【作者】 费军；

【导师】 王爱民；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 研究背景及研究目的随着材料科学和临床医学的发展,骨科手术率和术中各种内、外固定器械的使用率日益上升。但是相伴而生的骨和软组织感染作为骨科手术最常见和复杂的并发症,也早已成为骨科医生所面临的极富挑战性的难题之一。据AO组织2006年报道:每年在美国约有200万例院内感染病例,其中约50%与内植物有关。在使用内固定的人群中约5%发生了感染。同时,内固定术后感染可能还会导致包括由于全身应用抗生素、多次清创翻修手术、住院时间延长、截肢甚至死亡等对患者不利的严重后果。因此,内固定术后感染早已成为研究焦点之一。目前观点认为,与内固定术后感染主要表现为软组织感染和骨髓炎两种形式,是使用内固定所导致的严重并发症之一。尤其在开放性骨折中,各种固定器材包括钢板螺丝钉、外固定支架、髓内针等均可能和感染有关。资料显示:通常闭合性骨折的感染率约1.5%,而开放性骨折的感染率则高达3-40%。创伤所致的开放性骨折呈逐年上升的趋势。在开放性骨折中约60%以上的在损伤时受到了细菌的污染(5,10,11)。术后为预防感染,医生都会大剂量使用广谱抗生素。但是,尽管采取了上述积极的措施,不幸的是仍有一部分患者发生了感染(3)。对这部分病人的后期处理是骨科领域非常棘手的一个问题。骨折后通常会使用内固定或者外固定材料来固定骨折部位。而内固定具有固定效果确定,便于早期进行功能锻炼,便于护理的优点,在患者病情允许情况下,是骨科医生的首选。钢板往往被推荐在闭合性骨折和GustiloI型开放性骨折中使用。一般认为,伤口是否发生感染主要取决于伤口的性质、细菌的数量与毒力和宿主抵抗能力,以及伤口内异物的存留。最常见的导致“固定物与感染”的细菌为金黄色葡萄球菌和表皮葡萄球菌。金黄色葡萄球菌和表皮葡萄球菌均可以产生生物膜。生物膜的形成分两部进行(39),首先是细菌黏附于固定物的表面,然后细菌之间相互黏附形成多层状的细菌以进入细菌静止或休眠期,从而抵抗临床抗生素治疗和免疫系统的吞噬细胞清除细菌。金黄色葡萄球菌是导致手术或创伤后的主要致病菌(30%),是内植物相关的骨与关节、软组织感染的首要细菌。它一旦进入淋巴管或血液中,将导致脓毒症。耐甲氧西林金黄色葡萄球菌(methicillin-resistant S.aureus MRSAs)具有耐β-内酰胺类抗生素如青霉素、三代头孢、链霉素、四环素及磺胺类药物的特点,并且对氨基糖肽类抗生素和万古霉素的敏感性也在降低。目前已有对万古霉素和替考拉宁耐药的报道。当金黄色葡萄球菌黏附在固定物表面时,人体细胞并不能将其清除掉。骨感染的结果就是急性或慢性骨髓炎。其病理类型与细菌类型无关,但是金葡菌感染更易于产生慢性侵袭性骨髓炎。按照感染发生的时间,主要将其分为三类:早期感染(小于2周),主要在创伤或固定手术期间获得感染,是高致病性微生物感染(金葡菌、革兰氏阴性杆菌)所致。延迟感染(2-10周)和晚期感染(超过10周),主要在创伤或固定手术期间获得感染,低致病性微生物感染(凝固酶阴性葡萄球菌),偶尔由远隔部位的血源性播种所致。早期感染的临床症状出现得较早并持续出现局部疼痛、大血肿、发热,常见致病菌为高毒力型,当伤口愈合受到影响的时候,必须积极地寻找伤口的坏死边缘和术后的血肿及感染灶(96,97)。延迟和晚期感染的临床表现、治疗及预后相似。一般表现为持续或者加重的疼痛、假关节形成、固定物松动、有时出现的窦道是延迟感染的标志。但也有临床症状和体征缺乏的病例。其主要致病菌为低毒力型。其可能发生的原因是早期抗感染的力度不足以使微生物彻底根除,与假体关节感染相反,固定物所导致的晚期感染发生频率较低(98)。内固定术后感染的治疗包括手术治疗和抗生素治疗,抗生素的使用往往在配合手术翻修或取出固定物和彻底清创的基础上使用。针对抗生素的使用方法可以归纳为全身使用和局部使用两类。局部抗生素的使用是治疗固定物感染的重要措施之一,目前多采用缓释载体载抗生素以期在局部产生较长时间的局部高浓度抗生素。其基本理念均为:抗生素在特定的部位逐步地释放抗生素,获得较全身性使用抗生素更高的浓度。在目前内固定术后感染发生率高、抗生素耐药菌株日益增加和系统性全身应用抗生素局限性逐渐被认识的背景下,局部预防性或治疗性应用抗生素已成为在固定物相关感染的治疗中具有前景的主要措施之一。尤其是把固定物与抗生素组合或者合成在一起的途径,对医生来说极具吸引力。这种设想把固定物本身变成了“抗生素释放系统”,其好处是显而易见的:在同往常一样植入固定物的同时获得了额外的局部释放抗生素的益处,使局部聚集了高浓度的抗生素而很少有全身性的副作用,特别是在如髓内针固定术等闭合性复位的手术中,更有利于患者的治疗。同时,通过生物降解材料可以把抗生素物质限定在一定的时间段内释放以产生合理的药效学,最大程度的减少了对耐药菌株筛选的风险。因此被看作是骨科器械发展的“未来”。抗生素涂层的理论首先由Darouice于1998年提出并实施。目前应用较多的途径是生物降解材料如聚D,L-乳酸(poly D,L-Lactide PDLLA)负载抗生素以达到预防或治疗感染的目的。局部抗生素释放系统应该达到以下基本要求:缓释技术必须保证快速释放超过MIC浓度水平的抗生素;药物释放必须在一定的时间内,避免产生耐药性菌株;杀菌性抗生素应该产生杀菌效应,而不是仅仅抑菌;药物缓释系统应该自行降解。PDLLA涂层不仅具备上述条件,而且有利于阻止细菌黏附在固定物表面。MRSA对绝大多数抗生素交叉耐药,但是仍然对万古霉素或去甲万古霉素100%敏感。在体外药敏实验中,去甲万古霉素对MRSA非常有效,而且与其它种类的抗菌药物无交叉耐药。另外用MRSA在试管内进行传代培养试验,其对万古霉素的诱导耐药性也很低。去甲万古霉素能够抑制细菌细胞壁的合成,发挥杀菌作用。另外还可以通过改变细菌细胞壁的通透性,阻碍细菌RNA的合成的机制杀灭细菌。目前,去甲万古霉素对MRSA的MIC90为0.5-3.1mg/L。