VIBRATIONAL SPECTROSCOPICMETHODS: FTIRI & RAMAN.
E. P. Paschalis
HOW DOES VIBRATIONAL SPECTROSCOPY RECOGNIZE BONE?
FRACTURES vs COMMON WISDOM
SHOWCASE THE INFORMATION THAT VIBRATIONAL SPECTROSCOPY CAN PROVIDE IN RELATION TO BONE
HOW DOES THIS INFORMATION AGREE/DISAGREE WITH OSTEOPOROSIS MANTRA OF BMD AND TURNOVER
HOW CAN IT HELP US IMPROVE EXISTING DEFINITIONS
WHAT IS ITS PLACE IN THE BIGGER PICTURE?
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Mineral (~70%) Organic Matrix (~20%)
HOW DOES VIBRATIONAL SPECTROSCOPY RECOGNIZE BONE?
Bone = Collagen/Mineral Nano-Composite
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FRACTURES vs COMMON WISDOMContribution of Post-Treatment BMD Increases
to Vertebral Fracture Risk Reduction over 3 Years
Cummings et al, Am. J. Med. 2002Sarkar et al, JBMR 2002
Watts et al, J Clin Densitometry 2004
Author Treatment AnalysisMethod*
% Fx Reduction Explained by BMD
Cummings Alendronate(FIT)
IPD 16%
Sarkar Raloxifene(MORE)
IPD 4%
Watts Risedronate(VERT and HIP)
IPD 18%
* Analysis of individual patient data (IPD)LBIO
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Cortical and cancellous bone were independently evaluated in iliac crest biopsies from 54 women (32 with fractures, 22 without) who had significantly different spine but not hip BMDs.
JBMR in press
FRACTURES vs COMMON WISDOMOSTEOPOROSIS: WE ARE SHOOTING BUT ARE WE AIMING?
The ONLY “universal” correlation between fracture and bone properties was collagen cross-link ratio
Structural Properties:• Geometry
•Size•Shape
• Microarchitecture• Trabecular• Cortical / porosity
Material Properties:• Mineral
• Mineral : Matrix• Crystallite maturity/size
• Collagen• Type• Cross-links
• Microdamage
Bone Turnover
Bone strength & quality
P. Chem
LathyrogensFBPsSr
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FRACTURES vs COMMON WISDOM
Features of Bone TissueThat Reduce Strength and Fracture Toughness
Feature ModulusUltimate strain
Ultimatestress Toughness Cause/Example
Poorly mineralized bone
Hypermineralized bone
Increased crystallinity:
morphology of apatite crystal
Denaturation of collagen molecule
Debonding of mineral collagen
Osteomalacia
Reduced turnoverIncreased mean tissue age
Reduced turnoverIncreased mean tissue ageFluoride accumulation
Unclear
Fluoride accumulation
⇓ ⇑ ⇓ ⇓
⇑ ⇑ ⇓⇓
⇑ ⇓ ⇓
⇓ ⇓ ⇓ ⇓
⇑⇓ ⇓ ⇓
?
Burr DB and Turner CH Biomechanical Measurements in Age-Related Bone Loss. In:The Aging Skeleton. CJ Rosen, J Glowacki and JP Bilezikian (eds). San Diego:Academic Press 1999; Chapter 26, pp. 301-311
TISSUE AGE
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Mineral Crystallites
Na+1
Mg+2
Sr+2
CO3-3
HPO4-2
Cl-1
F-1
CO3-3
CRYSTAL LATTICE VACANCIES
Biological ApatitesBiological ApatitesDerived from Greek word meaning "to deceive"Derived from Greek word meaning "to deceive"
Crystallite chemical composition is changingCrystallite chemical composition is changing
MaturityMaturityphase purity, stoichiometry, solubil ityphase purity, stoichiometry, solubil ity
Concomitantly/as a result, crystallite sizechanges
Concomitantly/as a result, crystallite sizechanges
CrystallinityCrystallinity
white -- Hydrogen , Red --Oxygen, purple - Phosphorous ,
Blue - calciumhttp://www.ecs.syr.edu/faculty/schwarz/project1.htm
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MineralCrystallinity matters
TheoreticalConsiderations
Animalmodel data Human Clinical data
Fluoride treatment resulted inincreased BMD & bone fragility.Upon analysis, fluoride-treated
bone had higher crystallinitycompared to normal
Plenty of animal modelswith altered mineralcrystallinity & bone
mechanical properties
Study of biological composites atthe nano level reveals that
optimal mechanical propertiesare associated with a definiteplateau in mineral crystallinity.Crystllite sizes larger that theplateau value result in inferior
mechanical performance
Gao H, et al 2003 Proc NatlAcad Sci U S A 100(10): 5597-600, 2003.
