Systematic Evaluation of Skeletal Mechanical Function

Lauren Smith1, Erin M.R. Bigelow1, Karl J. Jepsen1

1 The University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo130027
Online Posting Date:  June, 2013
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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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Table 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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Materials

Basic Protocol 1: Harvesting Bone

  Materials
  • Mouse
  • 1× phosphate‐buffered saline (PBS)
  • 10% neutral buffered formalin (NBF)
  • 2‐ml microcentrifuge tubes
  • Permanent marker
  • Freezer storage boxes, cardboard or plastic
  • Gauze
  • Surgical tools
  • Tissue cassettes (Histosette II, Fisher Scientific)
  • No. 2 pencil
  • 1‐liter wide‐mouth HDPE bottles (Qorpak)
  • Absorbent pads
  • Additional reagents and equipment for euthanasia (Donovan and Brown, )

Basic Protocol 2: Embedding Bone in Plastic

  Materials
  • Bone samples in tissue cassettes (see protocol 1)
  • 10% neutral buffered formalin (NBF)
  • 1× phosphate‐buffered saline (PBS) or distilled water
  • Villanueva Osteochrome Bone Stain (Polysciences, optional)
  • Methanol (optional)
  • Ethylene glycol monoethyl ether (EGME)
  • 2‐Propanol
  • Methyl salicylate, 99%
  • Methyl methacrylate (MMA) solutions I‐IV (see protocol 3)
  • Cyanoacrylate
  • Diamond slurries (1‐2, 6, and 10 µm)
  • 1‐liter wide‐mouth HDPE bottles (Qorpak)
  • Agitator
  • 5‐ml plastic vials with caps
  • Vacuum oven (Isotemp or Eurotherm 91e Blue, Fisher Scientific)
  • Small drill
  • Small wooden dowel rod
  • Cutting device: commercial metallurgical cutting system with diamond wafering blade (e.g., Isomet Low‐Speed Saw, Buehler)
  • 1 × 3−in. plastic slides
  • Sandpaper ranging from 600 to 1000 grit
  • Light microscope (Axioplan2, Carl Zeiss IMT) or epifluorescence microscope
CAUTION: DO NOT open bottles of MMA II, III, or IV until they have been warmed completely to room temperature. Water vapor may condense on the surface of cold MMA when exposed to normal, humidified air, which will introduce water into the solution and compromise the embedding procedure.

Support Protocol 1: Preparation of Methyl Methacrylate Solutions

  Materials
  • Methyl methacrylate (MMA)
  • n‐Butyl phthalate (n‐BP)
  • Dry benzoyl peroxide
  • 1‐liter wide‐mouth HDPE bottles (Qorpak)
  • Orbital shaker

Basic Protocol 3: Computed Tomography

  Materials
  • Water or 1× phosphate‐buffered saline (PBS)
  • Frozen bone samples (see protocol 1)
  • Calibration standard
  • Nanotom S computed tomography system (GE Sensing & Inspection Technologies GmbH; Fig. )
  • Cylindrical specimen tube (made out of acrylic, polycarbonate, or other similar material)
  • Custom fixtures to hold specimen holder and acrylic equilibration bath (if needed, will depend on your design)
  • Bone holder (made out of acrylic, polycarbonate, or other similar material)
  • 1.69 mg/cc cortical bone equivalent rod, hydroxyapatite standard (Gammex)
  • Acrylic equilibration bath
  • Sonicator
  • Latex rubber bands
  • Acrylic pieces
  • Computers for acquisition and reconstruction (128 GB RAM; 8 CPUs; Intel Xeon 3.2 GHz; NVIDIA Graphics cards; 2 TB hard drive; Linux virtual machine)
  • Reconstruction software
  • VGStudio MAX 2.1 Software (Volume Graphics GmbH)
  • MicroView Advanced Bone Analysis (v. 2.2; GE Healthcare) software
  • Filter material (0.25, 0.3, or 0.762 mm aluminum)

Basic Protocol 4: Measurement of Whole‐Bone Mechanical Properties Using a Four‐Point Bending Test

  Materials
  • Whole frozen femurs (see protocol 1)
  • 1× phosphate‐buffered saline (PBS)
  • Mechanical testing system with four‐point fixture (Fig. A‐C)
  • Absorbent pads
  • Gauze
  • Small‐diameter (∼2‐mm) wooden dowel rods cut into 1‐inch (∼2.5‐cm) segments
  • Forceps
  • Glass Petri dish

Alternate Protocol 1: Measurement of Whole‐Bone Mechanical Properties Using a Three‐Point Bending Test

  • Whole frozen vertebrae (see protocol 1)
  • Appropriate standard, e.g., pencil‐top erasers
  • Lumbar or caudal testing fixtures (Fig. D,E)

