HighHigh--resolution xresolution x--ray diffraction microscopy ray diffraction microscopy

applied to engineered bone studyapplied to engineered bone study

A. CedolaA. Cedola, S. S. LagomarsinoLagomarsino

IFNIFN--CNR(CNR( RomeRome--ItalyItaly),),

M. Mastrogiacomo, R. Cancedda

CentroCentro BiotecnologieBiotecnologie AvanzateAvanzate-- UnivUniv. Genova. Genova

(Genova(Genova--ItalyItaly))

M. BurghammerESRF (ESRF (GrenobleGrenoble-- FranceFrance), ),

C. Giannini

A. Guagliardi ,M. Ladisa

ICIC-- CNR (BariCNR (Bari ItalyItaly))

V. Komlev, F. RustichelliUniv. AnconaUniv. Ancona (Ancona(Ancona--ItalyItaly))

OUTLINEOUTLINE

Qualitative analysis

Quantitative analysis

Set-up

Scientific problem

Mineralization distribution

Bone-Scaffold interaction

Engineered bone sample

Opened scientific problems

SAXS and WAXS combination

ICXOM ICXOM -- XVIII XVIII Frascati 25Frascati 25--30 30 SeptSept 20052005

Scientific Problem: Sample Scientific Problem: Sample

Bone marrow harvest

Amplification of osteoprogenitor cells

In vivo subcutaneously implantRemoved sample

Loading on bioceramic sub.

ICXOM ICXOM -- XVIII XVIII Frascati 25Frascati 25--30 30 SeptSept 20052005

grain shape and orientation of new bone

bone growth mechanism

AIM of X-ray μ-diffraction measurements is the determination of:

Opened Scientific Problems Opened Scientific Problems

bone-scaffold interaction

ICXOM ICXOM -- XVIII XVIII Frascati 25Frascati 25--30 30 SeptSept 20052005

XX--ray ray diffraction microscopydiffraction microscopy

standard diffraction + standard diffraction + subsub--micron micron beambeam

ICXOM ICXOM -- XVIII XVIII Frascati 25Frascati 25--30 30 SeptSept 20052005

MicroMicrometermeter--resolution mapping (or imaging) resolution mapping (or imaging)

of atomic structural informationof atomic structural information

Scanning microScanning micro--diffraction setdiffraction set--upup

Beam size:Beam size:3 μ

0.3 μ

detector

mirror slits

microscope

WG or KBmicroscope

beamstop

piezoscanner

STySTx

STz

SRz

SRxSRy

SPx SPy SPz

SR

ICXOM ICXOM -- XVIII XVIII Frascati 25Frascati 25--30 30 SeptSept 20052005

PrePre--opticsoptics

WG WG 1 μ

1 μKB KB

ID13 at ESRFID13 at ESRFSimultaneous acquisition of WAXS and SAXSSimultaneous acquisition of WAXS and SAXS

Scaf NB

ST

air

100 μm

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HA (HA (scaffoldscaffold)) new bone (HA)new bone (HA) SoftSoft tissuetissue

Spotty rings

Large grains (50 nm)

HA reflections:Continuous rings

Small grains ( 3-5 nm)

Diffuse scattering

Amorphous organictissue

ICXOM ICXOM -- XVIII XVIII Frascati 25Frascati 25--30 30 SeptSept 20052005

Qualitative Qualitative analysisanalysis

[002]

0 50 100 150 200 250 300 350

1,2

1,4

1,6

1,8

2,0

2,2

2,4 f13WAXS

AzimuthalAzimuthal intensityintensity

New bone New bone –– Orientation at pore surfaceOrientation at pore surface

ST

NBHA

HA

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WAXS WAXS fromfrom [002][002]

New bone New bone –– Orientation at pore surfaceOrientation at pore surface

c Unit cellUnit cell

Pore surface

Digital zoom at small angles of diffraction pattern

to obtain SAXS information

in the same sample point

ICXOM ICXOM -- XVIII XVIII Frascati 25Frascati 25--30 30 SeptSept 20052005

New bone New bone –– Orientation at pore surfaceOrientation at pore surfaceGrainGrain Anisotropy of SAXSSAXS shows that the elongated shape of bone mineral particles

are non-random distributed around the pore surface (// to the c axis ).

ICXOM ICXOM -- XVIII XVIII Frascati 25Frascati 25--30 30 SeptSept 20052005

Collagene Collagene fibres fibres

New bone New bone –– Growth mechanismGrowth mechanism

From From WAXS and SAXSWAXS and SAXS combinationcombination ::

ICXOM ICXOM -- XVIII XVIII Frascati 25Frascati 25--30 30 SeptSept 20052005

The The collagen collagen fibers fibers (bone seed) are oriented (bone seed) are oriented

parallel to the pore surface parallel to the pore surface

The The bone mineral crystalsbone mineral crystals are elongated along the collagen are elongated along the collagen fibersfiberswith the cwith the c--axis parallel to the elongation directionaxis parallel to the elongation direction

AS FOR NATURAL BONEAS FOR NATURAL BONE

Quantitative analysisQuantitative analysis

Study of a new scaffold composed of Study of a new scaffold composed of

two different phases: HA and TCPtwo different phases: HA and TCP

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The final goal of the Tissue Engineering is the replacement of the scaffold with the engineered tissue

We look for the best re-absorbable scaffold

The study of the Interaction scaffold-bone is necessary

RADIOGRAPHYRADIOGRAPHY XX--RAY DIFFRACTIONRAY DIFFRACTION

XX--RAY DIFFRACTION RAY DIFFRACTION -- MICROSCOPYMICROSCOPY

SAXS intensity distribution:

High resolution spatial

distribution of bone

mineral crystals

scaffold

bone

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0 8 16 2405

101520253035404550556065707580

%

Implantation time [weeks]

HA TCP NB

8w8w--no no cellscells

After implantation the scaffold is strongly modified After implantation the scaffold is strongly modified and a large amount of new bone is formed.and a large amount of new bone is formed.

What happens? What happens?

Depletion of TCP scaffold phase

when bone increases

bone growth mechanism

Conclusions Conclusions

Good degree of organisation

Orientation of the collagen fibers and c-axisas for natural bone

grain shape and orientation of new bone

ICXOM ICXOM -- XVIII XVIII Frascati 25Frascati 25--30 30 SeptSept 20052005

Interaction bone-scaffoldIn (HA+TCP) scaffoldstrong depletion of the TCP promotes bone formation