Quantitative analysis of bone and soft tissue by micro-computed tomography: applications to ex vivo and in vivo studies.

Micro-computed tomography (micro-CT) is a high-resolution imaging modality that is capable of analysing bone structure with a voxel size on the order of 10 μm. With the development of in vivo micro-CT, where disease progression and treatment can be monitored in a living animal over a period of time, this modality has become a standard tool for preclinical assessment of bone architecture during disease progression and treatment. For meaningful comparison between micro-CT studies, it is essential that the same parameters for data acquisition and analysis methods be used. This protocol outlines the common procedures that are currently used for sample preparation, scanning, reconstruction and analysis in micro-CT studies. Scan and analysis methods for trabecular and cortical bone are covered for the femur, tibia, vertebra and the full neonate body of small rodents. The analysis procedures using the software provided by ScancoMedical and Bruker are discussed, and the routinely used bone architectural parameters are outlined. This protocol also provides a section dedicated to in vivo scanning and analysis, which covers the topics of anaesthesia, radiation dose and image registration. Because of the expanding research using micro-CT to study other skeletal sites, as well as soft tissues, we also provide a review of current techniques to examine the skull and mandible, adipose tissue, vasculature, tumour severity and cartilage. Lists of recommended further reading and literature references are included to provide the reader with more detail on the methods described.

[1]  K. Balto,et al.  Quantification of Periapical Bone Destruction in Mice by Micro-computed Tomography , 2000, Journal of dental research.

[2]  Christoph Groden,et al.  Application of micro-CT in small animal imaging. , 2010, Methods.

[3]  J Daniel Bourland,et al.  Risedronate prevents early radiation-induced osteoporosis in mice at multiple skeletal locations. , 2010, Bone.

[4]  S. Boyd,et al.  Bone quality is partially recovered after the discontinuation of RANKL administration in rats by increased bone mass on existing trabeculae: an in vivo micro-CT study , 2011, Osteoporosis International.

[5]  M. Panjabi,et al.  The effect of radiation on the fracture repair process. A biomechanical evaluation of a closed fracture in a rat model , 1989, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[6]  M. Stolina,et al.  Sclerostin inhibition reverses systemic, periarticular and local bone loss in arthritis , 2013, Annals of the rheumatic diseases.

[7]  R Bogaerts,et al.  Development of micro-CT protocols for in vivo follow-up of mouse bone architecture without major radiation side effects. , 2011, Bone.

[8]  Ling Qin,et al.  3D image registration is critical to ensure accurate detection of longitudinal changes in trabecular bone density, microstructure, and stiffness measurements in rat tibiae by in vivo microcomputed tomography (μCT). , 2013, Bone.

[9]  R. Huiskes,et al.  Effects of PTH treatment on tibial bone of ovariectomized rats assessed by in vivo micro-CT , 2009, Osteoporosis International.

[10]  Robertson Js,et al.  Vertebrate Radiobiology (Lethal Actions and Associated Effects) , 1957 .

[11]  E. Fuchs,et al.  Targeted p120-Catenin Ablation Disrupts Dental Enamel Development , 2010, PloS one.

[12]  Marion Kee,et al.  Analysis , 2004, Machine Translation.

[13]  G. Lyritis,et al.  The laboratory rat as an animal model for osteoporosis research. , 2008, Comparative medicine.

[14]  L. Hofbauer,et al.  Sclerostin antibody treatment improves bone mass, bone strength, and bone defect regeneration in rats with type 2 diabetes mellitus , 2013, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[15]  TOR Hildebrand,et al.  Quantification of Bone Microarchitecture with the Structure Model Index. , 1997, Computer methods in biomechanics and biomedical engineering.

[16]  J. Nyman,et al.  Prostate cancer metastases alter bone mineral and matrix composition independent of effects on bone architecture in mice--a quantitative study using microCT and Raman spectroscopy. , 2013, Bone.

[17]  T Dufresne,et al.  Segmentation techniques for analysis of bone by three-dimensional computed tomographic imaging. , 1998, Technology and health care : official journal of the European Society for Engineering and Medicine.

[18]  Ralph Müller,et al.  In vivo micro-computed tomography allows direct three-dimensional quantification of both bone formation and bone resorption parameters using time-lapsed imaging. , 2011, Bone.

[19]  R. Müller,et al.  Micro-CT combined with bioluminescence imaging: a dynamic approach to detect early tumor-bone interaction in a tumor osteolysis murine model. , 2007, Bone.

