Bone Imaging and Mechanical Testing · Musculoskeletal Research Center 6/20/2014 . microCT – cancellous analysis • Cancellous Outcomes* – Bone volume fraction (BV/TV) – What

Bone Imaging and Mechanical Testing

WU Musculoskeletal Research Center

Musculoskeletal Structure &Strength Core

Matthew J. Silva, Ph.D.

June 20, 2014

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?

MJ Silva, Washington University Musculoskeletal Research Center 2 6/20/2014

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)

MJ Silva, Washington University Musculoskeletal Research Center

3 6/20/2014

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

5 MJ Silva, Washington University

Musculoskeletal Research Center 6/20/2014

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


6/20/2014

DXA

Outcomes:

• BMC (g), aBMD (g/cm2), %fat

GE/Lunar Piximus

Uses:

• Whole animal

• Phenotyping

• Systemic intervention

• Longitudinal studies

7

Piximus


6/20/2014

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



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



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



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



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

17

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)

18

Mechanical Testing:

Force-Displacement Plot

Fracture

Elastic Post-Yield

MJ Silva, Washington University

Musculoskeletal Research Center

6/20/2014

• 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)

19

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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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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Big: • Structure

• Whole-Bone

I-beam Steel

Femur

(whole-bone)

Bone

22

Small: • Material

• Tissue



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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

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

Asxl2-null mice: more cancellous bone, but less cortical bone

Wei Zou

Cancellous Cortical


28 6/20/2014

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


30 6/20/2014

Case Study 2: Tsp1 null mice

Sarah Amend, Kathy Weilbaecher

16

we

ek

wild type

TSP1-/- mice have increased trabecular bone

TSP1-/-


32 6/20/2014

Amend et al., In review

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**


33 6/20/2014


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%


34 6/20/2014


TSP1-null bone: damages more easily at material level

2 N

microindentation

Indentation distance increase

WT TSP1-/-0

5

10

15

*m

m


35 6/20/2014


TSP1-null mice: Summary

• Increased trabecular bone • Increased cortical bone size

• Bones are moderately stronger – disproportionate to size • Impaired material properties


36 6/20/2014

musculoskeletalcore.wustl.edu


37 6/20/2014

Acknowledgements

• Dan Leib

• Michael Brodt

• Tarpit Patel

• Steve Thomopoulos

• Simon Tang

• NIH/NIAMS P30AR057235


38 6/20/2014

Documents

Bone Imaging and Mechanical Testing · Musculoskeletal Research Center 6/20/2014 . microCT – cancellous analysis • Cancellous Outcomes* – Bone volume fraction (BV/TV) – What