万古霉素滴入硫酸钙和自体骨治疗兔骨感染的改进模型
1Department of Medicine,Zhejiang Academy of Traditional Chinese Medicine, 2Department of Orthopaedic Surgery,Tongde Hospital of Zhejiang Province, 3Institute of Orthopaedics and Traumatology,First Affiliated Hospital of Zhejiang Chinese Medical University, 4Department of Clinical Laboratory,Tongde Hospital of Zhejiang Province
Summary
本研究通过阻断骨髓中相同数量的细菌, 提出了一种感染金黄色葡萄球菌的改良兔模型。万古霉素负载硫酸钙和自体骨用于抗生素和骨修复治疗。该方案可为骨感染和再生的研究提供参考。
Abstract
骨感染是由细菌入侵引起的, 在临床、整形外科和创伤性手术中, 细菌感染是极其困难的。骨感染可能导致持续炎症、骨髓炎和最终骨不愈合。建立可行、可重复的动物模型对骨感染研究和抗生素治疗具有重要意义。作为体内模型, 兔模型在骨感染研究中得到了广泛的应用。然而, 此前对兔骨感染模型的研究表明, 由于细菌数量是可变的, 感染状态不一致。本研究通过阻断骨髓中的细菌, 提出了一种诱导兔骨感染的改进手术方法。然后, 进行多层次的评价, 验证建模方法。
一般情况下, 脱骨坏死组织和植入香康辛负载硫酸钙 (vcs) 是抗生素治疗的主要优势。尽管 vcs 中的硫酸钙有利于骨细胞的爬行和新的骨生长, 但大量的骨缺损发生后, 脱骨。自体骨 (ab) 是一种治疗坏死骨后骨缺损的一种吸引人的方法。
在这项研究中, 我们使用尾骨作为自体骨植入骨缺损。采用显微断层扫描 (微 ct) 和动物牺牲后的组织学分析测量骨修复。因此, 在 vcs 组中, 持续获得骨不愈合。相比之下, vcs-ab 组的骨缺损区明显减少。目前的建模方法描述了一种可重复、可行、稳定的骨感染模型制备方法。vcs-ab 治疗导致抗生素治疗后骨不愈合率降低。改进的骨感染模型以及 vcs 和自体骨的联合治疗有助于研究与创伤骨科应用相关的骨感染和骨再生的潜在机制。
Introduction
骨感染通常是由创伤、骨折或其他骨骼疾病后的细菌或其他微生物入侵引起的。骨感染可能导致高水平的炎症和骨组织破坏。在临床上,金黄色葡萄球菌(金黄色葡萄球菌) 是骨感染的主要病原体 2,3.骨感染是痛苦的, 使人虚弱的, 往往是慢性的, 是极难治疗的4。目前, 清创坏死组织和植入万古霉素负载钙 (vcs) 珠已被证实为控制局部感染5,6的有效策略。然而, 10% 至15% 的患者经历了长期的骨修复过程, 延迟愈合, 或抗感染治疗后不愈合 7。骨缺损的大部分是整形外科医生最困难的问题。自体骨移植被认为是骨不结合治疗8,9的最佳骨置换方法。
到目前为止, 关于骨感染和自体骨植入的研究大多是在各种动物模型中进行的, 如老鼠、兔子、狗、猪和羊10、11.兔子模型最常用于骨感染研究, 这是诺登和肯尼迪在 1970年12,13首次进行的。在我们之前的研究中, 我们采用了 nnoren 方法的兔子模型, 我们发现注入骨髓的金黄色葡萄球菌的数量无法准确量化, 因为从骨髓中漏出的血液导致细菌溶液溢出。
本文提出了一种改良的家兔骨感染的手术方法。在手术结束时, 进行血液生化检查、细菌学检查和组织病理学检查, 以验证骨感染模型。然后, 植入 vcs 抑制感染, 自体骨植入, 促进骨再生。
Protocol
本研究中使用的兔子按照《实验动物护理和使用指南》进行处理。所有的实验程序都遵循了浙江中医药研究院生物伦理委员会的规则。 1. 细菌悬浮液的制备 溶解0.5 毫克金黄色葡萄球菌冷冻干燥粉 (atcc 6538) 与0.3 毫升的卢里亚-伯塔尼培养基。将悬架完全混合。 将细菌悬浮液流到色氨酸大豆琼脂板上, 在37°c 孵育细菌菌落16小时。 选择单个菌落形成…
Representative Results
骨感染模型的评价兔感染金黄色葡萄球菌后, 其病理表现与临床上慢性骨髓炎的典型症状相似。在我们的研究中, 30只家兔被感染, 并作为模型组进行了研究, 10只兔子被作为对照动物。所有模型兔都感染了胫骨局部部位的鼻窦, 从鼻窦流出白色和黄色脓液 (图 3a)。h & e 染色结果表明, 模型组细菌聚集物位于死骨周围, ?…
Discussion
在以往的研究中, 构建了各种动物模型, 研究急性和慢性骨感染;然而, 对理想模型的寻找仍然持续到17,18。此外, 理想的骨感染模型有望模拟临床环境下骨感染的病理特征, 而建模期间, 成本仍然较低, 易于实施。目前, 兔骨感染模型是炎症性骨病研究中最常见的模型, 因为兔子是可用的、可行的、廉价的。在我们之前的研究中, 我们比较了不同体重的兔子的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金 (81803808, 81803808)、浙江省医疗卫生科学技术基金 (2017ky271) 和浙江省科技项目 (2017KY271) 的支持。
Materials
| absorbable surgical suture | Jinghuan | 18S0604A | |
| asepsis injector | Jinglong | 20170501 | |
| bone wax | ETHICON | JH5CQLM | |
| CCD camera | Olympus | DP72 | |
| EDTA-K2 anticoagulant blood vessel | XINGE | 20170802 | |
| Electric bone drill unit | Bao Kang | BKZ-1 | |
| Electric shaver | Codos | 3800 | |
| flexible silica gel mold | WRIGHT | 1527745 | |
| Hematoxylin and Eosin Staining Kit | Beyotime | 20170523 | |
| Luria-Bertani culture medium | Baisi Biothchnology | 20170306 | |
| Medical-grade calcium sulphate | WRIGHT | 1527745 | |
| microcomputed tomography (micro-CT) | Bruker | SkyScan 1172 | |
| Microscope | Olympus | CX41 | |
| New Zealand white rabbits | Zhejiang Experimental Animal Center | SCXK 2014-0047 | |
| No. 11 scalpel | Yuanlikang | 20170604 | |
| normal saline | Mingsheng | 20170903 | |
| PBS | TBD(Jingyi) | 20170703-0592 | |
| pentobarbital sodium | Merk | 2070124 | |
| povidone-iodinesolution | Lierkang | 20170114 | |
| S. aureus freeze drying powder | China General Microbiological Culture Collection Center | ATCC 6538 | |
| sheep blood agar | HuanKai Microbial | 3103210 | |
| tryptic soy agar plates | HuanKai Microbial | 3105697 | |
| tryptic soy broth tubes | HuanKai Microbial | 3104260 | |
| Vancomycin | Lilly | C599180 |
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Cite This Article
Zhang, Y., Shen, L., Wang, P., Xi, W., Yu, Z., Huang, X., Wang, X., Shou, D. Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection. J. Vis. Exp. (145), e57294, doi:10.3791/57294 (2019).