因此,在内固定表面涂层技术渐趋成熟的启示下,我们采用治疗金葡菌感染的特效药物去甲万古霉素,研制出“载去甲万古霉素聚乳酸缓释钢板”。自内而外的早期释放去甲万古霉素,直接杀灭金葡菌,破坏或阻碍固定物表面生物膜的形成,通过检测该钢板释放去甲万古霉素的释放动力学及其对金黄色葡萄球菌体外抑菌作用和新西兰大白兔胫骨中段横断模型,探讨其在创伤性骨髓炎的预防和治疗的应用前景。实验方法1.采用乙酸乙酯、丙酮、水的三元溶剂组合,运用溶胶-凝胶法制作载去甲万古霉素钢板。2.将两种钢板分别浸于1ml和10ml的浸提液中,应用凝胶渗透色谱和高效液相色谱仪分别检测PDLLA的降解和去甲万古霉素的局部释放浓度,了解该钢板释放去甲万古霉素的动力学过程。3.将载万古霉素钢板和普通钢板分别与不同浓度的金葡菌混悬液进行普通培养后计数金葡菌菌落数。扫描电镜检查钢板金葡菌在两种钢板表面的黏附情况。4.制备胫骨中段单侧皮质横断骨折并金葡菌污染模型,分别置入载去甲万古霉素缓释钢板和普通不锈钢钢板,比较两种钢板对兔胫骨金葡菌感染模型的预防作用。5.制备兔胫骨中段单侧皮质横断骨折并感染的模型。分别置入载去甲万古霉素缓释钢板和普通不锈钢钢板,比较两种钢板对兔胫骨金葡菌感染模型的治疗作用。实验结果1.采用乙酸乙酯、丙酮、水的三元溶剂组合,运用溶胶-凝胶法制作载去甲万古霉素聚乳酸缓释钢板。涂层中的聚D,L-乳酸(poly D,L-Lactide, PDLLA)降解失重率在第1、3、7、14、28天分别为0.4%、0.9%、1.75%、2.65%、4.76%。涂层中去甲万古霉素的局部释放浓度在35天内均高于去甲万古霉素对金葡菌的最低抑菌浓度。2.载去甲万古霉素缓释钢板可以有效降低兔胫骨金黄色葡萄球菌污染模型的感染率。提示涂层中释放的去甲万古霉素在可以有效杀灭伤口污染的金葡菌。3.载去甲万古霉素缓释钢板可以有效降低胫骨金黄色葡萄球菌污染模型大白兔术后第3、7、14天静脉血白细胞计数和CRP含量。但是对大白兔的肛温和体重没有影响。4.载去甲万古霉素钢板周围组织中去甲万古霉素的浓度在植入后28天仍然达到最低抑菌浓度。同时血浆中的浓度极低。可以有效避免全身使用去甲万古霉素的副作用。5.载去甲万古霉素缓释钢板对胫骨感染性骨折兔的感染控制率高于普通钢板。结论1.采用溶胶-凝胶法可以成功制作载去甲万古霉素聚乳酸钢板。在检测的35天内,该钢板在体外释放去甲万古霉素的释放动力学稳定,同时盐酸去甲万古霉素的载体PDLLA也能稳定、匀速地降解。2.释放的盐酸去甲万古霉素可以有效抑制金葡菌菌落的生长以及减少其金葡菌在钢板表面的黏附,说明载去甲万古霉素缓释钢板在体外可以有效抑菌。为该钢板在体内实验中的应用奠定基础。3.在预防兔胫骨感染的体内实验中,载去甲万古霉素缓释钢板组较普通钢板组为低,两组间感染率有明显区别;从白细胞计数、CRP含量、组织学和放射学角度观察,载去甲万古霉素缓释钢板不仅在体外可以有效抑菌,而且能够预防兔胫骨金黄色葡萄球菌污染模型发生感染。为该钢板过渡到临床应用提供了实验依据。4.载去甲万古霉素钢板治疗对内固定术后感染可能有一定的疗效。更多还原

【Abstract】 With the development of materials and medicine, it has increased greatly in the rate of operation and the rate of uesing the fixation device. But at the same time, the infection following with operations and fixation devices became a big tough problem. It was reported by AO that 50% of 2 millions infection cases in one year were cause by implants. The severity consequence are large dose antibiotics applicated all over the body, many times overhauling operations, prolongation of staying in hospital, amputation and death and so on. So, the infection following with the fixation devices has become a focus .Soft tissue infection and carious osteitis are the two kinds of appearances which are accompany by the fixation devices. Especially in open fracture, all kinds of fixation devices , e.g. steel plate, screw, external fixatior and intramedullary nail et al, can accompany bone infection. Some date were reported that the infection rate was 1.5% in closed fracture. But in compound fracture, the infection rate increased to 3-40%. With the development of transportation, the proportation of compound fracture has been increasing gradually. Above 60% of those fracture were polluted by bacteriums. So, for preventing