使用显微计算机断层扫描评估骨折愈合
1Center for Orthopaedic Research and Translational Science (CORTS),The Pennsylvania State University College of Medicine, 2Department of Orthopaedics and Rehabilitation,The Pennsylvania State University College of Medicine, 3Department of Pharmacology,The Pennsylvania State University College of Medicine, 4Department of Biochemistry and Molecular Biology,The Pennsylvania State University College of Medicine
Summary
显微计算机断层扫描（μCT）是一种无损成像工具，有助于评估临床前研究中的骨结构，但对用于分析骨愈合愈伤组织的μCT程序缺乏共识。本研究提供了一种循序渐进的μCT方案，可以监测骨折愈合。
Abstract
显微计算机断层扫描 （μCT） 是转化科学研究中表征骨折愈合过程中骨骼和新形成骨骼三维 （3D） 形态的最常见成像方式。啮齿动物长骨折愈合的研究通常涉及二次愈合和矿化愈伤组织的形成。形成的愈伤组织的形状和新形成的骨的密度可能在时间点和治疗之间有很大差异。虽然量化完整皮质和小梁骨参数的标准方法被广泛使用并嵌入到市售软件中，但对分析愈合愈伤组织的程序缺乏共识。这项工作的目的是描述一种标准化方案，该方案定量愈合愈伤组织中的骨体积分数和愈伤组织矿物质密度。该协议描述了成像和分析过程中应考虑的不同参数，包括成像过程中的样品对齐、目标体积的大小以及轮廓化以定义愈伤组织的切片数量。
Introduction
显微计算机断层扫描（μCT）成像已广泛用于临床前骨骼研究，提供无创、高分辨率的图像来评估骨骼的微观结构1,2,3,4,5。μCT涉及大量X射线图像，这些图像来自旋转样品或使用旋转的X射线源和检测器获得。算法用于以图像切片堆栈的形式重建 3D 体积数据。临床CT是人体骨骼3D成像的金标准，μCT是评估实验动物骨愈合效率的常用技术1,2,3,4,6,7。矿化骨与X射线具有极好的对比度，而软组织的对比度相对较差，除非使用造影剂。在骨折愈合评估中，μCT生成的图像提供有关矿化愈伤组织3D结构和密度的详细信息。体内μCT扫描也可用于骨折愈合的纵向、时程评估。
使用μCT对完整皮质和小梁骨的定量通常是成熟和标准化的8。尽管临床前研究使用各种定量方法来分析骨折愈合9,10,11，但用于愈伤组织定量的μCT图像分析的详细方案尚未发表。因此，本研究的目的是为骨愈合愈伤组织的μCT成像和分析提供详细的分步方案。
Protocol
开发以下协议以表征从安乐死小鼠收获的长骨愈合愈伤组织。然而，大多数步骤可以应用于大鼠，也可用于骨折骨的 体内 扫描。该协议描述了特定的μCT系统和特定的图像处理、分析和可视化软件（参见 材料表），但该方法通常适用于其他扫描仪和软件。该协议已获得宾夕法尼亚州立大学医学院机构动物护理和使用委员会的批准。本研究中使用的小鼠是16周龄的雄性C57BL / 6J小?…
Representative Results
为了监测骨折愈合过程中的骨形成，在成年雄性C75BL / 6J小鼠中诱导了骨干开放性胫骨骨折。使用髓内钉稳定骨折，这是一种既定的二次愈合模型13。在骨折后第14、21和28天收获愈伤组织12。这些时间点代表了愈合的不同阶段。继发性骨愈合期间的软骨内骨形成通过纤维软骨（软）愈伤组织的初始形成 进行 ，愈伤组织在后期矿化以减少骨折间隙处的微运动…
Discussion
本研究的目的是描述μCT分析的详细方案，目的是准确量化3D矿化愈伤组织结构，这通常是骨和骨折愈合研究的基础。该协议利用通用的最先进的3D图像分析软件平台，该平台有助于图像可视化，分割/标记以及从简单到复杂的测量。
该方案中最耗时的任务是半自动分割愈伤组织，排除皮质骨和髓管。在以前的许多研究中，该区域也被排除在外9,16,17,18。</sup…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院（NIH）R01 DK121327 to R.A.E和R01 AR071968 to F.K.的支持。
Materials
| 10% neutral buffered formalin | Fisher chemical | SF100-20 | Used for bone tissue fixation |
| Avizo | Thermo Scientific | Image processing and analysis software | |
| Hydroxyapatite phantom | Micro-CT HA D4.5, QRM | QRM-70128 | |
| Image Processing Language | Scanco | Used to convert raw images to DICOM images | |
| Micro-Mosquito Straight Hemostatic Forceps | Medline | Used to remove the intramedullary pin | |
| Microsoft Excel | Microsoft | Spreadsheet software | |
| Scanco mCT system (vivaCT 40) | Scanco | Used for µCT imaging |
References
- Rüegsegger, P., Koller, B., Müller, R. A microtomographic system for the nondestructive evaluation of bone architecture. Calcified Tissue International. 58 (1), 24-29 (1996).
