低剂量伽马辐射灭菌治疗脱细胞气管移植物
1Thoracic Surgery Department,Hospital Universitari de la Ribera, 2Pathology Department,Universitat de Valencia Facultat de Medicina i Odontologia, 3Center for Biomaterials and Tissue Engineering,Universitat Politècnica de València, 4Radiation Oncology,Hospital Universitari de la Ribera, 5Thoracic Surgery Department,Hospitales Universitarios Virgen del Rocío, 6Department of Applied Statistics and Operations Research and Quality,Universitat Politècnica de València, 7Networking Research Center on Respiratory Diseases (CIBERER), 8INCLIVA Biomedical Research Institute
Summary
绝育对于气管组织移植至关重要。在这里，我们提出了一种使用器官完全耐受的低剂量伽马辐照的灭菌方案。
Abstract
确保移植正确发展的主要关键方面之一是培养基的无菌性。脱细胞气管移植涉及植入最初与环境接触的器官，因此从一开始就不是无菌的。虽然脱细胞方案（通过洗涤剂暴露[2%十二烷基硫酸钠]，连续搅拌和渗透冲击）是按照无菌措施进行的，但它不提供灭菌。因此，主要挑战之一是在 体内 植入之前确保无菌。虽然对无机材料有既定的伽马辐射灭菌规程，但对有机材料没有这样的措施。此外，无机材料的协议不能应用于有机材料，因为既定的辐射剂量（25 kGy）会完全破坏植入物。本文研究了辐射剂量递增对去细胞化兔气管的影响。我们维持剂量范围（kGy）并测试递增剂量，直到找到达到灭菌的最小剂量。确定剂量后，我们研究了它对器官的组织学和生物力学的影响。我们确定，虽然0.5 kGy没有达到无菌，但1 kGy和2 kGy的剂量都可以，因此1 kGy是实现灭菌所需的最小剂量。显微镜研究显示，与未消毒的器官相比，没有相关变化。轴向生物力学特性根本没有改变，并且仅观察到器官可以径向耐受的每单位长度的力略有减少。因此，我们可以得出结论，1 kGy实现了脱细胞兔气管的完全灭菌，对器官的影响很小（如果有的话）。
Introduction
植入物的灭菌是其生存能力的基本要求;事实上，已被证明成功的假肢是植入无菌区域（血管、心脏、骨骼等）的假肢。1.气管有两个表面：一个与外部环境接触的表面，因此不是无菌的，另一个是朝向纵隔的表面，它是无菌的。因此，从提取气管的那一刻起，它就不是无菌器官。尽管随后的脱细胞过程是在最大无菌条件下进行的，但它不是灭菌步骤2。异物的植入本身就存在感染的风险，因为它产生的前细菌微环境3，即使材料已经过灭菌，疾病从供体传播到受体的风险高达0.014%4。为了确保气管的正确血管化，在几乎所有实验移植方案中，它首先将异位植入物5,6,7到无菌区域（肌肉，筋膜，网膜，皮下等）;这是因为在该培养基中植入非无菌元件会导致区域3 的感染。
有一系列可能的策略可以获得无菌植入物。使用超临界CO2实现了终端灭菌8,9。其他方法，例如紫外线辐射或用过氧乙酸，乙醇，过氧化氧和电解水等物质进行处理，在灭菌中获得了不同的成功率，几乎总是取决于它们的剂量，但它们已被证明会影响植入物的生物力学特性。事实上，一些物质，如环氧乙烷，可以显著改变植入基质的结构，甚至会引起不良的免疫原性效应。因此，许多这些策略不能应用于生物模型2,10,11,12,13。
最广泛研究和接受的灭菌策略是由ISO 11737-1：2006标准建立的，用于对植入人体的医疗器械进行灭菌，伽马辐射剂量为25 kGy。但是，该法规仅侧重于惰性，非生物元素的灭菌14,15。此外，在癌的根治治疗中，放射治疗剂量比用于对医疗器械进行灭菌的剂量低三个数量级1。考虑到这一点，我们可以得出结论，所述剂量不仅会杀死微生物群，还会破坏并从根本上改变植入物的生物结构。它还有可能在降解时产生残留脂质，这可能具有细胞毒性并加速支架13,14,15,16,17的酶降解，即使使用低至1.9 kGy的剂量并且损害与接受的辐射剂量成正比17。
因此，本文的目的是尝试确定允许获得无菌植入物的辐射剂量，而辐射引起的有害影响最小2,18,19。我们遵循的策略涉及在千格雷范围内（0.5、1、2、3 kGy等）以不同的递增剂量照射脱细胞和辐照气管，直到培养结果为阴性。对培养结果为阴性的剂量进行了额外的测试，以确认灭菌。在确定获得灭菌的最小剂量后，检查照射对气管的结构和生物力学影响。将所有指标与对照组本地兔气管进行比较。然后将气管植入新西兰白兔体内，在体内测试结构的灭菌。
Protocol
遵守关于实验动物护理和使用的欧洲指令 20170/63/EU，研究方案得到瓦伦西亚大学伦理委员会的批准（西班牙瓦伦西亚政府第 86/609/EEC 和 214/1997 号法律以及代码 2018/VSC/PEA/0122 Type 2）。 1.气管脱细胞 注意：去细胞化方法已在其他地方报道20. 使用200 mg / mL通过边缘耳静脉注射，对体重3.5-4.1kg的雄性新西兰白兔（Oryctolagu…
Representative Results
去细胞化DAPI染色显示不存在DNA，并且通过电泳在任何气管中均未检测到高于50ng的DNA值，所有片段均小于200 bp20。 微生物培养在承受0.5 kGy的8件作品中，有2件在不到1周的时间内显示出颜色变化。在1 kGy和2 kGy照射的碎片均未显示任何颜色变化（图1）。 组织学分析在分?…
Discussion
有几种灭菌策略。超临界CO2完全穿透组织，酸化培养基并解构细胞磷脂双层，通过植入物的减压轻松消除8,14,25。紫外线辐射也被使用，其对啮齿动物气管的有效性已经发表，尽管文献中只有少数报道10。使用的其他方法包括应用过氧乙酸，乙醇，过氧化氧或电解水等物质，这些物质已产生不规则的结?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
本文得到了2018年西班牙胸外科学会对国家多中心研究的资助[授予Néstor J.Martínez-Hernández的180101号]和PI16-01315（授予Manuel Mata-Roig）的支持。CIBERER由VI National R&D&I Plan 2018-2011，Iniciativa Ingenio 2010，Consolider Program，CIBER Actions和Instituto de Salud Carlos III资助，并得到欧洲区域发展基金的援助。
Materials
| 6-0 nylon monofilament suture | Monosoft. Covidien; Mansfield, MA, USA | SN-5698G | |
