【作者】 崔宜栋；

【导师】 蔡锦方；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 研究背景:慢性骨髓炎难以治疗、复发率高,是骨科的难症之一。慢性骨髓炎的治疗基本原则之一就是在充分的抗菌素治疗基础上彻底清除病灶。尽管采用了彻底的清创方法,但局部仍有一定数量的细菌残留。在一定条件下,这些细菌易大量繁殖,常导致治疗失败。同时由于局部血供差,导致机体在局部发挥杀灭细菌免疫功能困难。抗生素的局部应用越来越受到人们的重视,抗生素的局部应用可以直接作用于病变部位,不需血液将药物抗生素携带至病变区域,可在局部获得持续的较高药物浓度,同时保持较低的血药浓度,避免抗生素全身系统用药的毒副作用。但是随着近年来抗生素的不合理应用,慢性骨髓炎致病菌种类发生变化,产生细菌变异,细菌耐药,抗生素的治疗效果逐渐下降。所以,寻找一种新型的抗微生物剂治疗骨髓炎,成为本课题的目的之一。臭氧有极好的杀菌、抗炎以及促进创面愈合作用,并且不易产生细菌耐药,封闭负压吸引起到很好的引流作用,臭氧水灌注负压吸引为慢性骨髓炎的治疗提供了一种有效的新方法。目的研究不同浓度臭氧水对金黄色葡萄球菌和铜绿假单胞菌的杀灭作用,以及对臭氧水抗炎作用的机理进行研究。制备金黄色葡萄球菌感染的大鼠胫骨慢性骨髓炎的动物模型,并在大鼠胫骨近端慢性骨髓炎动物模型的基础上,研究臭氧水灌洗负压吸引治疗大鼠胫骨慢性骨髓炎的治疗效果,为慢性骨髓炎的治疗提供一种既能杀菌又能抗炎的新方法,并为其进一步临床应用提供实验依据。方法本实验由4部分组成1、臭氧水对金黄色葡萄球菌和铜绿假单胞菌杀灭作用的实验研究取实验菌金黄色葡萄球菌、铜绿假单胞菌用无菌生理盐水制备成菌悬液,麦氏比浊法调整含菌量,分别用不同浓度的臭氧水溶液处理不同的时间作用于两种菌悬液,计算两种细菌的杀灭率。2、臭氧水抗炎作用的实验研究在二甲苯致小鼠耳肿胀模型的基础上,分别采用2mg/L臭氧水溶液、扶他林膏剂、及生理盐水湿敷创面,通过观察耳壳厚度差、重量差、血清TNF-a检测和组织病理学,探讨臭氧水抗炎作用的机理。3、大鼠胫骨慢性骨髓炎动物模型的建立及评价采用胫骨近段髓腔注射5%鱼肝油酸钠和不同浓度的金黄色葡萄球菌菌液的方法诱导骨髓炎,并探讨诱导的骨髓炎模型与菌液浓度的关系,为慢性骨髓炎的实验研究提供一种合适的动物模型。4、臭氧水灌注负压吸引治疗大鼠胫骨慢性骨髓炎的实验研究在大鼠慢性骨髓炎模型的基础上,常规病灶清创,分别采用2mg/L的臭氧水,庆大霉素溶液、生理盐水局部灌注骨髓炎创面,半透膜封闭,间断负压引流,通过大体标本观察、放射学、细菌学、ELISA法和组织病理学方法,阐明臭氧水局部应用治疗实验性大鼠胫骨慢性骨髓炎的效果。结果1、臭氧水对金黄色葡萄球菌和铜绿假单胞菌杀灭作用的实验研究2种主要的致骨髓炎的标准菌株金黄色葡萄球菌和铜绿假单胞菌所需的臭氧水的杀菌条件相近,2mg/L臭氧水溶液作用60s或者1mg/L臭氧水溶液作用60s (铜绿假单胞菌), 90s (金黄色葡萄球菌)均可100%杀死菌悬液中的细菌。2、臭氧水抗炎作用的实验研究臭氧水组(A组)、扶他林组(B组)、生理盐水组(C组)对小鼠耳肿胀模型耳壳重量差进行统计学分析示:A,B组明显小于C组(P<0.01),A、B组比较无明显统计学差异(P>0.05);血清TNF-a浓度检测示:A、B组明显小于C组(P<0.01),A、B组比较无明显统计学差异(P>0.05)。组织学观察显示:生理盐水组表皮细胞间及细胞内水肿,能发现较多的单一核细胞及少数多形核细胞的浸润,真皮结缔组织水肿,血管周围可见轻度炎症细胞浸润;臭氧水组及扶他林组水肿消退,炎性细胞浸润减少。3、大鼠胫骨慢性骨髓炎动物模型的建立及评价A组(注入0.lml生理盐水),B组(注入0.lml 1×106 CFU/ml细菌),C组(注入0.lml 1×108 CFU/ml细菌),D组(注入0.1 ml 1×1010 CFU/ml细菌),各组造模后4周,影像学显示出现不同程度的软组织肿胀,骨密度降低,骨小梁纤细,骨小梁间隙加宽,骨皮质变薄,骨溶解,死骨形成;组织学切片显示均出现不同程度的中性嗜酸性粒细胞浸润,骨髓腔内骨细胞不同程度坏死。注入0.lml 1×108 CFU/ml细菌,即可获得可靠稳定的大鼠胫骨慢性骨髓炎模型。4、臭氧水灌注负压吸引治疗大鼠胫骨慢性骨髓炎的实验研究术后2周各组Norden改良骨髓炎分值进行统计学分析显示:臭氧水组(A组)、庆大霉素溶液组(B组)、生理盐水组(C组)比较差异有非常显著性;术后2周,大体标本可见A、B组保持较正常外观形状,末发现明显的骨质破坏及髓腔脓液,C组标本外形粗大,骨质破坏明显,骨缺损边缘有不规则增生及硬化,其间可见脓性分泌物;术后7、14天时,每克骨组织标本细菌计数转换成对数后进行统计学分析结果显示:A、B组细菌计数明显小于C组(P<0.01),A、B组比较,A组细菌计数小于B组(P<0.05);术后7、14天时,局部骨组织TNF-a浓度示:A、B组TNF-a浓度明显小于C组(P<0.01),A、B组比较,A组浓度小于B组(P<0.05);组织学观察显示:A、B组炎症反应明显较C组轻,A组又较B组炎症反应轻。结论:2mg/L的臭氧水的能够有效杀灭金黄色葡萄球菌和铜绿假单胞菌,并且能抑制炎症反应的进程,促进炎症反应的消退;臭氧灌注负压吸引系统在大鼠胫骨慢性骨髓炎的治疗中,臭氧水能够控制感染、抑制局部炎症反应、促进组织修复,改善骨髓炎的骨组织病理改变,负压吸引能通畅引流,避免臭氧泄漏造成的环境污染,可逐渐替代局部抗生素的应用,为慢性骨髓炎的治疗提供了一种新的途径。更多还原

【Abstract】 Background:Chronic osteomyelitis is a significant problem of patients and orthopedic surgeon. It is very difficult in treatment of this kind of disorder because of its character. One of the basic principles of the treatment of chronic osteomyelitis is adequate antibiotic treatment on the basis of completely clear of lesions. Despite the use of a thorough debridement method, there are still a number of bacterial residues in local osteomyelitis region. The treatment is easy to fail because of the amount of reproduction of bacterium. Because of bad blood supply in the local region, it is difficult for the body immune function to kill the bacterium. Local drug delivery system can be directly on the lesion site, and didn’t carry the drug to the lesions by blood and can be sustained in a high local drug concentration. With the unreasonable application of antibiotics in recent years, the types of pathogens are changing in chronic