Bone Imaging and Mechanical Testing · Musculoskeletal Research Center 6/20/2014 . microCT –...
Transcript of Bone Imaging and Mechanical Testing · Musculoskeletal Research Center 6/20/2014 . microCT –...
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Bone Imaging and Mechanical Testing
WU Musculoskeletal Research Center
Musculoskeletal Structure &Strength Core
Matthew J. Silva, Ph.D.
June 20, 2014
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Questions
• Does my mouse have a skeletal phenotype?
• I treated rats with drug XYZ … what was the effect on their bones?
• What outcomes do I need to measure?
• How do I integrate microCT and mechanical testing outcomes?
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Structure & Strength
• Imaging → Structure – how much bone is there? how is it distributed?
– density, morphology (geometry)
– whole-bone, cortical, cancellous
• Mechanical Testing → Strength – stiffness, strength, toughness
– length scale • big: ‘structural’ (whole-bone, organ)
• small: ‘material’ (tissue)
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Imaging → Structure
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X-Ray Based Imaging
• Based on x-ray absorption/attenuation
• Attenuation depends on thickness (t), density
• Can calibrate x-ray absorption to:
– Equivalent density of hydroxyapatite (HA) phantom
X-ray source Bone
X-ray Detector
(film, digital) t
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Musculoskeletal Research Center 6/20/2014
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Radiography • Outcomes: qualitative phenotype, spontaneous
fractures, fracture callus
Kai Yu, Dave Ornitz
Jenny McKenzie, Evan Buettmann, Mike Gardner
10 week
6
2 week post-fracture
5 month
18 month
Faxitron
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DXA
Outcomes:
• BMC (g), aBMD (g/cm2), %fat
GE/Lunar Piximus
Uses:
• Whole animal
• Phenotyping
• Systemic intervention
• Longitudinal studies
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Piximus
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DXA shows decreased post-natal bone mass in MAGP-deficient mice
– Phenotyping
– Longitudinal
– Whole animal
Craft, Mecham et al., 2010 8
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QCT – Quantitative Computed Tomography
• grayscale image reflects local attenuation, i.e., “mineral density map” [whiter = denser]
• morphology & density
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microCT
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Musculoskeletal Research Center 6/20/2014
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microCT – regions of interest Cancellous/Trabecular/Metaphyseal Scan region
• 100 slices = 2 mm Analysis region
• landmark (eg, growth plate) • 40 slices = 0.8 mm
Cortical/Diaphyseal Scan region = Analysis region
• Mid-point (50% of length), or X mm from TFJ • 35 slices = 0.7 mm
TFJ
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Musculoskeletal Research Center 6/20/2014
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microCT – cortical analysis
Original Ideal
Total Volume Bone Volume
Original (grayscale) Segmented
Cortical Outcomes
• Total area (or volume) – How big is the bone? → periosteal apposition
• Bone (cortical) area – How much bone is there? → formation & resorption
• Marrow area → resorption
• Cortical thickness → formation & resorption
• Area moment of inertia → How is bone distributed? Resists bending loads.
• Tissue mineral density (“mean2”) → mineralization
Binary • white=bone • black=not bone
Contour Threshold/ Segment
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Musculoskeletal Research Center 6/20/2014
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microCT – cancellous analysis
• Cancellous Outcomes* – Bone volume fraction (BV/TV) – What fraction of marrow cavity is filled
with bone?
