high-resolution x-ray microtomography for 3d imaging of

XCVI Congresso Nazionale della Società Italiana di Fisica,XCVI Congresso Nazionale della Società Italiana di Fisica,Bologna, 20 - 24 Settembre, 2010Bologna, 20 - 24 Settembre, 2010

High-resolution X-ray Microtomography High-resolution X-ray Microtomography for 3D Imaging of Cardiac Progenitor Cells Homing for 3D Imaging of Cardiac Progenitor Cells Homing

in Infarcted Rat Hearts. in Infarcted Rat Hearts.

Alessandra Giuliani, PhD

Section of Physical Sciences Department S.A.I.F.E.T.

Polytechnic University of Marche

[email protected]

2

Basic Studies on Stem Cells


Cellule pancreatiche

Cellule emopoietiche

Cellulemiocardiche

Cellule epatiche

Cellule nervose

Cellulestaminali

3



4



Le cellule staminali possono essere classificate anche in:

Totipotenti quando possono dare origine ad un individuo completo.

Pluripotenti quando sono in grado di dare origine a tutte le linee cellulari tra cui quelle germinali ma non ad un individuo.

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Multipotenti quando possono differenziarsi solo in alcuni tipi di cellule. Esempio ne sono le cellule staminali del midollo osseo che possono differenziarsi sia in eritrociti che in tutte le cellule bianche del sangue ed anche in osteociti.

Unipotenti possono differenziarsi in una sola linea cellulare


The recent introduction of stem cells in cardiology provides new tools in understanding the regenerative processes of the normal and pathologic heart and has opened the search of new therapeutic strategies.

Recent published reports have contributed to identify the possible approaches of cellular therapy to generate new myocardium involving transcoronary and intramyocardial injection of progenitor cells.

Cellular Therapy for the Myocardium Regeneration. Basic Principles.


Cellular Therapy for the Myocardium Regeneration. Basic Principles.

One of the limiting factors in the overall interpretation of clinical results obtained by cell therapy is represented by the lack of in vivo visualization of the injected cells and of their fate within the myocardium.

X-ray computed microtomography may offer the unique possibility to detect with high definition and resolution the 3D spatial distribution of rat Cardiac Progenitor Cells, labeled with iron oxide nanoparticles, inside the infarcted rat heart early after injection.

X-Ray Computed Microtomography. Basic Principles.

www.esrf.fwww.esrf.frr

ESRFESRF



Schematic set up of microCT system installed at ID19 in ESRFSchematic set up of microCT system installed at ID19 in ESRF




The total attenuation of a ray at position r, on the projection at an angle θ, is given by the line integral:

but:

so that eq.1 can be rewritten as:

(1)

(2)

where f(x,y) represents μ(x,y).

•Radon transform (or sinogram) of the 2D object



• The tomographic reconstruction is achieved by rotating the object in small angular steps over 180° and calculating tomographic slices using the Inverse Radon Transform.

• A stabilized and discretized version of the inverse Radon transform is used, known as the Filtered Back-Projection algorythm.



P.Cloetens: Tomography and holotomography. School on X-ray micro and nanoprobes.ESRF, Grenoble, France. June 13, 2007


Linked feature display - A linked cursor (or any given object such as points, lines, planes, etc.) can be used to indicate a location in a 3D volume visualization and its corresponding location in another (2D and/or 3D) image from a different source.

Integrated data display - Segmentation of objects in one data set allows the subsequent integration into another data set whereupon standard 3D rendering methods can be used to obtain an integrated 3D display.

Multimodal window display - Substitution of a section of a volume rendering of a source data set by the corresponding section of a volume rendering of another source data set is called multimodal window display.

X-Ray Computed Microtomography. Integrated 3D visualization.


Multimodal cutplane - The established use of cutplanes in volume visualization indicates that it is a powerful method for the analysis of image data.

Surface mapping, texturing, or painting - Information from one source can be mapped and (color) encoded onto the surface derived from another image data set. Typically these techniques are applied to target brain data, usually mapping, texturing, or painting functional information onto the surface of the brain.

Complementary visualization techniques - The aforementioned techniques for 3D integrated display can be augmented using standard presentation methods, such as movie sequences and stereo display, for an improved 3D perception.

X-Ray Computed Microtomography. Integrated 3D visualization.