osteomyelitis, which result in bacterial mutation and bacterial resistance. The therapeutic effect of antibiotics is decreasing. So one of the purposes of this project is to find an effective fungicide to treat chronic osteomyelitis. Ozone water can effectively treat wound infection and accelerate wound healing, and it is difficult to produce bacterial resistance. VSD is smooth for suction drainage. Ozone water infusion and vacuum aspiration is a new and effective method for treatment of osteomyelitis.Objective:To investigate bactericidal effect of different concentrations of ozone water on Staphylococcus aureus and Pseudomonas aeruginosa.The mechanism of anti-inflammatory effects of ozone water was studied. Rat models of chronic osteomyelitis at proximal tibia were established. The effect of ozone water was studied on the base of rabbit model of chronic osteomyelitis. Ozone water provide not only a new method of sterilization but also anti-inflammation to treat the chronic osteomyelitis. It can provide experimental evidence for further clinical applications.Method: The experiment was divided into four parts.1、The antimicrobial activity of ozone water on Staphylococcus aureus and Pseudomonas aeruginosa.Staphylococcus aureus, Pseudomonas aeruginosa bacteria were prepared in sterile saline suspension, respectively. Adjust the amount of bacteria by McNamara bacterial turbidimetric method. Different concentrations and time points of ozone water were used in bacteria suspended solution, and the bacteria killing rate were calculated.2、Experimental study of ozone water on Anti-inflammation.On the basis of models of xylene-induced by acute inflammatory edema of mice ear, the mice were devided randomly into 3 groups: ozone water group, voltaren group and normal saline group. The observations of model animals included thickness and weight difference of mice ear, detection of serum TNF-a, histopathology manifestation, which can illuminate the results of anti-inflammation.3、The establishment and evaluation of experimental chronic osteomyelitis induced by staphylococcus aureus in ratsFive percent sodium morrhuate and serial dilutions of the bacteria of Staphylococcus aureus suspended in saline were inoculated into the proximal metaphysis of the tibia.The relationship between the inoculation dose of the bacteria of Staphylococcus aureus and the severity of experimental Staphylococcus aureus osteomyelitis in rats was studied to provide a suitable animal model of chronic osteomyelitis.4、Experimental study of treatment of chronic osteomyelitis with ozone water infusion and vacuum aspiration.In order to observe the therapeutic effects of ozone water on osteomyelitis of rat models, modeled chronic osteomyelitis SD rats were devided randomly into 3 groups: ozone water group, antiseptic solution group and normal saline group. All were lavaged for 2 weeks. The observations of model animals included blood leukocyte count, bone X rays,detection of TNF-a and histopathology manifestation, which can illuminate the results of sterilization and anti-inflammation.Results:1、The antimicrobial activity of ozone water on Staphylococcus aureus and Pseudomonas aeruginosa.The ozone water has the similar bactericidal effect on the 2 bacteria .The bacteria in the suspension was completely killed with the treatment of 