Compromised Mechanical PropertiesCompromised Mechanical Properties
Ovariectomized animalsOvariectomized animals largerlarger
Osteogenesis ImperfectaOsteogenesis Imperfecta smallersmaller
Hyp miceHyp mice smallersmaller
Osteopontin KO miceOsteopontin KO mice largerlarger
Osteocalcin KO miceOsteocalcin KO mice smallersmaller
Animals & HumansAnimals & Humans
X-ray powder diffractionX-ray powder diffraction
Fourier transform infraredFourier transform infrared
Small angle x-ray scatteringSmall angle x-ray scattering
Fourier transform infrared imagingFourier transform infrared imaging
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COLLAGEN
• SCAFFOLD
• MECHANICAL PROPERTIES
• INITIATION OF MINERALIZATION?
• Cross-links are tissue- rather than collagen type-specific
Covalent Intermolecular Cross-linking
How Do Collagen Molecules Form a Functional/Stable Fibril?
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Importance of collagen cross-links
TheoreticalConsiderations
Animal modeldata
Human Clinicaldata
Osteogenesis Imperfecta
???
Animal models withaltered collagen cross-
links & bone mechanicalproperties
The matrix is proposed to play animportant role in alleviating impact damage
to mineral crystallites, and to matrix/mineral interfaces.
Osteogenesis ImperfectaOsteogenesis Imperfecta mousemouse
LathyrismLathyrism ratrat
Vitamin B6 deficiencyVitamin B6 deficiency chickenchicken
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Mineral & CollagenMineral & Collagen Tissue VariabilityTissue Variability
Tissue "Age"Tissue "Age"
Bone mineral crystallites are capable of sustained grow thw hen bathed in aqueous media, even in the absence of
cellular activity.
Bone mineral crystallites are capable of sustained grow thw hen bathed in aqueous media, even in the absence of
cellular activity.
Rey C., Glimcher M. et al; Cells & Mat. 5, 345-356, 1995Rey C., Glimcher M. et al; Cells & Mat. 5, 345-356, 1995
Post-translational modif ications may take place in biologicalf luids, even in the absence of cellular and/or enzymatic
activity.
Post-translational modif ications may take place in biologicalf luids, even in the absence of cellular and/or enzymatic
activity.
Yamauchi M 1996 Collagen: The major matrix molecule in mineralizedtissues. In: Anderson JJB, Garner SC (eds.) Calcium and Phosphorus inHealth and Disease. CRC Press, New York, pp 127-141.
Yamauchi M 1996 Collagen: The major matrix molecule in mineralizedtissues. In: Anderson JJB, Garner SC (eds.) Calcium and Phosphorus inHealth and Disease. CRC Press, New York, pp 127-141.
