Establishment of an animal model of open tibial fractures with infection in New Zealand rabbits

doi:10.3969/j.issn.2095-4344.0825

Abstract

Abstract:

BACKGROUND: Open facture is easy to induce infection, which is an urgent problem in clinic. Establishing a reliable animal model of open fracture with infection is of great significance for drug and instrument development and application.
OBEJCTIVE: To develop an open fracture with infection model in New Zealand white rabbits, and to identify the available number of bacteria that can cause infection.
METHODS: The amount of bacteria was determined by establishing open fracture structure and verifying the concentration of bacterial colonies. Thirty New Zealand white rabbits were randomly divided into control group and four experimental groups, and a transverse fracture at the middle part of tibia was established in all rabbits, followed by the injection of 1 mL of normal saline or 1mL of Staphylococcus aureus suspension at the concentrations of 1×105, 1×106, 5×106, and 1×107 CFU/mL. Afterwards, the optimal concentration of 1 mL of bacteria liquid causing infection was determined by gross observation, body temperature analysis and body mass measurement, white blood cell and C-reactive protein detection, bacterial culture and pathological observation.
RESULTS AND CONCLUSION: Rabbits in the 5×106 CFU/mL group were all infected and had higher survival rate. In the 1×105 and 1×106 CFU/mL groups, some rabbits showed no infection. One rabbit died due to infection in the 1×107 CFU/mL group. In summary, the reliable infection model of open fracture can be induced by injected with 1 mL of Staphylococcus aureus at the concentration of 5×106 CFU/mL in New Zealand white rabbits, which can be used as an effective model to guide drugs and instruments related anti-infective research.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Tibial Factures, Infection, Models, Animal, Tissue Engineering

CLC Number:

R318

Geng Fang1, Pan Li-cun1, Zhou Zi-hui2 . Establishment of an animal model of open tibial fractures with infection in New Zealand rabbits[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(8): 1223-1228.

Figures/Tables 3

2.1 实验动物数量分析实验选用新西兰兔30只，分为5组，全部进入结果分析。 2.2 造模后各组兔大体情况观察见表1。造模后2周内，30只新西兰兔存活28只，其中1×106 CFU/mL组的1只在4.5 d意外死亡，1×107 CFU/mL组1只严重感染在第3天死亡；其他28只术后2周存活良好，观察骨折部位外固定效果良好。无菌生理盐水组6只兔均未感染，1×105 CFU/mL组只有1只兔感染，1×106 CFU/mL组中2只兔未感染，5×106 CFU/mL和1×107 CFU/mL组均感染。未发生感染的新西兰兔手术切口干燥，缝合良好，无红肿及溢脓现象，体温保持正常，体质量略下降，饮食及活动正常；发生感染的新西兰兔部分手术切口缝线脱落，周围皮肤红肿，脓液渗出，体温升高，体质量下降幅度大，精神较差。造模后解剖发现感染的新西兰兔软组织恢复差，仍显肿胀，原缝合处切开在骨折线外部软组织有白色脓液，髓腔内部也有白色脓液；未发生感染的皮肤切口处缝合良好，轻微肿胀，切开后软组织部分愈合，部分新西兰兔骨折处有少量骨连接，见图2。 2.3 造模后各组兔体温体质量各组数据无统计学差异，在造模之后各组新西兰兔体质量随着时间变化总体呈下降趋势，下降幅度均在0.1 kg左右，见图3。图4显示各组动物手术前后不同时间体温的变化情况。各组新西兰兔在术后第1天体温都骤然升高，主要由于手术的创伤造成的影响，各组体温都比术前有极显著性差异；术后3，7和14 d体温结果显示生理盐水组和1×105 CFU/mL组体温较术后1 d明显下降，和术前比较差异无显著性意义，分析主要是由于自身抗感染免疫功能发挥作用，维持机体稳定，说明该浓度细菌数量少，不能引起炎症反应的发生；中等浓度1×106 CFU/mL组术后体温逐渐下降，第14天与术前体温无显著性差异，说明机体在恢复；而2个高浓度组中，5×106 CFU/mL组在第7天体温下降，但在第14天又反复升高，与术前比差异有非常显著性意义，说明细菌突破宿主防线，发生感染；最高浓度组1×107 CFU/mL组术后一直保持高温，说明细菌侵袭力和毒素作用强，感染的程度高。 2.4 血常规测试结果 (1)白细胞：图5白细胞的结果显示，生理盐水组和最低细菌浓度1×105 CFU/mL组，14 d内白细胞值都在10×109 L-1以下，并且在第7天和14天白细胞值明显下降，尤其第7天白细胞值与造模前相比显著性下降，说明在第7天时免疫系统已经明显战胜细菌，身体在恢复。而对于其他3个高细菌浓度组白细胞一直在升高，说明感染的发生，并逐渐严重化，造模后14 d与造模前对比，差异有非常显著性意义(P值均< 0.01)。 (2)C-反应蛋白：图6显示生理盐水组和2个低细菌浓度1×105 CFU/mL组和1×106 CFU/mL组造模前、造模后C-反应蛋白变化不明显，在7 d内略有下降，说明免疫系统在发挥防御作用；而5×106 CFU/mL组在造模后1 d和3 d C-反应蛋白上升，而造模后7 d有所降低，和体温表现一致，说明新西兰兔在7 d前的免疫防御系统比较强，但14 d C-反应蛋白又大幅提高，说明细菌突破宿主防线，严重的感染已经确定发生；最高浓度组1×107 CFU/mL组在术后不同时间C-反应蛋白持续上升，术后都在8 mg/L以上，说明初始细菌数量多，毒力作用大，表明感染较早发生并且感染程度不断加剧。 2.5 细菌培养结果解剖观察剖开骨折区域，用无菌棉棒粘取骨折附近组织液进行细菌培养鉴别，结果见表2，当注入细菌浓度5×106 CFU/mL及更高浓度时，经过14 d后在骨折处可发现大量细菌，说明感染存在，浓度更低时则不一定使新西兰兔感染。各组14 d细菌培养结果显示，在注射低浓度菌液时，1×105 CFU/mL组细菌感染1只兔， 1×106 CFU/mL组细菌感染3只，而高浓度5×106 CFU/mL组和1×107 CFU/mL组全部感染，并且感染程度高。 2.6 组织切片病理学结果第14天新西兰兔胫骨病理切片苏木精-伊红染色：空白对照组(生理盐水)，显示正常骨组织，增生骨组织，并有少量骨髓出现，表明骨组织在逐步愈合，无炎症反应；1×105 CFU/mL组有增生骨，软骨生成，无炎症，该骨组织良好；1×106 CFU/mL组显示出现异物巨噬细胞，炎症反应；5×106 CFU/mL组显示明显的炎症反应，大量巨噬细胞，组织坏死；5×106 CFU/mL组显示右侧整块组织出现炎症反应；1×107 CFU/mL组显示严重炎症反应和组织坏死。总体趋势是随着细菌浓度的增加，炎症反应加重，炎症细胞浸润增多，骨破坏增加，坏死组织增多，见图7。"

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