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High-resolution X-ray Microtomography for Three-High-resolution X-ray Microtomography for Three-dimensional Visualization of dimensional Visualization of

Cardiac Progenitor Cells HomingCardiac Progenitor Cells Homing in Infarcted Rat Heartsin Infarcted Rat Hearts

A. Giuliani, C. Frati, A. Rossini, V. Komlev, C. Lagrasta, M. Savi, S. Cavalli, A. Giuliani, C. Frati, A. Rossini, V. Komlev, C. Lagrasta, M. Savi, S. Cavalli, M. C. Capogrossi, F. Quaini, A. Manescu, F. RustichelliM. C. Capogrossi, F. Quaini, A. Manescu, F. Rustichelli

Submitted to the Submitted to the Journal of Tissue Engineering and Regenerative Medicine Journal of Tissue Engineering and Regenerative Medicine

Stem Cell Research related to Infarcted Heart.

Coronary binding to induce the infarct

Atria

Left VentricleHeart Apex

Aorta

Infarcted rat heart was injected with 5X105 rat Cardiac Progenitor Cells (CPCs)

after loading with super paramagnetic iron oxide nanoparticles (Feridex®).

A. Giuliani, C. Frati, A. Rossini, V. Komlev, C. Lagrasta, M. Savi, S. Cavalli, A. Giuliani, C. Frati, A. Rossini, V. Komlev, C. Lagrasta, M. Savi, S. Cavalli, M. C. Capogrossi, F. Quaini, A. Manescu, F. Rustichelli. Submitted to the M. C. Capogrossi, F. Quaini, A. Manescu, F. Rustichelli. Submitted to the Journal of Tissue Engineering and Regenerative Medicine Journal of Tissue Engineering and Regenerative Medicine

Stem Cell Research to treat Infarcted Heart.

Stem Cell Labeling by Iron Oxide Nanoparticles


• Superparamagnetic iron oxide (SPIO) nanoparticles have been originally developed to enhance the contrast of cellular targets in Magnetic Resonance Imaging (MRI).

• Among several types of nanoparticles approved for human use, Feridex® is one of the most used.

• The contrast agents are composed of a Feridex (FeO1.44 - ferumoxides injectable solution)-Poly-L-Lysine (PLL) complex composed of 25µgFe/ml + PLL 375 ng/ml.

• In our experiments rat CPCs were labeled with the contrast agent in RPMI 1640 medium for 24 hours. corresponding to an average value of 177 pg per cell.


(i)SPIO can enter the stem cell either by:(ii)- receptor-mediated interactions; (iii)-nonspecific internalization pathways.

In both cases, the nanomaterials become entrapped within endosomes and are then released in the cytoplasm or trafficked to the acidic environments of lysosomes for degradation.

Cytoplasm released nanomaterials might then be transported to the cell nucleus.

Stem Cell Labeling by Iron Oxide Nanoparticles


Results obtained

in Absorption Conditions

(spatial resolution: 5.42x5.42x5.42 µm3)


Infarcted Heart analyzed 1 week after injection with 5X105 Rat Cardiac Progenitor Cells

(previously labeled with FERIDEX nanoparticles)

EQUATORIALPORTION OF THE

Heart

APEX

BASE

1st Third of the Heart

1st Third of the Heart (just marked cells are visible)

2nd Third of the Heart

2nd Third of the Heart (just marked cells are visible)

3rd Third of the Heart

INFARCTEDAREA

RV

LV

SEPTUM

3rd Third of the Heart (just marked cells are visible)

Infarcted Area

International School on Advanced Materials Science and Technology "G.Occhialini", 12th CourseInternational School on Advanced Materials Science and Technology "G.Occhialini", 12th CoursePHYSICS AND CHEMISTRY IN NANOBIOTECHNOLOGY IIPHYSICS AND CHEMISTRY IN NANOBIOTECHNOLOGY II

7 - 10 September 2010, Jesi (An) - Italy7 - 10 September 2010, Jesi (An) - Italy

Stem Cell Research to treat Infarcted Heart

A

B

C

Portion of a reconstructed 2D slice. The X-ray attenuation produced by th e labeled stem cells is higher than attenuation referred to the other tissues of the injected hearts, allowing their visualization as bright spots in the 2D images ( magenta colored spots ).