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BONE
STRENGTH
CRYSTAL SIZE
COLLAGEN x-links
NORMAL RANGE
•Fluoride
•Ovx
•Osteopontin KO
•OI
•Hyp
•Osteocalcin KO •OI
•Lathyrism
•Vitamin B6-deficiency
INFRARED SPECTROSCOPYINFRARED SPECTROSCOPYBASISBASIS
TWISTING - BENDING - ROTATION - VIBRATIONTWISTING - BENDING - ROTATION - VIBRATION
ATOMS IN A MOLECULEATOMS IN A MOLECULE
ABSORPTION AT PARTICULAR WAVELENGTHSABSORPTION AT PARTICULAR WAVELENGTHS
CHARACTERISTIC OF FUNCTIONAL GROUPSCHARACTERISTIC OF FUNCTIONAL GROUPS
OVERALL CONFIGURATION OF ATOMSOVERALL CONFIGURATION OF ATOMS
SUBTLE INTERACTIONS WITH SURROUNDINGATOMS OF MOLECULE
SUBTLE INTERACTIONS WITH SURROUNDINGATOMS OF MOLECULE
STAMP OF INDIVIDUALITY ON THE SPECTRUM OF EACHCOMPOUNDSTAMP OF INDIVIDUALITY ON THE SPECTRUM OF EACHCOMPOUND
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800 1000 1200 1400 1600 1800 800 1000 1200 1400 1600 1800 Wavenumber (cm-1)
TYPICAL FTIRM SPECTRUM OF BONE
CO
3
AM
IDE
III
AM
IDE
I
AM
IDE
II
Abs
orba
nce
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Fourier Transform Infrared SpectroscopyFourier Transform Infrared SpectroscopyMajor Bone Relevant OutcomesMajor Bone Relevant Outcomes
MINERAL DENSITY (mineral : matrix)MINERAL DENSITY (mineral : matrix)May or may not agree with BMD or BMDDas it expresses mineral with respect tov olume AND matrix
May or may not agree with BMD or BMDDas it expresses mineral with respect tov olume AND matrix
CARBONATE CONTENT & TYPECARBONATE CONTENT & TYPE Important f or cry stal lattice considerationsand solubilityImportant f or cry stal lattice considerationsand solubility
MINERAL MATURITYMINERAL MATURITYDirect measure is chemistry . Cry stallinitymay be extrapolated, but caution should beexercised when interpreting results
Direct measure is chemistry . Cry stallinitymay be extrapolated, but caution should beexercised when interpreting results
COLLAGEN CROSS-LINKSCOLLAGEN CROSS-LINKSUNIQUE capability to describe them as af unction of tissue age AND surf acemetabolic activ ity
UNIQUE capability to describe them as af unction of tissue age AND surf acemetabolic activ ity
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0
.2
.4
.6
.8
1
950 1000 1050 1100 1150
-.004
-.002
0
.002
.004
.006
.008
950 1000 1050 1100 1150
.2
.4
.6
.8
1
1.2
1.4
950 1000 1050 1100 1150
RELEVANCE OF 1020 / 1030 RATIO
CTI, 59:480-487, 1996 LBIO
Statistically significant correlation of the FT-IRM parameterswith the SAXSparameter of crystal thickness.
Open and full symbols represent datafrom cortical and cancellous bone, respectively.
FTIRM parameter validation against SAXSChemistry vs Structure
Bone 25(3) 287-293, 1999
Human, L4 vertebrae
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MINERAL MATURITY / CRYSTALLINITY CHANGES IN OSTEOPOROSIS
% CHANGES IN MINERAL QUALITY WITHIN THE FIRST 60 um AT BONE FORMING SURFACES
Osteoporos Int (2005) 16: 2031–20LBIO
How Do Collagen Molecules Form a Functional / Stable Fibril?
L.H.
Lys
Hyl
Lysald
Hyl
L.O.
HisACP Lysald
Hylald
deH-HHMD
Hyl
Lys
Hyl
Intramolecular
Hylald
PrlLysald
deH-HLNL
deH-DHLNL
deH-HLNL
HisHHL
Pyr
Hylald
d-PrlLysald
d-Pyr
I
II
III
I: soft tissuesII: skin and corneaIII: skeletal tissues
1. dehydro-dihydroxylysinonorleucine (deH-DHLNL), 2. dehydro-hydroxylysinonorleucine (deH-HLNL), 3. dehydro-histidinohydroxymerodesmosine (deH-HHMD),
4. pyridinoline (Pyr), 5. deoxypyridinoline (lysyl analog of Pyr, d-Pyr), 6. histidinohydroxylysinonorleucine (HHL). 7. pyrrole
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Osteoblast OutsideOsteoblast
Genetic
LathyrogensCu
0
.05
.1
.15
.2
.25