– Trabecular thickness (Tb.Th)
– Separation (Tb.Sp)
– Trabecular Number (Tb.N)
– Density, vBMD (“mean1”) – Should correlate with BV/TV
* Use Direct Method values; prefix “VOX”; do not use Plate Model values; prefix “TRI”
Original (grayscale) Segmented (binary) Total volume (TV) Bone volume (BV)
Bouxsein et al. 2010 13
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Specimen microCT reveals osteopenia in diabetic rats
Parameter
Control
(n = 9)
Diabetic
(n = 8)
Tb.BV/TV
(mm3/mm3)
0.37
± 0.04
0.12 *
± 0.04
Tb.Th
(mm)
0.11
± 0.01
0.07 *
± 0.01
Tb.N
(1/mm)
4.4
± 0.2
2.6 *
± 0.5
Bone Area
(mm2)
5.85
± 0.30
4.46 *
± 0.27
Moment of
Inertia (mm4)
4.03
± 0.89
3.03 *
± 0.54
Cortical TMD
(mg/cm3)
1092
± 33
1111
± 51
Silva et al. 2009
Trab
ecu
lar
Co
rtic
al
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In Vivo CT: Mechanical Loading Stimulates Cortical Bone Accrual
(in Age-Dependent Manner)
Silva et al., 2012
BALB/c female mice
a*
a-5
0
5
10
15
0 3 6
Control
Loaded
12 months
Time (wk)
ab
*
b
*a
-5
0
5
10
15
0 3 6
Time (wk)
Ct.B
V (
% c
han
ge)
4 months
Control
Loaded
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Mechanical Testing → Strength
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How to measure bone mechanical properties
• No
• Yes
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Instron mechanical testing machine
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3-pt bending, Rat ulna
F
d Yield
Forc
e (
N)
0
10
20
30
40
0 0.02 0.04 0.06 0.08
Displacement (mm)
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Mechanical Testing:
Force-Displacement Plot
Fracture
Elastic Post-Yield
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Musculoskeletal Research Center
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• Stiffness (N/mm)
• Strength
– Yield Force (N)
– Ultimate Force (N)
• Post-yield
displacement
(ductility) (mm)
• Energy-to-fracture
(Nmm)
3-pt bending, Rat ulna F
d
L
Yield Stiffness
Ultimate Force (strength)
Fo
rce (
N)
0
10
20
30
40
0 0.02 0.04 0.06 0.08
Displacement (mm)
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Mechanical Properties
Energy-to-Fracture
Fracture
PYD (ductility)
Yield Force
(strength)
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Mechanical properties
0
5
10
15
20
25
0 0.2 0.4 0.6
Femur
Radius
Displacement (mm)
20
2-month female
BALB/c mice Femur vs. Radius:
– stiffer
– stronger
– less ductile
– requires more
energy to fracture
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Musculoskeletal Research Center
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Whole-bone properties depend on
morphology & material
0
5
10
15
20
25
0 0.2 0.4 0.6
Femur
Radius
Displacement (mm)
Femur
Radius
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Musculoskeletal Research Center
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Big: • Structure
• Whole-Bone
I-beam Steel
Femur
(whole-bone)
Bone
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Small: • Material
• Tissue
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Musculoskeletal Research Center
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Material Properties
• Estimate from whole-bone mechanical test
and bone size (microCT)
– Whole-bone strength: Ultimate Force (N)
– Material strength: Ultimate Stress (N/mm2)
• Measure with tissue-level test
– small piece of bone
– microindentation
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Microindentation
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Case Study 1: Asxl2 null mice
Wei Zou, Steve Teitelbaum
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Asxl2-null mice have low BMD
age- and body weight-adjusted Farber et al, 2011 MJ Silva, Washington University
Musculoskeletal Research Center 27 6/20/2014
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Asxl2-null mice: more cancellous bone, but less cortical bone
Wei Zou
Cancellous Cortical
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0
0.1
0.2
0.3
J (m
m4)
WT KO
*
0
10
20
30
Ult
imat
e Fo
rce
(N)
WT KO
*
0
50
100
150
200
250
Ult
imat
e St
ress
(M
Pa)
WT KO
Asxl2-null femurs: small & weak bones, normal material strength
Moment of Inertia Whole-bone strength Material strength
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Asxl2-null mice - Summary
• Increased trabecular bone (osteoclast defect) • Decreased bone size & whole-body BMD
• Bones are weaker in proportion to their smaller size • Normal material properties
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Case Study 2: Tsp1 null mice
Sarah Amend, Kathy Weilbaecher
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16
we
ek
wild type
TSP1-/- mice have increased trabecular bone
TSP1-/-
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Amend et al., In review
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TSP1-/- mice have larger bones w
ild t
yp
e
TS
P1
-/-
Cortical thickness
WT TSP1-/-0.00
0.05
0.10
0.15
0.20**
mm
Cortical thickness
WT TSP1-/-0.00
0.05
0.10
0.15
0.20**
mm
12 wk avg thickness
WT TSP1-/-0.0
0.1
0.2
0.3*
Total area
WT TSP1-/-0.0
0.5
1.0
1.5
***
mm
2
Total area
WT TSP1-/-0.0
0.5
1.0
1.5
***m
m2
12 wk T.ar
WT TSP1-/-0.0
0.5
1.0
1.5
2.0
2.5**
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Amend et al., In review
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Tsp1-null femurs: larger, moderately stronger, weaker material
Moment of Inertia Whole-bone strength Material strength
mm
4
Ult
imat
e Fo
rce
(N)
+17%
+60%
Yiel
d S
tres
s (N
/mm
2)
-35%
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Amend et al., In review
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TSP1-null bone: damages more easily at material level
2 N
microindentation
Indentation distance increase
WT TSP1-/-0
5
10
15
*m
m
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Amend et al., In review
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TSP1-null mice: Summary
• Increased trabecular bone • Increased cortical bone size
• Bones are moderately stronger – disproportionate to size • Impaired material properties
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musculoskeletalcore.wustl.edu
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Acknowledgements
• Dan Leib
• Michael Brodt
• Tarpit Patel
• Steve Thomopoulos
• Simon Tang
• NIH/NIAMS P30AR057235
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