Loeffler Henrike, Waletzko-Hellwig Janine, Fischer Ralf-Joerg, Basen Mirko, Frank Marcus, Jonitz-Heincke Anika, Bader Rainer, Klinder Annett
Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Rostock, Germany.
Microbiology, Institute of Biological Sciences, University of Rostock, Rostock, Germany.
J Biomed Mater Res B Appl Biomater. 2024 Feb;112(2):e35383. doi: 10.1002/jbm.b.35383.
To obtain bone allografts that are safe for transplantation, several processing steps for decellularization and decontamination have to be applied. Currently available processing methods, although well-established, may interfere with the biomechanical properties of the bone. High hydrostatic pressure (HHP) is known to devitalize tissues effectively while leaving the extracellular matrix intact. However, little is known about the inactivation of the contaminating microorganisms by HHP. This study aims to investigate the ability of high-pressure decontamination and to establish a treatment protocol that is able to successfully inactivate microorganisms with the final goal to sterilize bone specimens. Using Escherichia coli (E. coli) as a model organism, HHP treatment parameters like temperature and duration, pressurization medium, and the number of treatment cycles were systematically adjusted to maximize the efficiency of inactivating logarithmic and stationary phase bacteria. Towards that we quantified colony-forming units (cfu) after treatment and investigated morphological changes via Field Emission Scanning Electron Microscopy (FESEM). Additionally, we tested the decontamination efficiency of HHP in bovine cancellous bone blocks that were contaminated with bacteria. Finally, two further model organisms were evaluated, namely Pseudomonas fluorescens as a Gram-negative microorganism and Micrococcus luteus as a Gram-positive representative. A HHP protocol, using 350 MPa, was able to sterilize a suspension of stationary phase E. coli, leading to a logarithmic reduction factor (log RF) of at least -7.99 (±0.43). The decontamination of bone blocks was less successful, indicating a protective effect of the surrounding tissue. Sterilization of 100% of the samples was achieved when a protocol optimized in terms of treatment temperature, duration, pressurization medium, and number and/or interval of cycles, respectively, was applied to bone blocks artificially contaminated with a suspension containing 10 cfu/mL. Hence, we here successfully established protocols for inactivating Gram-negative model microorganisms by HHP of up to 350 MPa, while pressure levels of 600 MPa were needed to inactivate the Gram-positive model organism. Thus, this study provides a basis for further investigations on different pathogenic bacteria that could enable the use of HHP in the decontamination of bone grafts intended for transplantation.
为了获得安全可移植的骨移植物，必须应用几种脱细胞和去污的处理步骤。目前可用的处理方法虽然已确立，但可能会干扰骨的生物力学性能。已知高静水压（HHP）能有效使组织失活，同时保持细胞外基质完整。然而，关于HHP对污染微生物的灭活作用知之甚少。本研究旨在探讨高压去污的能力，并建立一种能够成功灭活微生物的处理方案，最终目标是对骨标本进行灭菌。以大肠杆菌（E. coli）作为模式生物，系统地调整HHP处理参数，如温度、持续时间、加压介质和处理循环次数，以最大限度地提高对数期和稳定期细菌的灭活效率。为此，我们在处理后对菌落形成单位（cfu）进行了定量，并通过场发射扫描电子显微镜（FESEM）研究了形态变化。此外，我们测试了HHP对被细菌污染的牛松质骨块的去污效率。最后，评估了另外两种模式生物，即革兰氏阴性微生物荧光假单胞菌和革兰氏阳性代表藤黄微球菌。使用350 MPa的HHP方案能够对稳定期大肠杆菌悬液进行灭菌，对数减少因子（log RF）至少为-7.99（±0.43）。骨块的去污效果较差，表明周围组织具有保护作用。当分别针对处理温度、持续时间、加压介质以及循环次数和/或间隔进行优化的方案应用于人工污染有含10 cfu/mL悬液的骨块时，100%的样本实现了灭菌。因此，我们在此成功建立了通过高达350 MPa的HHP灭活革兰氏阴性模式微生物的方案，而灭活革兰氏阳性模式生物则需要600 MPa的压力水平。因此，本研究为进一步研究不同病原菌提供了基础，这可能使HHP能够用于移植用骨移植物的去污。