| Amphotericin B 5% | Gibco Thermo Fisher Scientific; Waltham, MA USA | 15290018 | |
| Bioanalyzer | Agilent, Santa Clara, CA, USA | G2939BA | |
| Buprenorphine | Buprex. Reckitt Benckiser Healthcare; Hull, Reino Unido | N02AE01 | |
| Compression desktop UTM | Microtest, Madrid, Spain | EM1/10/FR | |
| Cryostate | Leyca CM3059, Leyca Biosystems, Wetzlar, Alemania | CM3059 | |
| DAPI (4',6-diamino-2-phenylindole) | DAPI. Sigma-Aldrich, Missouri, USA | D9542 | |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich; MO, USA | D2650 | |
| DMEM | Thermo Fisher Scientific; Waltham, MA, USA | 11965084 | |
| DNA extraction kit | DNeasy extraction kit Quiagen, Hilden, Germany | 4368814 | |
| Enrofloxacin, 2.5% | Boehringer Ingelheim, Ingelheim am Rhein, Germany | 0035-0002 | |
| Fetal bovine serum (FBS) | GE Healthcare Hyclone; Madrid, Spain | SH20898.03IR | |
| Fluorescence microscope | Leyca DM2500 (Leica, Wetzlar, Germany) | DM2500?? | |
| Freezing Container | Mr Frosty. Thermo Fisher; Madrid, Spain | 5100-0001 | |
| Isofluorane | Isoflo; Proyma Ganadera; Ciudad Real, Spain | 8.43603E+12 | |
| Ketamin | Imalgene. Merial; Toulouse, Francia | BOE127823 | |
| Linear accelerator | "True Beam". Varian, Palo Alto, California, USA | H191001 | |
| Magnetic stirrer | Orbital Shaker PSU-10i. Biosan; Riga, Letonia | BS-010144-AAN | |
| Meloxicam 5 mg/ml | Boehringer Ingelheim, Ingelheim am Rhein, Germany | 6283-MV | |
| OCT (Optimal Cutting Temperature Compound) | Fischer Scientific, Madrid, Spain | 12678646 | |
| Penicillin-streptomycin 5% | Gibco Thermo Fisher Scientific; Waltham, MA USA | 15140122 | |
| Pentobarbital sodium | Dolethal. Vetoquinol; Madrid, España | 3.60587E+12 | |
| Phosphate buffered saline (PBS) | Sigma-Aldrich; MO, USA | P2272 | |
| Propofol | Propofol Lipuro. B. Braun Melsungen AG; Melsungen, Alemania | G 151030 | |
| Proteinase K | Gibco Thermo Fisher Scientific; Waltham, Massachussetts, USA | S3020 | |
| PVC hollow tubes | Cristallo Extra; FITT, Sandrigo, Italy | hhdddyyZ | |
| PVC stent | ArgyleTM Medtronic; Istanbul, Turkey | 019 5305 1 | |
| R software, Version 3.5.3 R Core | R Foundation for Statistical Computing | R 3.5.3 | |
| Sodium dodecyl sulfate (SDS) | Sigma-Aldrich; MO, USA | 8,17,034 | |
| Spectrophotometer | Nanodrop, Life Technologies; Isogen Life Science. Utrech, Netherlands | ND-ONEC-W | |
| Spreadsheet | Microsoft Excel for Mac, Version 16.23, Redmond, WA, USA | 2864993241 | |
| Traction Universal Testing Machine | Testing Machines, Veenendaal, Netherlands | 84-01 | |
| UTM Software | TestWorks 4, MTS Systems Corporation, Eden Prairie, MN, USA | 100-093-627 F | |
| VECTASHIELD Mounting Medium | Vector Labs, Burlingame; CA; USA | H-1000-10 | |
| Xylacine | Xilagesic. Calier; Barcelona, España | 20102-003 |
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Citer Cet Article
Martínez-Hernández, N. J., Milián-Medina, L., Mas-Estellés, J., Monroy-Antón, J. L., López-Villalobos, J. L., Hervás-Marín, D., Roig-Bataller, A., Mata-Roig, M. Low-Dose Gamma Radiation Sterilization for Decellularized Tracheal Grafts. J. Vis. Exp. (194), e64432, doi:10.3791/64432 (2023).