NANOPHYSICAL ANALYSIS TO STUDY EVOLUTION OF

NANOPHYSICAL ANALYSIS TO STUDY EVOLUTION OF

VASCULAR AND ARTICULAR INFLAMMATORY PATHOLOGIES

Doctorant: MIREA Dragoş Alexandru

Directeurs de thèse:

BLANCHIN Marie-Geneviève - LPMCN

ŞABAN Rami - UPB

Co-encadrants:

TRUNFIO-SFARGHIU Ana-Maria - Lamcos, INSA Lyon

CIUCĂ Sorin - UPB

PlanGeneral introduction

Inflammatory pathologiesChosen strategies

Vascular pathologiesAtherosclerosisResults

Articular pathologiesOsteoarthritisResults

General discussion

2

General IntroductionInflammatory Pathologies

Vascularmortality 37% (2010)

Articularinvalidity 33% (2010)

Causes ? Only the risk factors are

known

HypertensionSmoking habitsAge

Patient's sexGenetically factorsHormonal factors

3

Presently medical treatments are available onlyin advanced stages of pathologies

Surgery Antalgic medicines

Prothesis implant surgeryNo synthetic synovial

fluid available(efficient as biolubricant)

restore blood flowtissue rupture risks

Detection of early inflammatory stage (vascular pathologies especially)

and follow up of subsequent stages of pathology evolution

Need for

Objectives

4

Testing new mimetics of antibodies to detect

vascular inflammatory markers

Correlating synovial fluid structure with rheological and tribological properties

RISKSDEATH INVALIDITY

New physical approaches to study the evolution of vascular and articularinflammatory pathologies

VASCULAR PATHOLOGIES ARTICULAR PATHOLOGIES

Measuring mechanical/elastic

properties of healthy and pathological

vascular tissues(what can improve surgical

gesture)

1V

2A2V

1A

Objectives for Vascular Pathologies

Objectives for Articular Pathologies

Improving knowledge ofsynovial fluid’s structure

5

Strategy1V

1A

2V

Measuring the affinity between antibodiesand inflammation markers

Measuring the affinity between different molecularcomponents of the synovial fluid

Measuring changes in elasticity of healthy or pathological vascular tissues

ELISA test can not : detect weak adhesion forcesbe used for lipids

Atomic Force Microscopy:Force Spectroscopy

Rheometric analysis:requires large and not ruguous samples

Atomic Force Microscopy:Indentation analysis

6

Strategy

2A Studying the rheological and tribological properties of synovial lubricant

Fluorescence RecoveryAfter Photobleaching (FRAP):

used to measure coefficient of diffusion of different

molecules with respect to other components

(towards rheological properties)

Tribological analysis:

used to determine friction coefficients between different fluid

componentsand lipids

(towards analysis of joint lubrication mechanism)

Atomic Force Microscopy – Force Spectroscopy

7

Principle

AFM functionalization techniques to determine interaction forces

Principle: Generating chemical interactions between free radicals from thesubstances of interest and the radicals from the cantilever binding

the substances to the cantilevers

8

Separator. Offers a higher freedomdegree to the linked molecules

AFM Force Spectroscopy to measure mechanical properties of biological samples

Calibration needed

9

Nanoindentation will be used here to determine the elastic properties of vascular tissues

Analytical models used to calculate the contact stress repartition

Hertz model

JKR model DMT model

Can be applied for very rigid materials for which elastic deformations are very low or in the lack of adhesion

10

The Hertz model with respect to indenter’s geometry

Data obtained in the experiments to determine the elastic modulus are usually several force-distance curves

The elastic modulus determined using equations specific to indenter’s geometry

11

Fluorescence Recovery After Photobleaching (FRAP)

12

Tribological Analysis

x

Moving table (v = 0.6 mm/s)

Fluorescence Microscope

Foucault sensor

Measurement of T

Normal load(NL = 2.5N)

