鼠股颈悬臂弯曲
Summary
本方案描述了在悬臂弯曲设置中为小鼠股骨颈开发可重复测试平台。使用定制的3D打印导轨以最佳对齐方式一致且刚性地固定股骨。
Abstract
股骨颈骨折在骨质疏松症患者中很常见。已经开发了许多小鼠模型来评估疾病状态和疗法，并将生物力学测试作为主要结果测量。然而，传统的生物力学测试侧重于应用于长骨中轴的扭转或弯曲测试。这通常不是骨质疏松症个体中高危骨折的部位。因此，开发了一种生物力学测试方案，在悬臂弯曲负荷下测试小鼠股骨颈，以更好地复制骨质疏松症患者所经历的骨折类型。由于生物力学结果高度依赖于相对于股骨颈的弯曲载荷方向，因此创建了3D打印导轨以将股骨轴保持在相对于载荷方向的20°角。新方案通过降低对准的可变性（21.6°±1.5°，COV = 7.1%，n = 20）和提高测量生物力学结果的再现性（平均COV = 26.7%）来简化测试。使用3D打印导轨进行可靠标本对准的新方法通过减少由于标本不对准引起的测量误差来提高严谨性和再现性，这应该可以最大限度地减少小鼠骨质疏松症研究中的样本量。
Introduction
骨折风险是与骨质疏松症相关的严重医学问题。仅在美国，每年就报告了超过150万例脆性骨折，其中髋部骨折，特别是股骨颈骨折是主要骨折类型1。据估计，18% 的女性和 6% 的男性在其一生中会出现股骨颈骨折2， 骨折后 1 年的死亡率大于 20%1。因此，允许对股骨颈部进行生物力学测试的小鼠模型可能适用于研究脆性骨折。小鼠模型还提供了强大的工具来阐明可能涉及骨质疏松症的可翻译细胞和分子事件。这是由于遗传报告基因的可用性，功能模型的获得和丧失以及分子技术和试剂的广泛库。小鼠骨骼的机械测试可以提供必要的结局指标，以确定骨骼健康、基因型和表型变异，从而解释疾病的病因，并根据骨骼质量和骨折风险的结局指标评估治疗3。
股骨颈的解剖结构会产生独特的机械负荷情况，这通常会导致弯曲（弯曲）骨折。股骨头被装入股骨近端的髋臼窝。这在股骨颈上产生悬臂弯曲场景，该股骨颈部刚性地连接到远端的股骨轴4。这与传统的股骨中骺端 3 点或 4 点弯曲试验不同。虽然这些测试是有帮助的，但它们不会复制通常导致骨质减少和骨质疏松症个体在骨折位置或载荷情况下的脆性骨折的负荷。
为了更好地评估小鼠的脆性骨折风险，寻求提高小鼠股骨颈悬臂弯曲试验的再现性。正如理论上预测的那样，股骨头相对于股骨干的载荷角已被证明会显著影响结局指标5，从而对报告结局的可靠性和可重复性提出了挑战。为了确保在样品制备过程中股骨正确和一致的对准，设计了指南，并根据对C57BL / 6小鼠股骨的μCT扫描进行的解剖学测量进行了3D打印。这些导轨旨在帮助始终如一地灌封样品，使股骨轴保持在垂直加载方向的~20°。选择这个角度是因为它最大限度地提高了刚度，同时最小化了沿股骨轴的最大弯曲力矩，这增加了股骨颈骨折的可能性，并导致更一致和可重复的测试5。导轨以各种尺寸进行3D打印，以适应样品之间的解剖学差异，并用于在丙烯酸骨水泥中灌封时将样品保持在稳定位置。刚度、最大力、屈服力和最大能量均根据力-位移图计算得出。该测试方法显示了上述生物力学结果的一致结果。通过实践和3D打印指南的帮助，可以最大限度地减少由于不对中引起的测量误差，从而获得可靠的结果测量。
Protocol
Representative Results
Discussion
该协议概述了对小鼠股骨颈的可靠悬臂弯曲试验。发生在股颈的自然悬臂弯曲情况在标准的3点和4点弯曲试验中通常不显示5。这种测试方法更好，更可靠地复制了骨脆性患者所经历的股骨颈骨折类型。执行该协议时的主要重点是消除由于股骨轴灌封不一致而导致的可变性。至关重要的是，严格遵循协议第一和第四部分中概述的步骤将确保指南和加载协议的创建将复制本出版物中?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
该研究得到了NIH P30AR069655和R01AR070613（H.A.A.）的支持。
Materials
| ¼” x ¼” square aluminum tubing | Grainger | 48KU67 | Cut to lengths of 1/2" to 1" lengths |
| 1 kN load cell | Instron | 2527-130 | Any load cell with sub 1 N resolution can be used. |
| 3.5x-45x Zoom Stereo Boom Microscope | Omano | OM2300S-GX4 | Microscope used to precisely line up samples with loading platen. |
| 3D printed guides | Custom made | Angled slots at 73.13°, with diameters between 1.9 mm and 2.2 mm | |
| 3D printed mount | Custom made | Tapped with M10 threads to fit the mount attachment and with 2 M4 threaded holes adjacent sides to hold the aluminum tubing with sample in place. | |
| Acrylic Base Plate Material Kit | Keystone Industries | 921392 | Mix 3.5 g of powder with 2 mL of liquid. This will be enough for approximately 8 samples, and will begin to harden quickly. |
| Amira | ThermoFisher Scientific | Used to compile µCT scans | |
| Biaxial stage | Custom made | Used to center femoral head of sample under the loading platen. | |
| BioMed Amber Resin | formlabs | RS-F2-BMAM-01 | Any resin from formlabs could be used for this project. |
| Bluehill 3 | Instron | V3.66 | Software used to set up loading protocol and collect load, displacement and time data. |
| ElectroPuls 10000 | Instron | E10000 | Mechanical testing system |
| Faxitron UltraFocus | Faxitron BioOptics | 2327A40311 | X-ray imaging system |
| Form 2 | formlabs | F2 | Used to print the mount and guides |
| Form 2 Resin Tank LT | formlabs | RT-F2-02 | LT Tank was used to be compatible with the BioMed Resin |
| ImageJ | National Institutes of Health | ImageJ | Used to assess µCT and X-ray images |
| Laxco iLED Series LED Light Source | ThermoFisher Scientific | AMPSILED30W | Light source used in conjugtion with microscope. |
| Loading platen | Custom made | This can be any metal rod that is tapered to a diameter of approximately 2.5 mm. We used an M6 screw that was tapered on a lathe. | |
| Mount attachment | Custom made | To secure the 3D printed mount to the load cell. We used a M10/M6 threaded rod | |
| Phosphate Buffer Saline (PBS) | ThermoFisher Scientific | 10010031 | Need to rehydrate the samples once acrylic base plate material has set. |
| Plumber's putty | Oatey | 31174 | Used to seal the end of the aluminum tubing when pouring acrylic base plate material in. Any clay or putty could be used. |
| PreForm | formlabs | Preform 3.15.2 | Formlabs software |
| Tissue Culture Dish | Corning | 353003 | Samples can be laid flat in culture dish and covered in PBS to rehydrate. |
| vivaCT 40 | Scanco | µCT 40 | Representative set or actual samples can be scanned prior to printing of guides to calculate femoral shaft angle and diameter. |
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