[20]  Steven K. Boyd,et al.  Quantitative Ex-Vivo Micro-Computed Tomographic Imaging of Blood Vessels and Necrotic Regions within Tumors , 2012, PloS one.

[21]  Rik Huiskes,et al.  No effects of in vivo micro‐CT radiation on structural parameters and bone marrow cells in proximal tibia of wistar rats detected after eight weekly scans , 2007, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[22]  R. Huiskes,et al.  The influence of microcomputed tomography threshold variations on the assessment of structural and mechanical trabecular bone properties. , 2002, Bone.

[23]  Erik L Ritman,et al.  Micro-computed tomography of the lungs and pulmonary-vascular system. , 2005, Proceedings of the American Thoracic Society.

[24]  Vertebrate radiobiology (lethal actions and associated effects). , 1954, Annual review of nuclear science.

[25]  A Odgaard,et al.  Three-dimensional methods for quantification of cancellous bone architecture. , 1997, Bone.

[26]  S. Kimmel.  Architecture , 2013, Arsham-isms.

[27]  T. Garland,et al.  The Relative Importance of Genetics and Phenotypic Plasticity in Dictating Bone Morphology and Mechanics in Aged Mice: Evidence from an Artificial Selection Experiment , 2022 .

[28]  Steven K Boyd,et al.  Automatic segmentation of cortical and trabecular compartments based on a dual threshold technique for in vivo micro-CT bone analysis. , 2007, Bone.

[29]  Steven K Boyd,et al.  Radiation effects on bone architecture in mice and rats resulting from in vivo micro-computed tomography scanning. , 2008, Medical engineering & physics.

[30]  D. Chappard,et al.  Bone mass and microarchitecture of irradiated and bone marrow-transplanted mice: influences of the donor strain , 2009, Osteoporosis International.

[31]  Ralph Müller,et al.  Guidelines for assessment of bone microstructure in rodents using micro–computed tomography , 2010, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[32]  I. H. Parkinson,et al.  Variation in segmentation of bone from micro-CT imaging: implications for quantitative morphometric analysis , 2008, Australasian Physics & Engineering Sciences in Medicine.

[33]  M. Robinson,et al.  Sclerostin antibody treatment enhances bone strength but does not prevent growth retardation in young mice treated with dexamethasone. , 2011, Arthritis and rheumatism.

[34]  Ralph Müller,et al.  Monitoring individual morphological changes over time in ovariectomized rats by in vivo micro-computed tomography. , 2006, Bone.

[35]  W. R. Taylor,et al.  Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury. , 2004, American journal of physiology. Heart and circulatory physiology.

[36]  S. K. Boyd,et al.  Signs of irreversible architectural changes occur early in the development of experimental osteoporosis as assessed by in vivo micro-CT , 2008, Osteoporosis International.

[37]  Jack Lee,et al.  Automatic segmentation of 3D micro-CT coronary vascular images , 2007, Medical Image Anal..

[38]  S. Ralston,et al.  Genetic Background Modifies the Effects of Type 2 Cannabinoid Receptor Deficiency on Bone Mass and Bone Turnover , 2013, Calcified Tissue International.

[39]  S. Boyd,et al.  The Magnitude and Rate of Bone Loss in Ovariectomized Mice Differs Among Inbred Strains as Determined by Longitudinal In vivo Micro-Computed Tomography , 2008, Calcified Tissue International.

[40]  B. Hallgrímsson,et al.  Effects of growth hormone on the ontogenetic allometry of craniofacial bones , 2013, Evolution & development.

[41]  M. Akhter,et al.  Gender specific LRP5 influences on trabecular bone structure and strength. , 2007, Journal of musculoskeletal & neuronal interactions.

[42]  Ralph Müller,et al.  Micro-computed tomography: a method for the non-destructive evaluation of the three-dimensional structure of biological specimens. , 2008, Methods in molecular biology.

[43]  Craig L Duvall,et al.  The Role of Osteopontin in Recovery from Hind Limb Ischemia , 2007, Arteriosclerosis, thrombosis, and vascular biology.

[44]  Three-dimensional Image Registration Improves the Long-term Precision of In Vivo Micro-Computed Tomographic Measurements in Anabolic and Catabolic Mouse Models , 2014, Calcified Tissue International.

[45]  J. Stock,et al.  Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta. , 2003, The Journal of clinical investigation.

[46]  H. Weinans,et al.  In vivo imaging of cartilage degeneration using microCT-arthrography. , 2007, Osteoarthritis and cartilage.

