【作者】 唐良华；

【导师】 王爱民；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 研究背景AO组织2006年报道开放性骨折的感染率是3~40%(闭合性骨折的感染率为1.5%),在受伤当时,高于60%的开放性骨折就受到了细菌的污染。而在战时火器伤中,严重的软组织损坏和污染导致在伤道周围0.5cm范围内出现了大量的细菌,即使经过了充分的伤口清创,骨和软组织的感染率仍有25% ~ 40%。Maurer等认为在开放性胫骨骨折中使用内固定后,感染率又再增高了30%。例如,在美国,每年大约200万医院感染病例中,有几乎一半与内植物有关;在英国,每年用于植入物相关感染的费用达到1100万英镑。除了消耗巨大社会财富,还给病人带来巨大的痛苦,甚至导致截肢、死亡等严重后果。因此研制具有抗感染作用的内固定植入物,预防和治疗内植入物相关感染,具有重要的意义。过去的三十多年中,在一系列新的生物相容性较好的无机材料和有机高分子材料基础上逐渐发展起来一种新型局部给药方式--抗生素缓释系统,在治疗开放性骨折、骨髓炎和感染性骨缺损等方面有着广泛的应用前景。万古霉素属于糖肽类抗生素,对各种革兰氏阳性菌都具有高度抗菌活性,是目前治疗耐甲氧西林金葡菌(MRSA)和耐甲氧西林表皮葡萄球菌(MRSE)等所致重症感染的首选抗生素,在临床治疗学上占据重要地位。因此被认为是较理想的的局部应用的抗生素之一;聚-DL-乳酸(PDLLA)是具有良好生物相容性、可降解吸收性、良好的力学稳定性和良好的成骨诱导能力,被广泛应用于生物医学工程领域,尤其是在药物控制释放体系中,它在植入物表面通过水解作用而降解,需要3~6个月时间,最终降解产物是CO2和H2O,中间代谢产物乳酸也是体内正常糖代谢产物,其降解产物最终通过柠檬酸循环而代谢,因此在体内不会对人体产生明显的不良影响;羟基磷灰石(HA)也具有良好的生物相容性和可降解吸收性,骨外科上可用作内固定材料。若能将PDLLA或HA与内固定材料及抗生素结合起来,制作出载抗生素缓释系统,从而在病灶局部释放出抗生素以使得治疗开放性骨折和火器伤等所致感染的效果能够得到大大提高。目的以PDLLA为载体,采用solvent casting技术,研制出载万古霉素-PDLLA钢板;同时以HA为载体,采用仿生溶液法研制出载万古霉素-HA钢板,通过体内体外试验的研究,希望此两种钢板在骨折愈合过程中能够在局部发挥强力的抗感染性能,起到预防和治疗感染的双重作用,为开放性骨折和火器伤提供早期的较好的治疗措施。方法第一部分:实验一:1.采用solvent casting技术制作载万古霉素-PDLLA钢板。2.通过扫描电镜对载万古霉素-PDLLA钢板涂层进行观察。3.测定载万古霉素-PDLLA钢板涂层中万古霉素的含量及在20ml磷酸盐缓冲液中不同时相点的万古霉素释放浓度,了解该钢板涂层中万古霉素的释放动力学过程。4.将载万古霉素-PDLLA钢板置入均匀涂有1.5×108 CFU/ml金黄色葡萄球菌(S. aureus)的Mueller-Hinton(MH)琼脂培养皿中培养,于不同时相点测量抑菌圈大小,检测其对金黄色葡萄球菌的抑制作用。实验二:参考医疗器械生物学评价标准,通过热原试验,溶血试验,肌肉植入试验,细胞毒性试验等来检测载万古霉素-PDLLA钢板的生物相容性。第二部分:1.采用仿生溶液(Simulated body fluid ,SBF)技术,以羟基磷灰石(HA)为载体,制作载万古霉素-HA钢板。2.通过扫描电镜、X射线能谱仪及傅里叶变换红外检测仪等对载万古霉素-HA钢板表面涂层进行检测。3.将载万古霉素-HA钢板置入均匀涂有1.5×108 CFU/ml金黄色葡萄球菌的MH琼脂培养皿中培养,于不同时相点测量抑菌圈大小,检测其对金黄色葡萄球菌的抑制作用。第三部分:1.采用solvent casting技术制备载万古霉素-PDLLA钢板。2.制备新西兰大白兔胫骨中段横断骨折金黄色葡萄球菌污染模型,分别置入载万古霉素-PDLLA钢板和普通裸钢板,在骨折断端注射1×106CFU/ml金黄色葡萄球菌,通过不同时相点的新西兰大白兔的体重、体温变化,白细胞、血沉和C反应蛋白检测,病理组织学检测、X光片检测及周围组织细菌培养等来比较两种钢板的抗感染效果。结果第一部分:实验一:1.成功制备出载万古霉素-PDLLA钢板,扫描电镜观察显示在钢板表面获得了较均匀、致密的涂层,万古霉素分子以小颗粒状分布于PDLLA涂层中。2.体外释放试验显示:载万古霉素-PDLLA钢板具有良好的缓释作用,在磷酸盐缓冲液中持续释放万古霉素20天以上。3.抑菌试验证明:载万古霉素-PDLLA钢板对金黄色葡萄球菌具有超过15天的抑制作用。实验二:载万古霉素-PDLLA钢板各项生物学指标均合格,其组织相容性、血液相容性良好,无细胞毒性,不含热原物质。第二部分:1.成功制备出载万古霉素-羟基磷灰石钢板;2. SEM、EDX及FT-IR检测显示载万古霉素-HA钢板表面形成了均匀、致密的涂层,其结构为HA。2.体外抑菌试验证明:载万古霉素-HA钢板对金黄色葡萄球菌具有一定的抑制作用,第一天载万古霉素-HA钢板周围出现了明显的圆形抑菌圈,但第二天的抑菌圈却迅速缩小,第三天基本未见抑菌圈,显示其经过第一天的爆发性释放后,第二天释放的万古霉素已不能达到有效抑菌浓度。我们根据实验结果发现,载万古霉素-HA钢板抗生素释放太快,与临床上治疗感染需要的时间不相适应。可能与我们的制备技术等各种因素有一定关系。所以我们暂时取消了载万古霉素-HA钢板的进一步研究。第三部分:载万古霉素-PDLLA钢板可以有效降低新西兰大白兔胫骨骨折金黄色葡萄球菌污染模型术后第1、3、7、14、28、56天静脉血中白细胞、血沉和C反应蛋白含量;对兔体温有一定影响,但对体重没有明显影响;X光片、病理学检查及细菌培养结果等显示可以有效降低新西兰大白兔胫骨骨折金黄色葡萄球菌污染模型的感染率。结论1.成功制备出了载万古霉素-PDLLA钢板,载万古霉素-PDLLA钢板的体外释放动力学稳定,并对金黄色葡萄球菌具有稳定的体外抑菌性能;载万古霉素-PDLLA钢板具有良好的生物相容性;载万古霉素-PDLLA钢板对新西兰大白兔胫骨骨折金黄色葡萄球菌污染模型具有明确的抗感染效果。2.载万古霉素-HA钢板能有效的携载万古霉素,且保持了其抑菌活性,但其有效抑菌效果仅维持24h。更多还原

