Compositional Changes of Bone Mineral and Matrix Have Correlations with Mechanical Properties that Depend on Bone Age ND 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
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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