Bone Mineral Content and Density

Christopher T. Esapa1, J.H. Duncan Bassett2, Holly Evans3, Peter I. Croucher4, Graham R. Williams2, Rajesh V. Thakker1

1 Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Headington, Oxford, United Kingdom, 2 Molecular Endocrinology Group, Department of Medicine, Imperial College London, Hammersmith Campus, London, United Kingdom, 3 The Mellanby Centre for Bone Research, Department of Human Metabolism, University of Sheffield, Sheffield, United Kingdom, 4 Garvan Institute for Medical Research, Sydney, Australia
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo120124
Online Posting Date:  December, 2012
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Abstract

The availability of high‐throughput biochemical and imaging techniques that can be used on live mice has increased the possibility of undertaking longitudinal studies to characterize skeletal changes such as bone mineral content and density. Further characterization of bone morphology, bone quality, and bone strength can also be achieved by analyzing dissected bones using techniques that provide higher resolution. Thus, the combined use of high‐throughput [e.g., biochemical analysis of plasma, radiography and dual‐energy X‐ray absorptiometry (DEXA)] and secondary phenotyping techniques (e.g., histology, histomorphometry, Faxitron digital X‐ray point projection microradiography, biomechanical testing, and micro‐computed tomography) can be utilized for comprehensive characterization of bone structure and quality and to elucidate the underlying molecular mechanisms giving rise to musculoskeletal disorders. Curr. Protoc. Mouse Biol. 2:365‐400 © 2012 by John Wiley & Sons, Inc.

Keywords: bone density; mouse models; radiography; dual‐energy X‐ray absorptiometry; Faxitron; biomechanical testing; micro‐computed tomography

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Biochemical Analysis
  • Basic Protocol 2: Radiography
  • Basic Protocol 3: Dual‐Energy X‐Ray Absorptiometry (DEXA)
  • Basic Protocol 4: Skeletal Sample Preparation and Fixation
  • Basic Protocol 5: Histology and Histomorphometry
  • Basic Protocol 6: Quantitative Faxitron Digital X‐Ray Microradiography
  • Basic Protocol 7: Biomechanical Testing
  • Basic Protocol 8: Quantitative Micro‐Computed Tomography (Micro‐CT)
  • Support Protocol 1: Use of the Batch Manager
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Biochemical Analysis

  Materials
  • Mice (control and experimental mice of the same strain, age, and sex)
  • Eutectic Mixture of Lidocaine and Prilocaine (EMLA) topical cream (Astra Zeneca), or other suitable topical anesthetic cream
  • Silver nitrate pencil to stem blood flow from tail vein
  • Personal protective equipment (PPE) including gloves and face mask
  • Vertical laminar air flow cabinet
  • Microvette tubes (Sarstedt) for collecting blood samples, e.g., lithium/heparin or EDTA‐coated tubes for plasma, and uncoated/clot activator tubes for serum
  • Permanent marker
  • Weighing scale
  • Heating box set at 37°C
  • Mouse restrainer
  • Scalpel blades
  • Ice box to chill blood samples prior to centrifugation
  • Microcentrifuge, chilled at 4°C
  • 1.5‐ml microcentrifuge tubes to hold plasma or serum samples

Basic Protocol 2: Radiography

  Materials
  • Mice
  • Anesthetic agent (ketamine at 100 mg/kg body weight)
  • Eye lubricant
  • Anesthetic reversal agent (xylazine at 10 mg/kg body weight)
  • Personal protective equipment (PPE) including gloves and face mask
  • Faxitron MX‐20 digital X‐ray system (Faxitron X‐ray Corporation)
  • Vertical laminar air flow cabinet
  • Weighing scale
  • Selotape
  • DicomWorks software (http://dicomworks.com)
  • Heating box at 37°C

Basic Protocol 3: Dual‐Energy X‐Ray Absorptiometry (DEXA)

  Materials
  • Mice
  • Anesthetic agent (ketamine at 100 mg/kg body weight)
  • Eye lubricant
  • Anesthetic reversal agent (xylazine at 10 mg/kg body weight)
  • Personal protective equipment (PPE) including gloves and face mask
  • Lunar PIXImus DEXA scanner (GE Lunar Piximus II X‐ray bone densitometer)
  • DEXA phantom mouse
  • Vertical laminar air flow cabinet
  • Weighing scale
  • Animal cages
  • Ruler
  • Mouse specimen trays
  • Selotape
  • Heating box set at 37°C