【Abstract】 BackgroundAs the report of AO in 2006 suggests,those patients with opened fractures are known to be at higher risk of infection ranged from 3~40% (the rate of infection in closed fractures is 1.5%). Up to 60% of open fractures are contaminated with bacteria at the time of injury . In wartime firearm injuries, serious tissue damage and pollution allow great quantities of bacteria to survive in the area (0.5 cm) around the wound. The infection rate of bone and soft tissue ranged from 25% to 40%, and even followed by adequate wound debridement. Maurer et al reported the rate of infection in open tibia fractures increased to greater than 30% when internal fixation was used. For instance, implant-associated infections account for nearly 50% of the estimated 2 million nosocomial infections in the United States per year. In Britain, the loss was 7-11 million pounds per year. Meanwhile, the infection raised the expense of patients and increased the suffering of the human body and psychology of patients. Infection can even have such severe consequences as death. Therefore, it would be clinically advantageous to develop an internal fixation that possesses anti-infection properties to prevent and cure implant infections.During the past thirty years, a drug-delivery system was gradually developed on the basis of a series of original, fine biocompatibility inorganic material and organ-polymers. This system had a broad application perspective in the treatment directions of opened fracture, osteomyelitis and bone defects due to infection. Vancomycin (VCM) belongs to glycopeptide antibiotics. It has broad antimicrobial spectrum, covering most bacteria commonly involved in Gram-positive bacteria. Now it is the one of the top choice to cure severe infection which caused by MRSA or MRSE. And it is considered the one of the antibiotics for local application. Poly(D,L)-lactic acid (PDLLA) has excellent features with respect to implant-coating including high mechanical stability, good osteoinductive potential, biodegradability and excellent biocompatibility in vivo. The PDLLA coating is degraded by hydrolysis 3-6 months after implantation. The products of degradation are metabolized in the citric acid cycle. Otherwise, HA has excellent features with respect to implant-coating including biodegradability and excellent biocompatibility too. Because of these above mentioned reasons, PDLLA and HA can be used in orthopedic internal fixation. It is possible that the risk of infection can be reduced by developing an antiseptic surface coating for medical implants.ObjectiveWith PDLLA as a carrier, PDLLA coatings carrying VCM were prepared on the surface of titanium alloy plate substrates using solvent-casting technology. Meanwhile, HA coatings which carrying Vancomycin were prepared on the surface of titanium plate substrate using simulated body fluid technology with the HA as a carrier. Through vivo and vitro test, VCM-PDLLA loaded plates and VCM-HA loaded plates were expected to have dual effects of prophylaxis and cure infection in local and had a characteristic of fine therapeutic measure for opened fracture and firearm wound .MethodsPart 1Experiment 1: VCM-PDLLA loaded plates were prepared by solvent casting technology. To observe the coating of VCM-PDLLA loaded plates by SEM. The drug release profile of the plates were measured by immersing plates into 20ml PBS and measuring the content of VCM in different phase. The