Flexible lames

Flurescent Lipid

Bilayers

Hydrogel ~ few nm RMS

Glass0.2 nm RMS

13

Through a collaboration with B.Munteanu (Lamcos, INSA, Lyon)

Liquid environments

5mm





General discussion

14

Vascular PathologiesAtherosclerosis

I II IV V VIIII 5 mm

P-Selectin

15

MRI DetectionSurgical Treatment

Inflammatory marker

Targeting contrast agents to improve MRI detection

Inflammatory stage Plaque formation

Vascular PathologiesDetection of atherosclerosis through MRI using contrast

agents

Magnetic core

Polymeric coverAntibody

Collaboration with Cardiovascular Bioengineering

Laboratory (Inserm, U698), University Paris

7, F-75877, France

16

Antibody tested:Fucoidan - Mimetic of

PSGl-1 ligand of P-Selectin

F7200 and F50500 with different molecular

weights

AFM functionalization techniques to study interaction between Fucoidans and P-Selectin

AFM cantilever

ProteinsFucoidan

Glass substrateGlass substrate

P-Selectin

andBSA

17

Silanisation with APTES

CMA separatormolecule usedFucoidans

F7200F50500

AFM study of Fucoidan’s affinity for different proteins

AFM cantilever

Glass substrateGlass substrate

No adhesion AdhesionF7200and

F50500

P-SelectinandBSA

CMA control test 18

Experimental Procedure

Adhesion Peak

Microscope Veeco Multimode

Fucoidan Type Adhesion Force (nN) Adhesion percentage

1st seriesF7200

1st seriesF50500

2nd seriesF7200

2nd seriesF50500

3rd seriesF7200

3rd seriesF50500

0.05 43.89

0.08 61.33

CMABSA

P-SelectinCMA

BSAP-Selectin

CMABSA

P-SelectinCMA

BSA

P-SelectinCMA

BSAP-Selectin

BSAP-Selectin

0.05 99.01

0.07 91.98

0.06 74.580.08 97.05

0.11 32.670.06 44.49

0.29 100

0.31 100

0.33 100

0.22 24.790.28 47.22

0.47 95.66

0.21 23.36

0.05 43.890.08 61.33 19

Vascular inflammation: Affinity of Fucoidan for different proteins

Forces of adhesion between both Fucoidans and proteins are analyzed with respect to medium adhesion force in CMA control test

Noticeable differences appear between the 1st and 2nd series on one hand and the 3rd series of results on the other

hand The results may be considered as erratic due to low values in medium

adhesion force for P-Selectin with respect to CMA and BSA

Failure of purification process of Fucoidan?

Medium adhesion forces in control test are weakThe medium adhesion force values are lower in BSA with respect

to P-Selectin protein test

20

• Third series of experiments

Accounting for the fact that adhesion percentage for F7200 in third series was 96% this compound can be considered as a

good candidate to detect P-Selectin

• First and Second series of experiments

Study of mechanical properties for vascular tissues

AFM-FS was used to test the sample’s elasticity

Collaboration with the Department of Vascular andEndocrine Surgery, Hospital Henri Mondor, Rennes

Healthy and Pathologicalsamples of human aorta

affected by atherosclerosiswere obtained

Stored at -80°C immersed in DMSO 10%

21

Study of mechanical properties of vascular tissues

2 cm

5 mm

222 cm

Pathological and healthy vascular tissue harvesting


AFM MicroscopePark Systems XE

70

SphericalIndenter

PyramidalIndenter

23

Study of mechanical properties of vascular tissues

24

Indentation in tissue determined from the force distance curves recorded by AFM

Elastic modulus of the tissues calculated using Hertz model’s equations

Force vs Indentation curves fitted with a simple power law: exponent

1.5 – spherical; 2 – pyramidal

From that fitting the correspondent elastic modulus was calculated

using equations according to the Hertz model

for example here,

Elastic modulus (MPa)Pathological tissue

Elastic modulus (MPa)