- Müller, R., et al. Morphometric analysis of human bone biopsies: a quantitative structural comparison of histological sections and micro-computed tomography. Bone. 23 (1), 59-66 (1998).
- Waarsing, J. H., et al. Detecting and tracking local changes in the tibiae of individual rats: a novel method to analyse longitudinal in vivo micro-CT data. Bone. 34 (1), 163-169 (2004).
- Boyd, S. K., Davison, P., Müller, R., Gasser, J. A. Monitoring individual morphological changes over time in ovariectomized rats by in vivo micro-computed tomography. Bone. 39 (4), 854-862 (2006).
- Christiansen, B. A. Effect of micro-computed tomography voxel size and segmentation method on trabecular bone microstructure measures in mice. Bone Reports. 5, 136-140 (2016).
- Holdsworth, D. W., Thornton, M. M. Micro-CT in small animal and specimen imaging. Trends in Biotechnology. 20 (8), 34-39 (2002).
- Schambach, S. J., Bag, S., Schilling, L., Groden, C., Brockmann, M. A. Application of micro-CT in small animal imaging. Methods. 50 (1), 2-13 (2010).
- Bouxsein, M. L., et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. Journal of Bone and Mineral Research. 25 (7), 1468-1486 (2010).
- Morgan, E. F., et al. Micro-computed tomography assessment of fracture healing: Relationships among callus structure, composition, and mechanical function. Bone. 44 (2), 335-344 (2009).
- O’Neill, K. R., et al. Micro-computed tomography assessment of the progression of fracture healing in mice. Bone. 50 (6), 1357-1367 (2012).
- Bissinger, O., et al. Fully automated segmentation of callus by micro-CT compared to biomechanics. Journal of Orthopaedic Surgery and Research. 12 (1), 108 (2017).
- Brown, M. L., et al. Delayed fracture healing and increased callus adiposity in a C57BL/6J murine model of obesity-associated type 2 diabetes mellitus. PLOS One. 9 (6), 99656 (2014).
- Khajuria, D. K., et al. Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model. Bone. 137, 115436 (2020).
- Sigurdsen, U., Reikeras, O., Hoiseth, A., Utvag, S. E. Correlations between strength and quantitative computed tomography measurement of callus mineralization in experimental tibial fractures. Clinical Biomechanics. 26 (1), 95-100 (2011).
- Duvall, C. L., Taylor, W. R., Weiss, D., Wojtowicz, A. M., Guldberg, R. E. Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontin-deficient mice. Journal of Bone and Mineral Research. 22 (2), 286-297 (2007).
- Gerstenfeld, L. C., et al. Comparison of effects of the bisphosphonate alendronate versus the RANKL inhibitor denosumab on murine fracture healing. Journal of Bone and Mineral Research. 24 (2), 196-208 (2009).
- Alentado, V. J., et al. Validation of the modified radiographic union score for tibia fractures (mRUST) in murine femoral fractures. Frontiers in Endocrinology. 13, 911058 (2022).
- Yu, K. E., et al. Enhancement of impaired MRSA-infected fracture healing by combinatorial antibiotics and modulation of sustained inflammation. Journal of Bone and Mineral Research. 37 (1), 1352-1365 (2022).
- Nyman, J. S., et al. Quantitative measures of femoral fracture repair in rats derived by micro-computed tomography. Journal of Biomechanics. 42 (7), 891-897 (2009).
- Fiset, S., et al. Experimental validation of the radiographic union score for tibial fractures (RUST) using micro-computed tomography scanning and biomechanical testing in an in-vivo rat model. The Journal of Bone and Joint Surgery. 100 (21), 1871-1878 (2018).
- Shefelbine, S. J., et al. Prediction of fracture callus mechanical properties using micro-CT images and voxel-based finite element analysis. Bone. 36 (3), 480-488 (2005).
- Liu, Y., et al. Glucocorticoid-induced delayed fracture healing and impaired bone biomechanical properties in mice. Clinical Interventions in Aging. 13, 1465-1474 (2018).
- Watson, P. J., Fitton, L. C., Meloro, C., Fagan, M. J., Gröning, F. Mechanical adaptation of trabecular bone morphology in the mammalian mandible. Scientific Reports. 8 (1), 7277 (2018).
- Nie, C., Wang, Z., Liu, X. The effect of depression on fracture healing and osteoblast differentiation in rats. Neuropsychiatric Disease and Treatment. 14, 1705-1713 (2018).
Cite This Article
Wee, H., Khajuria, D. K., Kamal, F., Lewis, G. S., Elbarbary, R. A. Assessment of Bone Fracture Healing Using Micro-Computed Tomography. J. Vis. Exp. (190), e64262, doi:10.3791/64262 (2022).