Percentage [%] of the Injected Cells in the different FE2 Heart Areas

78%

4%18%

FE2 - Heart injected with 5x10^5 labeled clonogenic cells

Infarcted Area Perinfarted Area Remaining Area

Results obtained

combining Absorption and

Phase Contrast Data (Fusion Method)(spatial resolution: 5.42x5.42x5.42 µm3)



International School on Advanced Materials Science and Technology "G.Occhialini", 12th CourseInternational School on Advanced Materials Science and Technology "G.Occhialini", 12th CoursePHYSICS AND CHEMISTRY IN NANOBIOTECHNOLOGY IIPHYSICS AND CHEMISTRY IN NANOBIOTECHNOLOGY II

7 - 10 September 2010, Jesi (An) - Italy7 - 10 September 2010, Jesi (An) - Italy

Application to Stem Cell Research related to Infarcted Heart.

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A B C

D

0.5 mm

I

II

III


33

I


34

III


Conclusions• The 3D MicroCT images represent a very innovative progress, as compared to the

usual histological images, which provide just 2D information and do not provide the correct position of the rat CPCs within the heart.

• The 3D MicroCT images guarantee an higher resolution with respect to images obtained by the Magnetic Resonance technique.

The obtained results confirmed that the labeled CPCs were distributed mostly around and within the infarcted area one week after their injection, demonstrating their migration from the injection site towards the damaged infarcted area.

Smaller cell units were observed in all parts of the heart, as in the atria, in the big vessels and in the right ventricle. This new information is a consequence of the high-level performances of the used technique.


But... Gianella et al. (Gianella et al., 2010) recently showed that MRI of Feridex-loaded

endothelial progenitors is not reliable to assess life span of injected progenitors into ischaemic tissues, providing evidences that iron released by dead endothelial progenitors may be taken up by host macrophages invading the ischaemic tissue.

We estimated the total number N of the cells found in the hearts one week after CPC injection.




We exploited the results of the investigation performed by some of the authors after the injection of GFPpos Cardiac Progenitor cells (Kajstura et al., 2005 ; Rota et al., 2008) in infarcted rat hearts.

Two dimensional distributions were found one week after CPC injection: I → attributed to the new myocytes, with an average dimension of 980µm3 (corresponding to the 15% of the total cell volume inside the heart);II → attributed to the remaining cells, with an average dimension of 455 µm3 (corresponding to the 85% of the total cell volume inside the heart).

[ ] 33 75.533)455()85.0()980()15.0( mmVcell µµ =⋅+⋅> =<




For each heart the Total Cell Number N was obtained by dividing the Total Volume Vtot, obtained by quantitative analysis of the microCT data, by the Average Cell Volume <Vcell>.

The number N of CPC-derived cells is more than doubled with respect to the number of injected CPCs, demonstrating that, at least for labeled CPCs and at a week from injection, the presence of Feridex does not dramatically affect the CPC life span.

In any case, the issue whether either iron particles, as detected here by MicroCT, could only represent unreliable signals of macrophages uptaking tracers released by dying cells, remains an unresolved problem especially on clinical ground.


Stem Cell Research related to Infarcted Heart.

40

Basic Studies of Stem Cells

MicroCT imaging of the infarcted portion of a Rat Heart injected with Cardiac Progenitor Cells. CPCs are not labelled by any contrast agent.



Alessandra Giuliani PhD., Adrian Manescu PhD., Franco Rustichelli Prof.Università Politecnica delle Marche, Ancona, Italy;

Caterina Frati PhD., , Costanza Lagrasta Dr., Monia Savi PhD., Stefano Cavalli Dr., Federico Quaini MD.University of Parma, Parma, Italy;

Vladimir s. Komlev, Dr.A.A. Baikov Institute of Metallurgy and Materials Science, Russian Academy of Science, Moscow, Russia;

Maurizio C. Capogrossi MD.Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata, Rome, Italy;

Alessandra Rossini PhD.,Vascular Biology and Regenerative Medicine Lab, Centro Cardiologico Monzino, Milano, Italy;

The authors acknowledge the ESRF User Office for kindly providing beam-time and Dr. Paul Tafforeau for the technical support during the experiments.

The Authors of the Research Activity:The Authors of the Research Activity:

Thank You for Your Attention


high-resolution x-ray microtomography for 3d imaging of

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