VIBRATIONAL SPECTROSCOPIC METHODS: FTIRI & RAMAN

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


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.


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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




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


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


collx form

High Hcy

s

Low Hcy

s0

2

4

6

8

10




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.

Documents

VIBRATIONAL SPECTROSCOPIC METHODS: FTIRI & RAMAN