[47]  D. Chappard,et al.  Three-Dimensional Characterization of the Vascular Bed in Bone Metastasis of the Rat by Microcomputed Tomography (MicroCT) , 2011, PloS one.

[48]  R. Zernicke,et al.  Osteoblast Response to Ovariectomy Is Enhanced in Intrinsically High Aerobic-Capacity Rats , 2011, Calcified Tissue International.

[49]  P Rüegsegger,et al.  Resolution dependency of microstructural properties of cancellous bone based on three-dimensional mu-tomography. , 1996, Technology and health care : official journal of the European Society for Engineering and Medicine.

[50]  H. Gundersen,et al.  Quantification of connectivity in cancellous bone, with special emphasis on 3-D reconstructions. , 1993, Bone.

[51]  Peter Willett,et al.  What is a tutorial , 2013 .

[52]  S. A. Arrington,et al.  Temporal changes in bone mass and mechanical properties in a murine model of tumor osteolysis. , 2006, Bone.

[53]  Françoise Peyrin,et al.  A constrained region growing approach based on watershed for the segmentation of low contrast structures in bone micro-CT images , 2008, Pattern Recognit..

[54]  A. Laib,et al.  The use of micro-CT to evaluate cortical bone geometry and strength in nude rats: correlation with mechanical testing, pQCT and DXA. , 2006, Bone.

[55]  M. Almeida,et al.  The estrogen receptor-alpha in osteoclasts mediates the protective effects of estrogens on cancellous but not cortical bone. , 2010, Molecular endocrinology.

[56]  L. Hofbauer,et al.  Effects of parathyroid hormone on bone mass, bone strength, and bone regeneration in male rats with type 2 diabetes mellitus. , 2014, Endocrinology.

[57]  C H Turner,et al.  Basic biomechanical measurements of bone: a tutorial. , 1993, Bone.

[58]  A. Boskey,et al.  Variation in Mineral Properties in Normal and Mutant Bones and Teeth , 2006, Cells Tissues Organs.

[59]  L. Zheng,et al.  Skeletal site-specific response to ovariectomy in a rat model: change in bone density and microarchitecture. , 2015, Clinical oral implants research.

[60]  C T Rubin,et al.  Quantification of adiposity in small rodents using micro-CT. , 2010, Methods.

[61]  R E Guldberg,et al.  Quantitative assessment of articular cartilage morphology via EPIC-microCT. , 2009, Osteoarthritis and cartilage.

[62]  K. Dahlman-Wright,et al.  Estrogen receptor-α in osteocytes is important for trabecular bone formation in male mice , 2013, Proceedings of the National Academy of Sciences.

[63]  D W Holdsworth,et al.  Fundamental image quality limits for microcomputed tomography in small animals. , 2003, Medical physics.

[64]  R. Mole,et al.  Quantitative observations on recovery from whole body irradiation in mice. II. Recovery during and after daily irradiation. , 1957, The British journal of radiology.

[65]  D. Holdsworth,et al.  Micro-CT in small animal and specimen imaging , 2002 .

[66]  P. Rüegsegger,et al.  A new method for the model‐independent assessment of thickness in three‐dimensional images , 1997 .

[67]  F. Jirik,et al.  Micro-CT evaluation of bone defects: applications to osteolytic bone metastases, bone cysts, and fracture. , 2013, Medical engineering & physics.

[68]  Todd E Peterson,et al.  Longitudinal live animal micro-CT allows for quantitative analysis of tumor-induced bone destruction. , 2011, Bone.

[69]  R. J. van ‘t Hof.  Analysis of bone architecture in rodents using microcomputed tomography. , 2012, Methods in molecular biology.

[70]  Steven K Boyd,et al.  Reproducibility of bone micro-architecture measurements in rodents by in vivo micro-computed tomography is maximized with three-dimensional image registration. , 2010, Bone.

[71]  Rob van't Hof,et al.  Analysis of bone architecture in rodents using microcomputed tomography. , 2012 .

[72]  W. Jonat,et al.  Application of ex vivo micro-computed tomography for assessment of in vivo fluorescence and plain radiographic imaging for monitoring bone metastases and osteolytic lesions , 2012, Journal of Bone and Mineral Metabolism.

[73]  Steven K. Boyd,et al.  A Novel 3-D Image-Based Morphological Method for Phenotypic Analysis , 2008, IEEE Transactions on Biomedical Engineering.

[74]  E. Seeman,et al.  Insights into material and structural basis of bone fragility from diseases associated with fractures: how determinants of the biomechanical properties of bone are compromised by disease. , 2007, Endocrine reviews.