Skeletal Structure and Strength CoreSkeletal Structure and ... · Skeletal Structure and Strength CoreSkeletal Structure and Strength Core musculkltl tldloskeletalcore.wustl.edu Matthew

Skeletal Structure and Strength CoreSkeletal Structure and Strength Core

l k l t l tl dmusculoskeletalcore.wustl.edu

Matthew J. Silva, Ph.D.

N b 20 2009November 20, 2009

Washington University Core Center for Musculoskeletal Biology and Medicine (NIH P30)

M.J. Silva, November 2009

Musculoskeletal Structure and Strength CoreMusculoskeletal Structure and Strength Core

Focus: Animal modelsObjectives:

1) to increase access to existing resources for densitometry, imaging, and mechanical testing

2) to enhance expert oversight and quality control2) to enhance expert oversight and quality control3) to provide training and enrichment opportunities related to core

services4) f i i d h i i i i4) to foster new interactions and enhance existing interactions

between members of the Research Base of the Core Center for Musculoskeletal Biology and Medicine (CCMBM) at Washington University

(Research Base = 48 Principal Investigators)


Musculoskeletal Structure and Strength Core

RadiographyFaxitron


Imaging(post mortem

& in vivo)Densitometry

DXA

pQCT

3D MorphologymicroCT

“Standard” “Specialized”

Bone

Long-BoneBending

Vertebral

Standard SpecializedDemineralizedBone Tension

Trabecular

MechanicalTesting

Instron MaterialsTesting Systems

Tendon

Compression

Insertion SiteTension

Indentation

MidsubstanceTension

Grass Stimulator & Force Transducer Muscle Skeletal Muscle

Tension



• Matthew Silva – [email protected]

• Steve Thomopoulos Dwight Towler Assoc Directors• Steve Thomopoulos, Dwight Towler – Assoc. Directors• Staff

– Tarpit Patel – microCT (and mechanical testing)– Tarpit Patel – microCT (and mechanical testing)[email protected]

– Michael Brodt – mechanical [email protected]



• Fee structureFee structure– NIH requires cost sharing between Core and investigators

Description Charge Billed ByDescription Charge Billed ByRadiograph/Faxitron $4/exposure Ortho Surg

DXA/Piximus $5.50/scan Medicine

pQCT scanning $20/hr Ortho Surg

microCT scanning $25/hour – uCT40 (3rd floor)$20/hr – VivaCT (7th floor)

MedicineOrtho Surg$20/hr VivaCT (7 floor) Ortho Surg

CT tech. time $15/hr Ortho Surg

Mechanical testing $375/full-day$200/half-day

Ortho Surg$200/half-day


RadiographyRadiography

• Faxitron® Specimen Radiography System, Model MX-20Faxitron Specimen Radiography System, Model MX 20 – Yalem 810

• Kodak Xomat film, standard developer, pMouse Femur Fracture Healing

2 wks 5 wksS. Putnam, A. WongM.J. Silva, November 2009

DXADXA• Dual-energy x-ray absorptiometry (DXA)gy y p y ( )• GE/Lunar Piximus – Yalem 6

• Outcomes: BMC (g), BMD (g/cm2), %fatCaveat: BMD = “areal BMD”


DXA detects changes in whole body BMC with timeDXA detects changes in whole-body BMC with time

8 010

PTH Dose (ug/kg)

*line)

4

6 40 *

MC om

bas

e

0

2rBM

chan

ge fr

o

-2

0

0 4 8Ti ( k )

(% c

Time (weeks)

M. LynchM.J. Silva, November 2009

pQCT – Yalem 810pQA

Canine distal phalanx

Rat Ulna

1 mm1 mm

P i h l Q i i C d T h• Peripheral Quantitative Computed Tomography• Resolution

70 100 μm in plane resolution; 500 μm slice thickness– 70-100 μm in-plane resolution; 500 μm slice thickness • Outcomes

– total (or trabecular) BMC (g), BMD (g/cm3), bone area (cm2)( ) (g), (g ), ( )– BMD = “volumetric BMD”


pQCTpCanine distal phalanx

B • Pros – volumetric BMD– site-specific

can segment cortical– can segment cortical-trabecular compartments (sometimes)

d t bilitSilva et al. 2006 – good repeatability– moderate expense

• ConsRat Ulna

Cons– medium resolution (~100

um) not good for mouse bone– not good for mouse bone morphology

– not good for trabecular i t t

Silva et al. 2009

microstructure


microCT post mortem scanningmicroCT – post mortem scanningMouse Femora

SENCARScanco μCT 40

C57 DBA SENCAR

Mouse Vertebra (L4)

• Yalem 3rd floorResolution• Resolution– 6-72 μm isotropic voxel– 16 or 20 μm recommendedμ