Healthy tissue

SphericalIndenter

PyramidalIndenter 3.4

0.9

0.55

0.11

2.7

1.7

1.1

Results

4.1

3.7

25

SphericalIndenter

Results and discussion

26

A higher elastic modulus is found for the healthy zones (in agreement with literature values) compared to the

pathological ones. Decrease in elasticity of pathological tissues is thought to be related to increase in adipose and calcification

Significant differences are obtained with the different types of indenters: This may be due to the large roughness and the 3D heterogeneity of the

samples.Friction forces between spherical indenter and samples may be

important and cause errors

Vascular: conclusions and perspectives

27

These results suggest that F7200 can successfully detect P-selectinwhile F50500 exhibits lower performances

Elastic moduli were calculated for both healthy and pathological tissue samples in agreement with literature values and consistent with influence of pathology





General discussion

28

Articular pathologies

29

Osteoarthritis is the most common joint disease affecting especially people aged over 55. It involves the whole joint and can be associated with cartilage

loss, changes in the subchondral bone and development of osteophytes.

Rheumatoid Arthritis is a complex autoimmune disease that causes chronicinflammation of synovial joints.

2 cm

Synovial joints

30

Remarkable tribological performances:

Life expectancy over 80 years!

Synovial fluid

Joint Implants2 cm

Many of the current treatmentsavailable are based on the partial

or total replacement of the synovialjoint by an artificial one

Current research is focused on

determination of synovial fluid’s structure to obtain a

more efficient artificial lubricant

Physiologic serum

+

Glucides:Hyaluronic Acid 3g/l

Proteines: Albumin 18 g/lGlobulin 2 g/l

Lipids 3 g/l

+

+

Molecular chain

L ~ 12 000 nm

Globular protein

8 nm3 nm

Glycoproteic gelAlbumin

Hyaluronic acid

2,5 nm

0,5 nm

Lipid bilayer

Oates K.M.N. et all, 2005

The Synovial Fluid

31

Composition of the Synovial Fluid

AFM cantileverCMA – a

“separator” in order to keep the

molecular configuration in

solution

1. Hyaluronic acid

2. Globular proteins (BSA, globulin )

8 nm3 nm

3. Lubricin

Substances of interest

5 nm

Lipid bilayer

Measurement of

intermolecular affinity

AFM methods to detect affinities between molecular components of the synovial fluid

Polyelectrolyte

Mimicked here by Mucin III or Proteo-Glycan 4

Lubricin

32

AFM functionalization techniques to study affinity between molecular components of the synovial

fluidAFM cantilever

ProteinsHyaluronic Acid

Mucin III, Proteo-Glycan 4,BSA andγ-Globulin 33

Silanisation with APTES

CMA separatormolecule used

Lipid bilayer deposition techniques

34

5 nm

Co-adsorption technique

DOPC

DLPC

0.5 nm

0.5 nm

2.5 nm

2 nm

Te

Langmuir-Blodgett technique

Vesicle burst technique


35

AFM cantilever

5 nm

Lipid bilayer

Measurement of intermolecular

affinity

Affinity of the synovial fluid components for lipid bilayers measured from AFM force distance curves

Specific force versus distance curves recorded

Microscope Veeco Multimode

36

Functionalized

substance

Penetration percentage

Adhesion percentage

Medium Adhesion Force

(nN)

CMA

BSA

γ-Globulin

Hyaluronic Acid

Mucin III

CMA

BSA

γ-Globulin

Hyaluronic Acid

PG 4

0%

0%

0%

0%

0%

0%

0%

0%

21.54%

15.4%

9.14%

25.05%

23.67%

61.15%

82.85%

6.1%

19.8%

25.3%

67.62%

65.1%

0.21

0.31

0.32

1.45

0.58

0.18

0.36

0.23

1.06

0.56

ResultsF

irst

Se

ries

Se

con

d S

eri

es

36

Discussion

37

The seric proteins which exhibit low affinity for the lipid bilayers probably play a secondary role