bacteriostatic activity to Staphylococcus aureus of the plates were evaluated by put the plates into Mueller-Hinton (MH) agar Petri dishes and the inhibition zones forming around the discs were measured in different phase.Experiment 2: To consult the biological evaluation of medical devices, the biocompatibility of VCM-PDLLA loaded plates were investigated by pryogen test, hemolytic test, cytotoxic test, et al.Part 2VCM-HA loaded plates were prepared using simulated body fluid technology. VCM-HA loaded plates were detected by the test method of SEM, EDX and FT-IR. The bacteriostatic activity to Staphylococcus aureus of the plates were evaluated by put the plates into MH agar Petri dishes and the inhibition zones forming around the discs were measured in different phase.Part 3VCM-PDLLA loaded plates were inserted in model of rabits that the middle tibia were fracture and were stained with 1×106CFU/ml S. aureus. In different phase, WBC, CRP, ESR, et al were measured to evaluate the anti-infection effect of the plates.Results1. VCM-PDLLA loaded plates were obtained. Plates showed a sustained in vitro drug release character in the experiment, in which VCM released from the plate was sustained over 20 days. The in vitro inhibition of Staphylococcus aureus showed that the plates had an inhibitory effect on Staphylococcus aureus, and the antibacterial activity was maintained for at least 15 days. There were no toxicity for the VCM-PDLLA loaded plates, and the plates had good biocompatibility .2. VCM-HA loaded coatings were prepared on the plates. The test of SEM, EDX and FT-IR indicated that the smooth and uniform coating was formed on the surface of the plates. The test of bacteriostasis indicated that obvious inhibition zone was appeared around the plate and it showed that VCM was successfully loaded on the coating. But in the next day the inhibition zone quickly contracted and in 3d the inhibition zone vanished. The result showed it could not achive the valid inhibitive concentration through the explosive release of the first day. According this, we think it may be related to various factor such as the preparative technique and determined to cancel the next study of VCM-HA loaded plates.3. VCM-PDLLA loaded plates could effective cut down the content of WBC, ESR and CRP of the model of rabits that the middle tibia were fracture and were stained with S.aureus, and could affect the body temperature, but could not affect the body weight in different phase. X-ray, pathologic examine and bacterial culture showed the plates could effective cut down the infection rate of the model.Conclusion1. VCM-PDLLA loaded plates showed a sustained in vitro drug release character, and the plates had an stabile inhibitory effect on S.aureus in vivo. There was a good biocompatibility for the plates. The plates were effective at inhibiting infection in an model of rabits that the middle tibia were fracture and were stained with S. aureus.2. VCM-HA loaded plates carried Vancomycin effectively and kept their bacteriostatic activity, but only sustained 24h.更多还原