• Outcomes – morphology & density– Cortical: tissue area, bone area, TMD– Trabecular: BV/TV, Tb.N, Tb.Th, BMD

etc… Ramanadham et al. 2008M.J. Silva, November 2009

microCT: analysist thresholdcontour threshold

Binary• white=bone• black=not bone

tissue volume (TV) bone volume (BV)

• Morphology– BV/TV = bone volume fraction

tissue volume (TV) ( )

– Tb.N, etc. –• Density

“mean1” = “BMD” = BMC/TV– mean1 = BMD = BMC/TV• report for trabecular bone• should be ~ BV/TV

“mean2” = “TMD” = BMC/BV– mean2 = TMD = BMC/BV• report for cortical bone M.J. Silva, November 2009

microCT post mortem scanningmicroCT – post mortem scanning

C t l Di b tiParameter

Control(n = 9)

Diabetic(n = 8)

Tb.BV/TV (mm3/mm3)

0.37± 0 04

0.12 *± 0 04(mm3/mm3) ± 0.04 ± 0.04

Tb.Th(mm)

0.11± 0.01

0.07 *± 0.01

Tb N 4 4 2 6 *rabe

cula

r

Tb.N(1/mm)

4.4± 0.2

2.6 *± 0.5

Bone Area (mm2)

5.85± 0 30

4.46 *± 0 27

Tr

(mm2) ± 0.30 ± 0.27Moment of

Inertia (mm4)4.03

± 0.893.03 *± 0.54

Cortical TMD 1092 1111C

ortic

alCortical TMD

(mg/cm3)1092± 33

1111± 51

Silva et al. 2009


microCT post mortem scanningmicroCT – post mortem scanning

Supraspinatus Muscle Volume

50Botox

Vascularity – CT angiography

30354045

(mm

3 )

BotoxSalineNormal

BotoxSalineNormal BotoxSalineNormal

*

*10152025

Vol

ume

(

#

St F t*05

0 10 20 30 40 50 60

Timepoint

## #

J. McKenzie

Control Stress Fracture

Thomopoulos et al. 2007


microCTmicroCT• ProsPros

– high resolution (~6-70 um voxels)– 3D morphology– quantitative (mineral HA phantom)

• Cons– expensive/laborious– results depend on image settings (e.g., energy level) and

analysis settings (e g threshold filtering)analysis settings (e.g., threshold, filtering)• beware “black box” and “mfg. settings”


microCT auto contouringmicroCT – auto contouring• Cortical • Trabecular

manualmanualauto


microCT auto contouringmicroCT – auto contouring• Cortical • Trabecular

0.35y = 0.00 + 0.95x R2= 0.74 0.3

y = 0.03 + 1.08x R2= 0.87

0 25

0.3

0 2

0.2

0.25 0.2

0.150.15 0.2 0.25 0.3 0.35

3

0.1

0.1 0.2 0.3Auto BV (mm3) Auto BV/TV (%)


In vivo microCTIn vivo microCT

• Steinberg 7102Scanco VivaCT 40

• Steinberg 7102• Isofluorane anesthesia• ResolutionResolution

– 10-80 μm isotropic voxel resolution– 20 μm recommended

• Outcomes: morphology & density– Same as post mortem microCT


Tibial Regions of InterestTibial Regions of Interest

7-mo22-mo 7 mo22 mo


In vivo microCTIn vivo microCT%Change in Cortical BV

• Pros– serial scanning2 0

3 02 m o *%Change in Cortical BV

serial scanning– statistical power of repeated

(paired) measures0

1 0

**

g

2 0

-1 0C o ntro lL oa de d

g

-2 00 3 6

W e ek s


In vivo microCT: Radiation ExposureIn vivo microCT: Radiation Exposure • Data from Scanco

• Only scanned volume is exposed; t f i l i hi ld d

CTDI : CT dose index; 1 G 1 R drest of animal is shielded 1 cGy = 1 Rad


In vivo microCT: Radiation ExposureIn vivo microCT: Radiation Exposure • Weekly CT scans at 100 cGy affected bone morphology inWeekly CT scans at 100 cGy affected bone morphology in

growing mice (Klinck et al., 2006)• Whole-body dose at 50 cGy caused 25% reduction in CFU-GM

(G d t l 2006)(Grande et al. 2006)• Weekly CT scans at 50 cGy in adult rats did not affect bone

morphology or the number of viable marrow cells after 8 weeks p gy(Brouwers et al., 2006)

• Weekly scans did not affect bone morphology in growing mice (Buie et al 2008)(Buie et al, 2008)

• 1 or 2 scans of proximal tibia at our normal settings did not cause a reduction in CFU-F (D. Wood, unpublished)


In vivo microCT: RepeatabilityIn vivo microCT: Repeatability• Intra-rater agreementg

