group C that was80%, which inoculated1.0×10~7(CFU)/mlbacterial, while there was the highest mortality in the group D which was70%.Intramedullary fibrosis and sequestrum formation indicated infection on histology at the4th week after the operations.4. Evaluation of the anti-infective effects with implantation of modified metal implantsinto the rabbit infection model: Postoperative X-ray follow-up to the experimental animalsshowed that periosteum reaction can be seen in the two groups at2weeks; Thisperiosteum reaction was aggravated in the group B at the time of4and8weeks,accompanyed with osteolysis destruction and sequestrum. Bacterial culture showed thatinoculation with bone tissue and implants had no bacterial colonies on the culture plates ingroup A, which indicating that the anti-infective metal implants in group A played asignificant bacteriostatic and bactericidal role. While the white colonies almost covered allthe petri dish in group B, which indicating that there were a large number of bacteriaexisting in the bone tissue and on the implant surface. The difference between the twogroups were significant. Histological observation showed that the marrow fat cellsarranged regularly with hematopoietic cells distributed in the cells’ gap in group A.Structure of ortical layer of was complete and tightly packed. Cells were also in a goodcondition, nuclei can be obviously observed. Infarction and fibrosis could be observed inthe medullary cavity in group B, plasma cells and monocytes were accumulated in themedullary cavity. Normal vacuole fat cell structure disappeared and occupied by a largenumber of pus. Necrotic bone fragments existed in the medullary cavity and cortical bone,and range of necrotic bone chip expanded with time increased.Conclusion1. Surface modification by using poly-dopamine coating to the titanium alloy willcause a slight negative impact on cell adhesion in vitro. The calcein-AM and PI stainingindicated that there was no obvious biological toxicity. Composite materials contain gelatin microsphere exhibit a good biocompatibility in vivo.2. An animal model of IAOMI had been successfully established，which was easilyprepared and had a high survival rate. It could be a good animal model for the study ofIAOMI.3. The antibiotic drug delivery system on titanium surface showed a goodanti-infection effect in the rabbit osteomyelitis model. Combined with good mechanicalcompatibility from low elastic titanium alloy and good biocompatibility from surfacemodification, this method is expected to providing a effective solutions for the preventionand treatment of IAOMI by achieving the two advantages of bone integration andsterilization.更多还原