Systematic Evaluation of Skeletal Mechanical Function
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- Abstract
- Table of Contents
- Materials
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- Literature Cited
Abstract
Many genetic and environmental perturbations lead to measurable changes in bone morphology, matrix composition, and matrix organization. Here, straightforward biomechanical methods are described that can be used to determine whether a genetic or environmental perturbation affects bone strength. A systematic method is described for evaluating how bone strength is altered in the context of morphology and tissue?level mechanical properties, which are determined in large part from matrix composition, matrix organization, and porosity. The methods described include computed tomography, whole?bone mechanical tests (bending and compression), tissue?level mechanical tests, and determination of ash content, water content, and bone density. This strategy is intended as a first step toward screening mice for phenotypic effects on bone and establishing the associated biomechanical mechanism by which function has been altered, and can be conducted without a background in engineering. The outcome of these analyses generally provides insight into the next set of experiments required to further connect cellular perturbation with functional change. Curr. Protoc. Mouse Biol. 3:39?67 © 2013 by John Wiley & Sons, Inc.
Keywords: bone; biomechanics; strength; nanocomputed tomography; cortical bone; trabecular bone; adaptation
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Harvesting Bone
- Basic Protocol 2: Embedding Bone in Plastic
- Support Protocol 1: Preparation of Methyl Methacrylate Solutions
- Basic Protocol 3: Computed Tomography
- Basic Protocol 4: Measurement of Whole‐Bone Mechanical Properties Using a Four‐Point Bending Test
- Alternate Protocol 1: Measurement of Whole‐Bone Mechanical Properties Using a Three‐Point Bending Test
- Alternate Protocol 2: Measurement of Spine Compression
- Basic Protocol 5: Measurement of Tissue‐Level Mechanical Properties
- Basic Protocol 6: Measurement of Ash Weight, Water Content, and Bone Density
- Basic Protocol 7: Systematic Data Analysis
- Commentary
- Literature Cited
- Figures
- Tables
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PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: Harvesting Bone
Materials
Basic Protocol 2: Embedding Bone in Plastic
Materials
Support Protocol 1: Preparation of Methyl Methacrylate Solutions
Materials
Basic Protocol 3: Computed Tomography
Materials
Basic Protocol 4: Measurement of Whole‐Bone Mechanical Properties Using a Four‐Point Bending Test
Materials
Alternate Protocol 1: Measurement of Whole‐Bone Mechanical Properties Using a Three‐Point Bending Test
Alternate Protocol 2: Measurement of Spine Compression
Materials
Basic Protocol 5: Measurement of Tissue‐Level Mechanical Properties
Materials
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Figure 1. Proper marking of microcentrifuge tube with permanent marker and tissue cassette with no. 2 pencil. View Image -
Figure 2. Sectioning of mouse femora. (A ) Wide view of IsoMet diamond wafering saw. (B ) Enlarged view of chuck, specimen, and blade interface. (C ) Single femur embedded in polymerized MMA. View Image -
Figure 3. External view of Nanotom S nanoCT (approximately 64 x 56 x 29 inches and 2750 pounds). View Image -
Figure 4. Five‐bone scanning unit. (A ) Acrylic cylindrical tube with a threaded insert. (B ) Top view of six‐well acrylic specimen holder. (C ) Side view of same six‐well specimen holder showing threads (bottom) that screw into the insert of the tube to maintain stability. This view also shows the center in‐cut where a latex rubber band can be placed to further secure the bones. (D ) Filled calibration insert, with hydroxyapatite standard embedded in center. (E ) Empty calibration insert with a small air hole on top and solid acrylic bottom that represents water. View Image -
Figure 5. Acrylic equilibration block with drilled‐out center for placement of the loaded cylindrical tube. Metal stick inserts on the bottom to stabilize holder on the stage of the nanoCT. View Image -
Figure 6. Inside cabinet of Nanotom S. Cylindrical tube fits securely into the three‐jaw chuck between the X‐ray source (right) and detector panel (left). In this system, the tube is stationary, the detector can shift to the right or left to allow a larger field of view, and the specimen rotates on the stage in the center. The laser is used to center the specimen between the tube and detector. View Image -
Figure 7. Iso‐surface rendering of a single 16‐week chromosome substitution strain male femur using VGStudio MAX 2.1 scaled in Hounsfield. View Image -
Figure 8. Images from MicroView Advanced Bone Analysis on a mouse femur. (A ) Cylindrical region of interest (yellow) selected just below the third trochanter. (B ) Selected region of interest with cortical bone segmented from trabeculae. (C ) Distal femur slice just proximal to the growth plate. Yellow line shows spline separation of trabeculae and cortical shell. View Image -
Figure 9. Mechanical testing setup used for whole‐bone testing. (A ) Entire system is shown with a small custom frame to hold testing fixtures. (B ) Closer view of smaller frame on the testing machine. This is adjustable to incorporate different load cells and testing fixtures. (C ) Four‐point bending fixture with bone ready for testing. (1) Screw that adjusts the vertical height of the upper two points; (2) upper two points resting on the bone; (3) lower two points; (4) screws used to individually adjust height of the bottom points; (5) additional screw for adjusting lower points, with the ability to rotate them and lock them into place. (D ) Compression testing fixture for lumbar vertebrae. (6,7) Upper and lower platens. Latter has a peg to help position the vertebra. (E ) Compression testing fixture for caudal vertebrae. (8,9) Top and bottom platens have a small depression in the middle to help position the vertebra. Bottom platen rests on a peg so that it can be tilted slightly, if needed, to align the vertebra. View Image -
Figure 10. Typical load‐displacement curve. Stiffness is indicated by the blue line. The green line is stiffness multiplied by 0.9, which is useful when calculating yield point. View Image -
Figure 11. Density kit for use with an analytical balance. This kit contains a pan to measure dry weight as well a water bath and suspension wires to measure wet weight. View Image -
Figure 12. ANCOVAs of body size (body mass × length) and various traits for chromosome substitution strain 5 (CSS5) and control (B6) mice at 16 weeks. (A ) Stiffness, (B ) robustness, (C ) cortical area, and (D ) tissue mineral density (TMD). p < 0.008 is considered significant. View Image
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Literature Cited
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