构建内固定术后早期感染细菌生物膜形成灌注冲洗的动物模型

Construction of an animal model for treating early postoperative infected bacterial biofilms by irrigating after internal fixation

背景:骨科内固定术后感染细菌生物膜形成的治疗是临床非常棘手的问题,建立内固定术后早期感染细菌生物膜形成的灌注冲洗动物模型对探索该病的治疗方法具有重要意义。目的:建立可用不同药物灌注冲洗治疗内固定术后早期感染细菌膜形成的动物模型。方法:选取6只新西兰大白兔,暴露双侧股骨表面并钻孔,将定植铜绿假单胞菌的接骨板(实验组)和空白接骨板(空白对照组)各植入一侧钻孔周围,留置2条引流管并固定分别作为“入口”和“出口”,模拟灌注后保留浸泡一定时间后再进行冲洗。术后5 d,观察兔体温、伤口情况、引流液细菌培养及接骨板结晶紫染色、扫描电镜检查结果。结果与结论:①6只兔术后患肢活动不便,术后第2-4天出现体温升高,实验组部分伤口处可触及局部肿胀,空白对照组伤口愈合良好;②引流液细菌培养结果显示,实验组铜绿假单胞菌向周边扩散或蔓延;术后5 d,实验组接骨板结晶紫染色后呈紫色,酶标仪检测570 nm处吸光度值为2.621±0.088,提示存在细菌;③术后5 d的扫描电镜显示,实验组接骨板表面可见大量细菌微菌落,形成类似“磨菇样”“塔状”的高度组织不均匀性三维立体结构,高倍镜下可见“磨菇样”结构之间有丝状连接的水通道,是典型的生物膜结构且致密度极高,空白对照组未见明显菌落形成;④结果显示该动物模型模拟了内固定术后早期感染生物膜形成后的状态,提供了一种可用不同药物进行灌注冲洗的方法。

BACKGROUND:The treatment for bacterial biofilms after internal fixation surgery is a very difficult problem in clinic.It is a great significance to establish an animal model of irrigation for treating bacterial biofilms in the early stage after internal fixation surgery.OBJECTIVE:To establish an animal model for treating bacterial biofilms with different drugs through irrigation in early stage after internal fixation surgery.METHODS:Six New Zealand white rabbits were selected.Bilateral femoral surfaces were exposed and drilled holes were made,and bone plates colonized with Pseudomonas aeruginosa(experimental group)and blank bone plates(blank control group)were implanted around the drilled holes on one side,and two drainage tubes were retained and fixed to serve as the“inlet”and“outlet,”respectively.The model was immersed for a certain period of time after simulated perfusion before rinsing.After the simulated irrigation,the plates were soaked for a certain time before washing.At 5 days postoperatively,the rabbits were observed for body temperature,wound condition,bacterial culture of drainage fluid,and crystalline violet staining and scanning electron microscopy of the bone plate.RESULTS AND CONCLUSION:Six rabbits had difficulty in moving the affected limbs after surgery and showed elevated body temperature at 2-4 days after surgery.Local swelling could be touched at some wounds in the experimental group,and the wounds in the blank control group healed well.The results of bacterial culture of drainage fluid showed that Pseudomonas aeruginosa diffused or spread in the experimental group.At 5 days after surgery,the plate in the experimental group became purple shown by crystalline violet staining,and the absorbance value at 570 nm detected by the microplate reader was 2.621±0.088,indicating the presence of bacteria.Scanning electron microscopy at 5 days after surgery showed that a large number of bacterial microcolonies appeared on the surface of the plate in the experimental group,forming a highly inhomogeneous three-dimensional structure similar to the“mushroom-like”and“tower-like”structures,with filamentous water channels connecting the“mushroom-like”structures,which were typical biofilm structures with high densities,while no obvious colonies were seen in the blank control group.Overall,this animal model simulates the state of infected biofilm formation due to early infection after internal fixation and provides an available method of irrigation with different drugs.