Basic Protocol 4: Skeletal Sample Preparation and Fixation

  Materials
  • Control and experimental mice of the same strain, age, and sex
  • 70% (v/v) ethanol
  • 10% Neutral buffered formalin (NBF; Sigma Aldrich)
  • Dissecting instruments including:
    • Scissors, fine
    • Scalpel
    • Tweezer
  • 20‐ml polystyrene tubes
  • 4°C incubator
  • Pencil
  • Additional reagents and equipment for euthanasia (Donovan and Brown, )

Basic Protocol 5: Histology and Histomorphometry

  Materials
  • Dissected bones (see protocol 4)
  • 10% EDTA, pH 7.4
  • Graded ethanol series
  • Paraffin wax (Leica Microsystems)
  • Pathcentre (Thermo Shandon)
  • Xylene (VWR) or Sub‐X (Leica Microsystems)
  • Scott's tap water (17.5 g sodium bicarbonate, 100 g magnesium sulfate, 25 ml formaldehyde, made up to 5 liters with distilled water)
  • Gill 3 Hematoxylin (Fisher Scientific)
  • Acid alcohol (1% hydrochloric acid in 70% Ethanol)
  • 20% lithium carbonate
  • Eosin (Fisher)
  • Isopropyl alcohol
  • Clearium mounting medium (Leica Microsystems)
  • 2% Alcian Blue 8GX (Fisher): prepared in 3% acetic acid, pH 2.5
  • Weigerts Hematoxylin (see recipe)
  • van Gieson (Fisher)
  • Acetate buffer, pH 5.2 (see recipe)
  • Acetate‐tartrate buffer (see recipe)
  • Naphthol AS‐BI phosphate solution (see recipe)
  • Pararsosaniline (see recipe)
  • 4% sodium nitrite (see recipe)
  • DPX (VWR)
  • LRWhite medium resin (Taab Laboratories)
  • Automated tissue processing system, optional
  • Paraffin embedding system (Leica), optional
  • Microtome (Leica Microsystems)
  • SuperFrost Plus slides (VWR)
  • 37°C incubator
  • Coplin jar, 37°C
  • Measuring cylinder, 37°C
  • Glass bottles
  • Leica DMRB microscope
  • Osteomeasure bone histomorphometry system (OsteoMetrics)
  • UV lamp

Basic Protocol 6: Quantitative Faxitron Digital X‐Ray Microradiography

  Materials
  • Bones in 70% ethanol (see protocol 4)
  • Faxitron MX20 variable kV point projection X‐ray source and digital image system (Qados, Cross Technologies)
  • ImageJ 1.45 software (download at http://rsb.info.nih.gov/ij/)
  • Calibration standards including:
    • 1‐mm diameter steel wire
    • 1‐mm diameter aluminum wire (Hollinbrow Precision Products))
    • 1‐mm diameter polyester fiber
  • Mitutoyo CD‐6 CP Digital calipers (Mitutoyo)
  • Paper towels
  • Microsoft Excel
  • Adobe Photoshop CS5 (Adobe Systems)

Basic Protocol 7: Biomechanical Testing

  Materials
  • Cleaned long bones or vertebrae from experimental mice of the same strain, age and sex, fixed and stored in 70% ethanol at 4°C ( protocol 4)
  • 70% ethanol
  • Cyano‐acrylate glue (Loctite Precision)
  • Instron 5543 load frame using 100 N or 500 N load cells and Bluehills2 software (Instron Limited)
  • Custom mounts for destructive 3‐point bend and compression testing of mouse bones (Quality Test Solutions)
  • Mitutoyo CD‐6 CP Digital calipers (Mitutoyo)
  • Aluminum foil
  • Safety glasses
  • Paper towels
  • Dissecting instruments including a scalpel with size 22 disposable blades (Swann‐Morton)
  • 6‐cm polystyrene petri dish
  • Fine forceps

Basic Protocol 8: Quantitative Micro‐Computed Tomography (Micro‐CT)