infection, large dose of broad-spectrum was used. A lot of active steps were adopted, but a part of patiences were infected. How to treat those patiences? The orthopedists have no good ideas.After fracture, internal or external fixations are used. The internal fixation is the first choice in suitable cases. The steel plate is prefered in closed fracture and Gustilo I compound fracture. Many matters can caused infection. For example, the wound, the quantity and toxcity of bacteriums and the extraneous matter in wound. The straphylococcus aureus (SA) and straphylococcus epidermidis (SE) are detected usually in infected wound implanted in fixation device. All of SA and SE can produce biological membrane. Firstly, the bacteriums acctete the surface of fixation device. Secondly, those bacteriums can possess each other to form multilayer bacterium lamellar for resist the antibiotics and phagocyte. The SA is the main pathogenic bacteria after operation or trauma. At same time it is chief bacteria related with infection in bone, joint and soft tissue also. Methicillin-resistant S.aureus (MRSAs) are kind of SA which are drug fast to peniciline, tert-cephalosporin, phytomycin, ambramycin.The phagocyte can not kill the SA adhering at surface of fixation device. The infection caused by SA can induce facilely to chronic invasive carious osteitis.According to the time of infection, the infection can be classified to III species. The early period infection which genesis in 2 weeks is caused by trauma or operation. The pathogenic bacteria is hypsi-invasive organism, e.g. SA, gram-negative bacillus. The delay infection which genesis in 2nd to 10th week and the advanced stage infection which genesis exceed 10 weeks are caused by trauma and operation also. The pathogenic bacteria is hypo-invasive organism, e.g. coagulase negative staphylococcus (CN-S). The clinical symptom of early infection is pain, blood tumor, fever and so on. The delay and advanced stage infection have same clinical symptom. The patients are contiue and aggravated pain, pseudoarticulation formation, fixation device cinch. Some there is sinuses which is the mark of delay infection. Be different with infection of prosthesis joint, the infection connected with fixation device is early infection mainly.To treat the infection connected with fixation device, an operation must to be done is to take out of the device. At same time, a large dose antibiotics is used. This is an important measure that antibiotics is used at regions. Now, the delayed release antibiotics