2mg/L ozone water for 30s or 1mg/L ozone water for 60s(Pseudomonas aeruginosa) and 90s (Staphylococcus aureus).2、Experimental study of ozone water on Anti-inflammation Statistical analysis indicate that there was a significant difference between group A (ozone water group),group B(voltaren group) and group C(normal saline group), no significant difference between group A and group B of mice ear weight difference after 2 hours. Concentration of TNF-a of mice blood serum indicates that group A and group B significantly less than group C(P<0.01) , no significant difference between group A and group B (P>0.05). Histological observation group C showed that there were significant epidermal cells and intracell- ular edema infiltration of mononuclear cells, dermal connective tissue edema. Histological observation group A and B find the reduction of edema and inflammatory cell infiltration3、The establishment and evaluation of experimental chronic osteomyelitis induced by staphylococcus aureus in ratsFour weeks after modeling of A group(0.1ml Normal Sodium was inoculated)、B group(0.1ml 1×106 cfu/ml staphylococcus aureus )、C group (0.1ml 1×108 cfu/ml staphylococcus aureus)、D group (0.1ml 1×106 cfu/ml staphylococcus aureus), radiography show that there were different degrees of soft tissue swelling, bone mineral density reduced, bone trabecular rare, cortical thinning, osteolysis, sequestra formation in experimental groups;histomorphology indicated that there were varying degree of neutrophil infiltration, necrosis of marrow cells and interstitial hemorrhage。It can establish successfully the rat model of acute staphylococcal osteomyelitis with the staphylococcus aureus of 1.0×l08CFU inoculating directly into tibial bone of rat.4、Experimental study of treatment of chronic osteomyelitis with ozone water infusion and vacuum aspirationStatistical analysis indicate that there was a significant difference between group A(ozone water group),group B(antiseptic solution group) and group C(normal saline group), no significant difference between group A and group B of Norden osteomyelitis scores after 2 weeks. Bacterial count per gram indicate that group A and group B significantly less than group C(P<0.01), group B less than group A,,and there was a significant difference(P<0.05). Concentration of TNF-a of local bone indicates that group A and group B significantly less than group C(P<0.01), group B less than group A, and there was a significantly difference(P<0.05). Histological observation showed that the inflammation response of group A and group B was significantly lighter than group C, group A lighter than group B. ConclusionOzone water can effectively kill Staphylococcus aureus and Pseudomonas aeruginosa. Ozone water can also inhibit the inflammatory response process and promote the extinction of inflammation. In the treatment of osteomyelitis with ozone water infusion and vacuum aspiration, ozone water can skill the bacterium, prohibit local inflammation response, promote tissue reparation and the bone pathological changes of chronic osteomyelitis. VSD is easy and smooth for suction drainage, which can avoid environmental pollution caused by ozone leak. Ozone water can gradually replace the application of antibiotics and provides a new method for the treatment of chronic osteomyelitis.更多还原