  Materials
  • Bone samples
  • SkyScan 1172 high‐resolution micro‐CT system (e2v technologies)
  • Computer for image reconstruction
  • Small BMD phantom set (SP‐4002; e2v technologies)
  • Cling‐film
  • Plastic straws or pipet tips or polystyrene tubes
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Figures

Videos

Literature Cited

Literature Cited
   Acevedo‐Arozena, A., Wells, S., Potter, P., Kelly, M., Cox, R.D., and Brown, S.D. 2008. ENU mutagenesis, a way forward to understand gene function. Annu. Rev. Genomics Hum. Genet. 9:49‐69.
   Barbaric, I., Perry, M.J., Dear, T.N., Rodrigues Da Costa, A., Salopek, D., Marusic, A., Hough, T., Wells, S., Hunter, A.J., Cheeseman, M., and Brown, S.D. 2008. An ENU‐induced mutation in the Ankrd11 gene results in an osteopenia‐like phenotype in the mouse mutant Yoda. Physiol. Genomics 32:311‐321.
   Bassett, J.H., Boyde, A., Howell, P.G., Bassett, R.H., Galliford, T.M., Archanco, M., Evans, H., Lawson, M.A., Croucher, P., St Germain, D.L., Galton, V.A., and Williams, G.R. 2010. Optimal bone strength and mineralization requires the type 2 iodothyronine deiodinase in osteoblasts. Proc. Natl. Acad. Sci. U.S.A. 107:7604‐7609.
   Beamer, W.G., Donahue, L.R., Rosen, C.J., and Baylink, D.J. 1996. Genetic variability in adult bone density among inbred strains of mice. Bone 18:397‐403.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Esapa, C.T., Hough, T.A., Testori, S., Head, R.A., Crane, E.A., Chan, C.P., Evans, H., Bassett, J.H., Tylzanowski, P., McNally, E.G., Carr, A.J., Boyde, A., Howell, P.G., Clark, A., Williams, G.R., Brown, M.A., Croucher, P.I., Nesbit, M.A., Brown, S.D., Cox, R.D., Cheeseman, M.T., and Thakker, R.V. 2012. A mouse model for spondyloepiphyseal dysplasia congenita with secondary osteoarthritis due to a Col2a1 mutation. J Bone Miner Res. 27:413‐428.
   Franco, G.E., Litscher, S.J., O'Neil, T.K., Piette, M., Demant, P., and Blank, R.D. 2005. Dual energy X ray absorptiometry of ex vivo HcB/Dem mouse long bones: left are denser than right. Calcif. Tissue Int. 76:26‐31.
   Hough, T.A., Nolan, P.M., Tsipouri, V., Toye, A.A., Gray, I.C., Goldsworthy, M., Moir, L., Cox, R.D., Clements, S., Glenister, P.H., Wood, J., Selley, R.L., Strivens, M.A., Vizor, L., McCormack, S.L., Peters, J., Fisher, E.M., Spurr, N., Rastan, S., Martin, J.E., Brown, S.D., and Hunter, A.J. 2002. Novel phenotypes identified by plasma biochemical screening in the mouse. Mamm. Genome 13:595‐602.
   Hough, T.A., Bogani, D., Cheeseman, M.T., Favor, J., Nesbit, M.A., Thakker, R.V., and Lyon, M.F. 2004. Activating calcium‐sensing receptor mutation in the mouse is associated with cataracts and ectopic calcification. Proc. Natl. Acad. Sci. U.S.A. 101:13566‐13571.
   Karunaratne, A., Esapa, C.R., Hiller, J., Boyde, A., Head, R., Bassett, J.H., Terrill, N.J., Williams, G.R., Brown, M.A., Croucher, P.I., Brown, S.D., Cox, R.D., Barber, A.H., Thakker, R.V., and Gupta, H.S. 2012. Significant deterioration in nanomechanical quality occurs through incomplete extrafibrillar mineralization in rachitic bone: evidence from in‐situ synchrotron X‐ray scattering and backscattered electron imaging. J. Bone Miner Res. 27:876‐890.
   Parfitt, A.M., Drezner, M.K., Glorieux, F.H., Kanis, J.A., Malluche, H., Meunier, P.J., Ott, S.M., and Recker, R.R. 1987. Bone histomorphometry: Standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner Res. 2:595‐610.
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