Alternate Protocol 2: Measurement of Spine Compression

  Materials
  • Fresh or frozen bone samples, e.g., femurs (see protocol 1)
  • Scotch‐Weld acrylic adhesive DP‐810
  • Basic fuchsin
  • Caroplastic (Carolina Biological Supply Co.)
  • Alignment device
  • Brass pots
  • CNC milling machine (Modela MDX‐20, Roland DGA)
  • Light microscope (e.g., Zeiss Axioplan2, Carl Zeiss IMT)
  • Micro‐scissors
  • Four‐point bending fixture (see protocol 5)
  • Servo‐hydraulic materials testing system (Instron model 8872)
  • High‐resolution digital video camera (RH1100, Duncan Tech)
  • Video zoom microscope lens (Edmund Industrial Optics)
  • 16‐µm silicon carbide particles (McMaster‐Carr, optional)
  • Low‐speed diamond‐coated wafering saw (Buehler)
  • Acrylic slides
  • Digital Exwave HAD 3CCD Camera (Sony)
  • Digital‐image correlation (IMAQ Vision Builder 6.0, National Instruments Corp.)

Basic Protocol 5: Measurement of Tissue‐Level Mechanical Properties

  Materials
  • Fresh or frozen bone samples (see protocol 1) or broken bones from four‐point bending tests (see protocol 5)
  • Distilled water
  • Stereomicroscope
  • Vacuum oven (Fisher Scientific Isotemp or Eurotherm 91e Blue)
  • Analytical balance with strain gauge wire (Fig. )
  • Delicate task wipes
  • 2‐ml microcentrifuge tubes
  • Microcentrifuge (Fisher Scientific accuSpin Micro 17)
  • Ashing oven (Thermo Lindberg Blue M)
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Figures

Videos

Literature Cited

Literature Cited
   Bonadio, J., Jepsen, K.J., Mansoura, M.K., Jaenisch, R., Kuhn, J.L., and Goldstein, S.A. 1993. A murine skeletal adaptation that significantly increases cortical bone mechanical properties. Implications for human skeletal fragility. J. Clin. Invest. 92:1697‐1705.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Furuya, K., Nifuji, A., Rosen, V., and Noda, M. 1999. Effects of GDF7/BMP12 on proliferation and alkaline phosphatase expression in rat osteoblastic osteosarcoma ROS 17/2.8 cells. J. Cell. Biochem. 72:177‐180.
   Jepsen, K.J. 2009. Systems analysis of bone. Wiley Interdiscip. Rev. Syst. Biol. Med. 1:73‐88.
   Jepsen, K.J., Schaffler, M.B., Kuhn, J.L., Goulet, R.W., Bonadio, J., and Goldstein, S.A. 1997. Type I collagen mutation alters the strength and fatigue behavior of Mov13 cortical tissue. J. Biomech. 30:1141‐1147.
   Jepsen, K.J., Davy, D.T., and Krzypow, D.J. 1999. The role of the lamellar interface during torsional yielding of human cortical bone. J. Biomech. 32:303‐310.
   Jepsen, K.J., Hu, B., Tommasini, S.M., Courtland, H.‐W., Price, C., Terranova, C.J., and Nadeau, J.H. 2007. Genetic randomization reveals functional relationships among morphologic and tissue‐quality traits that contribute to bone strength and fragility. Mamm. Genome 18:492‐507.
   Jepsen, K.J., Hu, B., Tommasini, S.M., Courtland, H.‐W., Price, C., Cordova, M., and Nadeau, J.H. 2009. Phenotypic integration of skeletal traits during growth buffers genetic variants affecting the slenderness of femora in inbred mouse strains. Mamm. Genome 20:21‐33.
   Jepsen, K.J., Courtland, H.‐W., and Nadeau, J.H. 2010. Genetically‐determined phenotype covariation networks control bone strength. J. Bone Miner. Res. 25:1581‐1593.
   Lou, J., Tu, Y., Burns, M., Silva, M.J., and Manske, P. 2001. BMP‐12 gene transfer augmentation of lacerated tendon repair. J. Orthop. Res. 19:1199‐1202.
   Maloul, A., Rossmeier, K., Mikic, B., Pogue, V., and Battaglia, T. 2006. Geometric and material contributions to whole‐bone structural behavior in GDF‐7‐deficient mice. Connect. Tissue Res. 47:157‐162.
   Marder, E. and Goaillard, J.M. 2006. Variability, compensation and homeostasis in neuron and network function. Nat. Rev. Neurosci. 7:563‐574.
   Nadeau, J.H., Burrage, L.C., Restivo, J., Pao, Y.H., Churchill, G., and Hoit, B.D. 2003. Pleiotropy, homeostasis, and functional networks based on assays of cardiovascular traits in genetically randomized populations. Genome Res. 13:2082‐2091.
   Waddington, C.H. 1942. Canalization of development and the inheritance of acquired characters. Nature 14:563‐565.
   Wright, S. 1921. Correlation and causation. J. Agric. Res. 20:557‐585.
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