Vesper Evan O, Hammond Max A, Allen Matthew R, Wallace Joseph M
Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, IN, United States.
Department of Biomedical Engineering, Indiana University-Purdue University Indianapolis, IN, United States; Department of Anatomy and Cell Biology, Indiana University School of Medicine, IN, United States; Roudebush Veterans Administration Medical Center, Indianapolis, IN, United States; Department of Orthopaedic Surgery, Indiana University School of Medicine, IN, United States.
Bone. 2017 Apr;97:49-53. doi: 10.1016/j.bone.2017.01.001. Epub 2017 Jan 3.
Typically, bones are harvested at the time of animal euthanasia and stored until mechanical testing. However, storage methods are not standardized, and differential effects on mechanical properties are possible between methods. The goal of this study was to investigate the effects that two common preservation methods (freezing wrapped in saline-soaked gauze and refrigerating ethanol fixed samples) have on bone mechanical properties in the context of an in vitro ribosylation treatment designed to modify mechanical integrity. It was hypothesized that there would be an interactive effect between ribose treatment and preservation method. Tibiae from twenty five 11week old female C57BL/6 mice were separated into 2 preservation groups. Micro-CT scans of contralateral pairs assessed differences in geometry prior to storage. After 7weeks of storage, bones in each pair of tibiae were soaked in a solution containing either 0M or 0.6M ribose for 1week prior to 4 point bending tests. There were no differences in any cortical geometric parameters between contralateral tibiae. There was a significant main effect of ethanol fixation on displacement to yield (-16.3%), stiffness (+24.5%), strain to yield (-13.9%), and elastic modulus (+18.5%) relative to frozen specimens. There was a significant main effect of ribose treatment for yield force (+13.9%), ultimate force (+9.2%), work to yield (+22.2%), yield stress (+14.1%), and resilience (+21.9%) relative to control-soaked bones. Postyield displacement, total displacement, postyield work, total work, total strain, and toughness were analyzed separately within each preservation method due to significant interactions. For samples stored frozen, all six properties were lower in the ribose-soaked group (49%-68%) while no significant effects of ribose were observed in ethanol fixed bones. Storage in ethanol likely caused changes to the collagen matrix which prevented or masked the embrittling effects of ribosylation that were seen in samples stored frozen wrapped in saline-soaked gauze. These data illustrate the clear importance of maintaining hydration if the eventual goal is to use bones for mechanical assessments and further show that storage in ethanol can alter potential to detect effects of experimental manipulation (in this case ribosylation).
通常情况下，骨骼在动物安乐死时采集并储存，直至进行力学测试。然而，储存方法并未标准化，不同方法对力学性能可能产生不同影响。本研究的目的是在旨在改变力学完整性的体外核糖基化处理背景下，研究两种常见保存方法（用浸有生理盐水的纱布包裹冷冻和冷藏乙醇固定样本）对骨骼力学性能的影响。研究假设核糖处理和保存方法之间会存在交互作用。将25只11周龄雌性C57BL/6小鼠的胫骨分为2个保存组。在储存前，对侧胫骨对的显微CT扫描评估几何形状差异。储存7周后，在进行四点弯曲试验前，将每对胫骨中的骨骼浸泡在含有0M或0.6M核糖的溶液中1周。对侧胫骨之间的任何皮质几何参数均无差异。相对于冷冻标本，乙醇固定对屈服位移（-16.3%）、刚度（+24.5%）、屈服应变（-13.9%）和弹性模量（+18.5%）有显著的主要影响。相对于用对照溶液浸泡的骨骼，核糖处理对屈服力（+13.9%）、极限力（+9.2%）、屈服功（+22.2%）、屈服应力（+14.1%）和弹性恢复力（+21.9%）有显著的主要影响。由于存在显著的交互作用，在每种保存方法中分别分析屈服后位移、总位移、屈服后功、总功、总应变和韧性。对于冷冻保存的样本，核糖浸泡组的所有六个属性均较低（49%-68%），而在乙醇固定的骨骼中未观察到核糖的显著影响。储存在乙醇中可能导致胶原基质发生变化，从而阻止或掩盖了在用浸有生理盐水的纱布包裹冷冻保存的样本中观察到的核糖基化脆化效应。这些数据表明，如果最终目标是将骨骼用于力学评估，保持水合作用显然非常重要，并且进一步表明储存在乙醇中会改变检测实验操作（在本案例中为核糖基化）效果的可能性。