connected with delayed release supporter are uesd for longer hypso-density in regions.The antibiotics coating was raised and practiced firstly by Darouice.Now, the poly D,L-Lactide (PDLLA) is used generally. The relayed release qntibiotics coating must be to achieve requirements: 1) The antibiotics in coating must be released that density of regional antibiotics exceeds the minimal inhibitory concentration (MIC);2) The release must keep on a long time to aviod drug resistance strain; 3) The bactericidal antibiotic must be bactericidal effect, but bacteriostasis; 4) The supporter must degradate initiativly. The PDLLA not only achieve all requirements, but also can prevent bacterial adhesion. Though MRSA is drug fast to many antibiotics, it is sensitive to vancomycin and norvancomycin. In vitro, norvancomycin is effective extremely to MRSA. And there is achiasmate drug fast with others. Norvancomycin can inhibit composition of cell wall and RNA. The MIC90 OF norvancomycin to MRSA is 0.5-3.1mg/L.In this study, we advanced developed the steel plate coating with delayed release PDLLA- norvancomycin and research the function of preventing infection in open fracture.There are some results.1.Acetoacetate, acetone and water were used to manufacture coating by collosol-gel method. The degradation agravity rate of PDLLA is 0.4%,0.9%,1.75%,2.65%,4.76% at 1st,3rd,7th,14th and 28th day. In 35 days, the released norvancomycin are higher than MIC90 to SA.2. The infection rate in expermental group was lower than that in contral. It is suggested that the released norvancomycin from steel plate’s coating can kill the SA in tibia. But the steel plate coating with delayed release PDLLA- norvancomycin can not treat the infection in open fracture.3. The WBC and CRP were lower in experimental group at 3rd, 7th, 14th day after operation than that in contral. But there were difference in rectal temperature and weight among two groups.4. In 28th day after steel plate implanted into rabbits, the norvancomycin concentration of surrounding tissue was not lower than MIC90. At same time, the norvancomycin concentration of blood plasm was very low. So, the side effect of norvancomycin could be avoided.5. The steel plate coating with delayed release PDLLA- norvancomycin controlled 13 of 25 rabbits treated the infection in open fracture.While the stainless steel only controlled 5 of 25 rabbits treated the infection in open fracture.The controlled rate were 52% vs 20%. But there was not statistically significant between the two groups.更多还原