1750 1700 1650 1600 1550 1500 1450
0
.05
.1
.15
.2
.25
1750 1700 1650 1600 1550 1500 1450
0
.05
.1
.15
1850 1800 1750 1700 1650 1600 1550 1500 1450 1400
0
.05
.1
.15
1850 1800 1750 1700 1650 1600 1550 1500 1450 1400
PYRIDINOLINE
0
.2
.4
.6
.8
1
1.2
1800 1750 1700 1650 1600 1550 1500 1450
0
.2
.4
.6
.8
1
1.2
1800 1750 1700 1650 1600 1550 1500 1450
DHLNL
COLLAGEN
J of Bone and Mineral Research, 16(10), 1821-8, 2001. LBIO
0 10 20 30 40 50-2
0
2
4
6
8
10
12
14
16
18
20
22
Time (hrs)
Data: Data1_BModel: LogisticEquation: y = A2 + (A1-A2)/(1 + (x/x0) p)Weighting: y No weighting Chi 2/DoF = 1.19791R^2 = 0.9852 A1 20.01549 ±1.03992A2 -1702514.9366 ±104119243870.75128x0 617607941.88614 ±5.4279E13p 0.69592 ±0.32166
Aver
age
% o
f 166
0
INFRARED IMAGING
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0
1.300
2.600
3.900
5.200
6.500
0.3200
0.5760
0.8320
1.088
1.344
1.600
0
0.4400
0.8800
1.320
1.760
2.200
Mineral / Matrix Mineral Crystallinity Pyr / DHLNL
Min
eral
Col
lage
n
PMM
A
FTIR IMAGING DATA
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In case of collagen analysis, histologically stained sections may be employed
0
0.4000
0.8000
1.200
1.600
2.000
von
Kos
sa
coun
ters
tain
ed w
/ne
utra
l red
Tric
hrom
e
Actual Picture Pyr / DHLNLJ of Bone and Mineral Research, 16(10), 1821-8, 2001 LBIO
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Match spatial distribution of collagen cross-links with mineralization
Collagen cross-links & bone turnoverMild Primary Hyperparathyroidism
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p< 0.01
p< 0.001
NL pHPT PostPTX PostMF0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
HyperPT
J Clin Endocrinol Metab. 2008 Sep;93(9):3484-9.
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0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.40
50
100
150
200
250
300
350
400
450
500
550
PIXE
L PO
PULA
TION
POP
ULAT
ION
Pyr / DHLNL
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.40
20
40
60
80
100
120
140
160
180
200
PIXE
L PO
PULA
TION
DIS
TRIB
UTIO
N
Pyr / DHLNL
0.3000
0.7400
1.180
1.620
2.060
2.500
0.3000
0.7400
1.180
1.620
2.060
2.500
NO
RM
AL
OP
HOW IS COLLAGEN QUALITY IN OSTEOPOROSIS?
HOW IS COLLAGEN QUALITY IN OSTEOPOROSIS?
0 1 0 2 0 3 0 4 0 5 0
0
2
4
6
8
1 0
Py
r /
de
H-D
HL
NL
A n a t o m ic a l L o c a t io n ( u m )
N o r m a l H T O P L T O P
Normal N = 10Low-turnover (LTOP) = 10High-turnover (HTOP) = 10
JBMR 18(11): 1942-1946 2003JBMR (2004)
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HOW IS COLLAGEN QUALITY IN FRAGILE BONE?
0 1 0 2 0 3 0 4 0 5 0- 0 .5
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
3 .0
3 .5
4 .0
4 .5
5 .0
5 .5
6 .0
Py
r /
de
H-D
HL
NL
A n a t o m ic a l L o c a t io n ( u m )
n f c o l l x l t o p f c o lx S F f c o l l x
JBMR (2004) LBIO
What is the clinical evidence / suggestions?
J Bone Miner Res. 2007 May;22(5):747-56.
Bone. 2007 Mar;40(3):730-6.
J Bone Miner Res. 2007 Jan;22(1):127-34
CalcifTissue Int. 2006 Sep;79(3):160-8.
Am J Med. 2005 Nov;118(11):1250-5
JAMA. 2005 Mar 2;293(9):1121-2
N Engl J Med. 2004 May 13;350(20):2042-9.
N Engl J Med. 2004 May 13;350(20):2033-41
HOMOCYSTEINE & FRACTURE
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Contour Graph 1
X Data
10 20 30 40 50 60
Y D
ata
10
20
30
40
50
60
0 1 2 3 4 5
Hcys = 43.99 uM/L
Contour Graph 4
X Data
10 20 30 40 50 60
Y D
ata
10
20
30
40
50
60
0 1 2 3 4
Hcys = 6.0 uM/L
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PLASMA HOMOCYSTEINE LEVELS & COLLAGEN CROSS-LINKS
0.3000
0.7400
1.180
1.620
2.060
2.500
0.3000
0.7400
1.180
1.620
2.060
2.500
NO
RM
ALO
P
???
Bone. 2009 May;44(5):959-64.