– four different raters analyzing same scans twice– ICC = 0.97-0.98 for trabecular BV/TVICC 0.97 0.98 for trabecular BV/TV

• Inter-rater agreementcomparing four raters analyzing the same scans– comparing four raters analyzing the same scans

– ICC = 0.95-0.97 for trabecular BV/TVI t t t• Intra-operator agreement– two people scanned the same set of mice twice– ICC = 0.99 for trabecular BV/TV

• ICCs were lower for other outcomes (eg, Tb.N)


Mechanical AssessmentMechanical AssessmentHow strong How big, dense

Whole-bone mechanicalt t (b di t i ) Mi CT

gis bone?

gis bone?

test (bending, torsion) MicroCT

Structural mechanicalStructural mechanicalproperties: Stiffness,

Failure Load, etc.Morphology: Area,

Mom. Inertia, BV/TV

EngineeringMechanicsIs strength normal

relative to size?

Material (tissue) mechanicalproperties: Modulus, p p ,

Ult. Stress, etc.M.J. Silva, November 2009

Long Bone BendingLong Bone Bending

4040

Yi ld M t

Ultimate Moment

20

30

20

30 FemurRadius

t (N

mm

) Yield Moment

Post-Yield Displacement

Fracture

Rigidity

10

20

10

20

Mom

en

Energy-to-Fracture

Post Yield Displacement

00 0.02 0.04 0.06 0.08

Normalized Displacement ( / 2)

00 0.05 0.1 0.15

Normalized Displacement( / 2) (mm/mm2) (mm/mm2)


Aged bones are stiffer, stronger but more brittle than young bones


Aged bones are bigger, stronger but more brittle than young bones


Mechanical Testing Vertebral CompressionMechanical Testing – Vertebral Compression

40

Yi ld F

Ultimate Force

20

30

e (N

)

Yield Force

Stiffness

10

20

Forc

e

Energy-to-Ultimate

00 0.05 0.1 0.15 0.2

Displacement (mm)

Ultimate

Tommasini et al. 2005


Mechanical Testing Demineralized Bone TensionMechanical Testing – Demineralized Bone Tension

Silva et al. 2006


Mechanical Testing – Canine Flexor (FDP) Tendon & Tendon-Bone

50 Ulti t F50 Ulti t F

(N)

30

40

50 Ultimate Force

30

40

50 Ultimate Force

Rigidity

Forc

e (

10

20

30

10

20

30

0

10

0 5 10 15 20

0

10

0 5 10 15 20Repair Site Strain (%)Repair Site Strain (%)


Mechanical Testing Muscle ForceMechanical Testing – Muscle Force

Force-frequency plot

10

12Botox

6

8

10

e (m

N) Saline

2

4

6

Forc

e

0

2

0 30 60 90 120 150F (H )Frequency (Hz)

Das, Thomopoulos et al.



RadiographyFaxitron


Imaging(post mortem

& in vivo)Densitometry

DXA

pQCT

3D MorphologymicroCT

“Standard” “Specialized”

Bone

Long-BoneBending

Vertebral

Standard SpecializedDemineralizedBone Tension

Trabecular

MechanicalTesting

Instron MaterialsTesting Systems

Tendon

Compression

Insertion SiteTension

Indentation

MidsubstanceTension

Grass Stimulator & Force Transducer Muscle Skeletal Muscle

Tension


Study DesignStudy Design• Sample Size

– Average coefficients of variation (CV = 100x[mean/SD])• 8% - femoral diaphyseal cortical bone area (microCT)• 23% - tibial metaphyseal trabecular bone volume (BV/TV) (microCT)• 12% - femoral diaphyseal ultimate moment (“whole-bone strength”, 3-pt

bending)– sample size (n) for power = 0.8, p = 0.05 (for unpaired t-test)

Effect Size (% diff between means)

Cortical Bone Area

Trabecular Bone Volume

Ultimate Moment

10 11 84 24 20 4 22 730 3 10 4 40 2 6 4

– n = 10 will allow for detection of differences of 11% for bone area, 30% for BV/TV, 16% for ultimate moment


AcknowledgementsAcknowledgements• Michael BrodtMichael Brodt• Akhilesh Kotiya

M k Willi h

• NIH/NIAMS P30AR057235

• Mark Willingham• Abby Stephens• Michelle Lynch• Dan WoodDan Wood• Jenny McKenzie

T it P t l• Tarpit Patel• Steve Thomopoulos• Dwight Towler M.J. Silva, November 2009

Documents

Skeletal Structure and Strength CoreSkeletal Structure and ... · Skeletal Structure and Strength CoreSkeletal Structure and Strength Core musculkltl tldloskeletalcore.wustl.edu Matthew