Bone Mineral Content and Density

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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

Figure 1. Equipment for blood collection from mouse tail vein. (A ) Mouse restrainer. (B ) Microvette tube and sleeve assembly. (C ) Introduction of incision in the tail of a mouse held in the restrainer. (D ) Microvette tube used to collect blood by capillary action following incision of mouse tail vein.

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Figure 2. Equipment for radiography and DEXA analysis of mice. (A ) Faxitron MX‐20 digital X‐ray system with appropriate shielding. (B ) Lunar PIXImus DEXA scanner without shielding and (C ) with shielding and connected to a computer. (D ) Sample data readout from the DEXA scanner.

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Figure 3. Sample collection, fixation, and analysis in mouse skeletal phenotyping.

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Figure 4. Histology and histomorphometric analysis. Proximal tibia from a 22‐week‐old mouse stained with (A ) Hematoxylin and eosin and (B ) Alcian blue (cartilage) and van Gieson (osteoid) revealing the articular cartilage and growth plate. (C ) Histomorphometry, using calcein to label new bone.

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Figure 5. Quantitative Faxitron digital X‐ray microradiography. (A ) Use of a Faxitron MX‐20 showing PC imaging software below to illustrate recommended organization of limbs and vertebrae alongside polyester, aluminum, and steel calibration standards. (B ) Original 16‐bit DICOM image. The histogram below shows the grayscale pixel distribution with location of the three standards relative to skeletal samples. The large peak on the left represents the background. (C ) Pseudo‐colored 8‐bit TIFF image following stretch processing. The histogram below shows the stretched grayscale distribution in relation to the 16 color bins. (D ) Pseudo‐colored 8‐bit TIFF image of two representative, cleaned femurs from wild‐type and mutant montages. Relative and cumulative frequency histograms show reduced bone mineral content in mutant mice ( n = 4). Kolmogorov‐Smirnov test, mutant 1 versus wild‐type, *** p <0.001. (E ) Faxitron image of digital micrometer set at 15 mm for ImageJ calibration. (F ) Grayscale images of two representative, cleaned femurs from wild‐type and mutant montages showing determination of femur length. (G ) Graphs illustrating reduced bone length and cortical thickness in mutant mice ( n = 4). Student's t test (mutant 2 versus wild‐type), * p <0.05, ** p <0.01. (H ) Images showing determination cortical bone thickness by measurement of the external and internal diameter at five separate mid diaphyseal locations.

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Figure 6. Biomechanical analysis. (A ) Instron 5543 load frame. (B ) Custom mount for destructive 3‐point bend testing of mouse bones incorporating rounded support and loading pins to minimize cutting and shear forces. The location of a femur during testing is shown. (C ) Custom mounts for mouse vertebral compression testing showing upper and lower anvils. The location of proximal caudal vertebrae during testing is shown. (D ) Instron Bluehills 2 software Illustrating load displacement values and curves. (E ) Grayscale Faxitron images showing medio‐lateral (ML) and anterior‐posterior (AP) views of a femur prior to fracture and an AP view post fracture. Small black and white arrows show the ML and AP cortical thickness and the red lines indicate how the femur cross‐section, shown above, was determined. The larger white arrows indicate the site of fracture. (F ) Femur load displacement curves from wild‐type and mutant mice showing a strong but brittle phenotype in mutant 1. (G‐H ) Femur load displacement curves showing the stored (orange) and dissipated (purple) energy at maximum load and at fracture. (I ) Grayscale images showing a Ca5 before and after fracture. White arrow shows the cylindrical height ( B ), which excludes the vertebral end plates. Small black arrows indicate internal diameter ( D _int ) and small white arrows the external diameter ( D _ext ). The red lines indicate how the cross‐section, shown above, was determined. Larger white arrows indicate the site of the crush fracture (J ). Caudal vertebrae load displacement curves from wild‐type and mutant mice showing a weak and flexible phenotype in mutant 2 and the lack of a clear point of fracture. (K ) Caudal vertebra load‐displacement curve showing elastic stored energy at maximum load [(ESE_m ) in orange] and dissipated energy at maximum load ( DE _m ) in purple. Initial cartilage compression is shown in dark gray. (L ) Determination of “Toughness” by calculating the dissipated energy per unit strain around maximum load. Dissipated energy ( DE ) is shown in purple and elastic stored energy ( ESE ) in orange. The compressive extension (δh) is indicated by the double‐headed black arrow.

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Figure 7. Micro‐computed tomography. (A ) SkyScan 1172 micro‐CT scanner. (B ) Plastic straw used to hold specimens in the sample holder and mouse tibia (specimen) wrapped in cling‐film to prevent drying. (C ) Tibia mounted within the straw inside the sample chamber. (D ) 3‐dimensional reconstruction of the tibia. (E ) X‐ray projection of the tibia through the longitudinal axis. Gray bar shows the region of interest (ROI) with arrow indicating extremes. The red line is the position of the transverse slice shown in F. (F ) Single transverse slice showing the ROI in red. (G ) 3‐dimensional reconstruction of the trabecular bone ROI upon which assessment of structural characteristics is undertaken.

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Videos

Literature Cited

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