PLASMA HOMOCYSTEINE LEVELS & COLLAGEN CROSS-LINKS
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Patient data
70 80 90 100-10
0
10
20
30
40
50
Spearman r95% confidence interval
P value (two-tailed)P value summary
0.3715-0.1139 to 0.71380.1173ns
Patient Age (yrs)
Hcy
s le
vel (µM
)
collx form
High Hcy
s
Low Hcy
s0
2
4
6
8
10
Unpaired t testP value
P value summaryAre means signif. different? (P < 0.05)
One- or two-tailed P value?t, df
0.0007***YesTwo-tailedt=4.129 df=17
pyr/
diva
lent
collx res
High Hcy
s
Low Hcy
s0
5
10
15
Unpaired t testP value
P value summaryAre means signif. different? (P < 0.05)
One- or two-tailed P value?t, df
0.1197nsNoTwo-tailedt=1.638 df=17
pyr
/ div
alen
t
Bone. 2009 May;44(5):959-64.
0 1 0 2 0 3 0 4 0 5 0
0
2
4
6
8
1 0
Py
r /
de
H-D
HL
NL
A n a t o m ic a l L o c a t io n ( u m )
N o r m a l H T O P L T O P
???
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Bone. 2009 May;44(5):959-64.
0 10 20 30 40 500
20
40
60
80
hcys levels (µM)
% A
rea
of 1
660
band
10 20 30 40 50-2
0
2
4
6
8
10
hcys levels (µM)
% A
rea
of 1
690
band
PLASMA HOMOCYSTEINE LEVELS & COLLAGEN CROSS-LINKS
collx form
High Hcy
s
Low Hcy
s0
2
4
6
8
10
Unpaired t testP value
P value summaryAre means signif. different? (P < 0.05)
One- or two-tailed P value?t, df
0.0007***YesTwo-tailedt=4.129 df=17
pyr/
diva
lent
FTIRI RAMANSpatial resolution 7 um 0.6 umTissue preparation Dehydration &
embeddingNone
Background literature Solid ChaoticAcquisition times 5 minutes Seconds – 2 minutesSignal to noise Superior InferiorMineral information A lot of information SomeCarbonate information A lot of calculations
involvedStraightforward
Collagen A lot of information SomeOrientation Natively none A lot of information
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Vibrational Spectroscopy vs Vibrational Spectroscopy
-0.25 0.00 0.25 0.50 0.75 1.00 1.253
4
5
6
7
8
sin2 ϕ
ratio
v1P
O4/
am
ide
I
Amer, MS (2009) Raman Spectroscopy for Soft Matter Applications, Chapter 9, Raman Applications in Bone Imaging (S. Gamsjäger, M. Kazanci, E.P. Paschalis, and P. Fratzl) Wiley
RAMAN & ORIENTATION DEPENDENCE : turkey leg tendon
LBIOP045 CORTICAL BONE ORIENTATION AND COMPOSITION IN A MOUSE MODEL AS A FUNCTION OF TISSUE AGE VS ANIMAL AGE S. Gamsjaeger, P. Roschger, K. Klaushofer, E. P. Paschalis, P. Fratzl
amide I
amide III
v1PO4
v2PO4CO3 B-type
50 x objective
RAMAN & TISSUE AGE
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amide III
amide I
phenylalanine
DNA
100x water immersion objective
RAMAN & CELL CULTURES
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SUMMARY
FTIRI & Raman vibrational spectroscopic techniques are capable of providing information on material properties
Information on all tissue components at the same time
No probe molecule required. Non-destructive
Employed parameters have been / are validated against “golden standard” techniques such as XRD, and Biochemical analyses
To date, mineral maturity & the ratio of two of the major mineralizing collagen cross-links, may be deduced in thin tissue sections with optimal spatial resolution of ~ 0.6-6.3
um. Moreover, structure vs orientation effects may be discernedIts major strength is that it may provide answer to the question: what is the difference
between normal and diseased/treated tissue at EQUIVALENT tissue age anatomical locations?
As a result, effects due to alterations in turnover and/or other factors may be readily discerned
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SUMMARYClinical Value
Information provided in spatially resolved manner thus can compare material properties as a function of tissue age and bone surface activity
Not likely to be used in everyday clinical practice as biopsy is required
Excellent tool in cases where BMD does not fully account for fracture risk (SF)
Excellent tool in animal model experiments
Excellent tool for determining material properties (clinical trials)
Excellent tool for establishing effect of therapies on bone material properties.