Compositional Changes of Bone Mineral and Matrix Have Correlations with Mechanical Properties that Depend on Bone AgeND Sahar, M Reghavan, MD Morris, DH Kohn

Funding Sources:- DoD/US Army DAMD17-030100556- NIH R01-AR052010- U of M Regenerative Sciences Training Grant R90-DK071506

No conflicts of interest

Aging and Fracture RiskF

RA

CT

UR

E

RIS

K

PE

R

1000

PE

RS

ON

– Y

EA

RS

BONE MASS (g/cm)

AGE (years)

WHY?

Age is a better predictor of hip fracture than bone mass.

S.L. Hui, et al. 1988

Traditional Clinical View of Bone Health

RP Heaney, Bone 2003

BONESTRENGTH

MATERIALPROPERTIES

TISSUE COMPOSITION

GROWTH & AGING

ND Sahar, et al. ORS 2008

Previous Data: Material Properties Decreased with Age

4 MONTH 5 MONTH 19 MONTH0

50

100

150

200

250ULTIMATE STRENGTH (MPa)

4 MONTH 5 MONTH 19 MONTH0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0RESILIENCE (MPa)

* p < 0.05 compared to 4 & 5 month-old groups

* *

ND Sahar, et al. ORS 2008

Previous Data:Growth Slowed After 5 Months

ND Sahar, et al. ORS 2008

4 Months 5 Months 19 Months0

2

4

6

8

10

12

MINERAL / MATRIX RATIO

4 Months 5 Months 19 Months0.00

0.05

0.10

0.15

0.20

0.25

0.30

CARBONATE / PHOSPHATE RATIO

Previous Data: Aging Altered Tissue Composition

* p < 0.05 compared to 4 & 5 month-old groups

* *

ND Sahar, et al. ORS 2008

Experimental DesignHypothesis: Changes in bone material properties with aging are dependent on changes to bone composition.

Male C57Bl/6 mice• 4 Months (N = 8)

– Growing but reaching skeletal maturity *

• 5 Months (N = 12)– Skeletally mature *

• 19 Months (N = 13)– Aging bone tissue with compromised integrity *

MD Brodt, et al. 1999; VL Ferguson, et al. 2003; BP Halloran, et al. 2002; JM Somerville, et al. 2004

*

Experimental Design

1. Raman Microspectroscopy2. Nanoindentation

60 µm

Experimental Design

• Sample preparation:– Only exposed to Ca buffered saline– Kept hydrated until just before nanoindentation

• Matching depth of Raman & nanoindentation– 2000 nm

• Removal of orientation/polarization effects in Raman spectra

Raman Microspectroscopy

Raman Peak (cm-1) Assignment Phase of Bone

851 Hydroxyproline Matrix

873 Hydroxyproline Matrix

917 Proline Matrix

958 Phosphate Mineral

1070 Carbonate Mineral

1660 Amide-I Matrix

1690 Amide-I sub-band Matrix

A Carden, MD Morris 2000; EP Paschalis et al. 2001

Mineral/Matrix Ratio (Min/Mat)

Carbonate/Phosphate Ratio (Carb/Phos)

Collagen Cross-Linking Ratio (Cross-Link)

958 / (851+873+917)

1070 / 958

1660 / 1690

=

=

=

Modified Indentation Routine

Used to modelcreep behavior

Segment 1 Segment 2

Modeling Creep Behavior

AC Fisher-Cripps, Nanoindentation. 2004

Eparallel

hparallel

hseries

Modeling Creep Behavior

Time (sec)

Dis

plac

emen

t (n

m)

R2 > 0.999 for all tests

Raw Data

Model Fit

Elastic Recovery

WP

WE

Hardness Calculation

Bone

Aluminum

Hardness calculationswere corrected for elastic recovery.

Oyen ML, “Nanoindentation hardness of mineralized tissues.” J Biomech 2006

Modeling Correlations between Raman and Nanoindentation Metrics

Only predictors that contributed to each model with p < 0.05 were included

Nanoindentation Metric = β0 + β1 * Min/Mat + β2 * Carb/Phos + β3 * Cross-Link

4-Month Old Mice (Growing)

Partial Correlation Overall Model Fit

Carb/Phos Min/Mat Cross-Link p Adj. R2

Modulus -0.73 0.041 0.45

Hardness -0.94 0.85 0.004 0.85

Elasticity Index -0.95 0.87 0.003 0.86

Eparallel N.S.

hparallel N.S.

hseries -0.86 0.014 0.68

Multivariate regressions with significant covariates

5-Month Old Mice (Mature)

Partial Correlation Overall Model Fit

Carb/Phos Min/Mat Cross-Link p Adj. R2

Modulus 0.64 -0.59 0.012 0.50

Hardness -0.68 0.011 0.41

Elasticity Index -0.77 0.002 0.55

Eparallel 0.67 0.023 0.39

hparallel 0.70 0.017 0.42

hseries N.S.

Multivariate regressions with significant covariates

19-Month Old Mice (Aging)

Partial Correlation Overall Model Fit

Carb/Phos Min/Mat Cross-Link p Adj. R2

Modulus N.S.

Hardness -0.88 0.001 0.76

Elasticity Index -0.91 0.001 0.82

Eparallel N.S.

hparallel N.S.

hseries -0.53* 0.075 0.21

* indicates 0.05 < p < 0.1

Multivariate regressions with significant covariates

Mineralization and Hardness

5 6 7 8 9 10 11 12 130.0

0.5

1.0

1.5

2.0

2.5

4 Months Linear (4 Months)

MINERAL / MATRIX RATIO

HAR

DN

ESS

(GPa

) R2 = 0.00 R2 = 0.78R2 = 0.17

Mineralization and Hardness

5 6 7 8 9 10 11 12 130.0

0.5

1.0

1.5

2.0

2.5

<10 Linear (<10) >10 Linear (>10)

MINERAL / MATRIX RATIO

HAR

DN

ESS

(GPa

)R2 = 0.54R2 = 0.01 R2 = 0.54R2 = 0.01

Aging of Bone Tissue

TISSUE MATURITYINCREASING MINERALIZATION

MEC

HAN

ICAL

CO

MPE

TEN

CE

BONE REMODELING

Summary

• Growing bone: crystal maturity positively correlated with modulus and resistance to plastic deformation

• Mature bone: mineralization positively correlated with resistance to elastic and visco-elastic deformation

• Aging bone: mineralization negatively correlated with resistance to plastic deformation

• Mechanical consequences of compositional changes depend on bone’s age and initial compositional state