The AFM study represents a static approach: a more “rheological” approach is needed to confirm these results

The results are analyzed in terms of adhesion force between substances of interest and lipid bilayers with respect to adhesion

force in CMA control test:Clearly Lubricin and Hyaluronic Acid exhibit the highest

affinities and are thought to play a key role

FRAP studies

38

Diffusion of the synovial fluid’s main components incubated on lipid bilayers was studied using FRAP techniques

Leica Confocal TSP3 microscope equipped with a 488nm line of the argon laser for photobleaching was used

The DLPC and DOPC lipid bilayers were deposited using both Langmuir-Blodgett (first series) and Vesicle burst (second series) techniques

The fluorescence of bilayers was obtained by addition of 1% NBDfluorescent molecules in the initial lipid solution

1mg/ml in PBS

0.25mg/ml

FRAP studies

2.5μm

50μm

ROI

Between 2 and 45 ROIs+ 1 Reference non-

bleached

5 nm

39

Diffusion coefficient

Displaced exponential law

Half-Life time

39

Results

40

Incubated substance of

interest

Number of measureme

nts

Diffusion coefficient

(cm2/s)

Immobile fraction

First series

DLPC 6 5.93 0.52DLPC + Mucin III 89 1.51 0.58

DLPC + Hyaluronic Acid 30 1.41 0.51DLPC + γ-Globulin 58 4.94 0.54

DLPC + BSA 55 4.56 0.59

Second series

DLPC 51 8.12 0.53DOPC 48 7.86 0.55

DOPC + Mucin III 89 2.77 0.52DOPC + Hyaluronic Acid 20 5.41 0.54

DOPC + γ-Globulin 527.41

0.51

DOPC + BSA 23 6.74 0.59

• 10

-9

40

Discussion

41

No dependence of diffusion coefficients on ROI dimension

Over the two series no significant difference in the diffusion coefficient values depending on lipid compound or bilayer deposition

technique, except in the case of Hyaluronic Acid whose diffusion coefficient remains lower

The substances that exhibit high affinity for the lipid bilayers as measured by AFM-Force Spectroscopy also exhibit here significantly

lower diffusion coefficients

Tribological Analysis

Normal pressure: 0.3 – 1 MPa (similar to knee)

Speed : 0.1 – 1 mm/s (no hydrodynamic phenomena)

x

Moving table (v = 0.6 mm/s)

Fluorescence

Microscope

Foucault sensor

Measurement of T

Normal load(NL = 2.5N)

Flexible lames

Flurescent Lipid

Bilayers

Hydrogel ~ few nm

RMS

Glass0.2 nm RMS

Friction coefficient (f) = T/N

42

1. Physiological serum salt

4. Lipid vesicles containing

glycoproteic gel

2. Lubricin solution 200 µg/ml

3. Glycoproteic gel: solution HA

3mg/ml + BSA 18mg/ml + Globulin 2mg/ml

Through a collaboration with B.Munteanu (Lamcos, INSA, Lyon)

Tribological resultsLubricant Fluorescence

microscopyFriction

coefficientVelocity

accommodation

43

0.008

0.035

0.1

0.008

80μm

Physiological salt

solution

Lubricinsolution

Glycoproteicgel

Lipid vesicles

LubricinCartilage

Hyaluronic acid + seric

proteins

Lipid layers

Hyaluronic acid (HA)High affinity for lipidSeric proteins – low

adhesion lipid and reticulation with HA

glycoproteic gelCOF non included glycoproteic

gel COF glycoproteic gel included

HA and seric proteins

remain inside the vesicles

44

lipid multilamellar

vesicles0.1µ

m

VOLUME

Presence of lipid

multilamellar layers

Hills A.B., Internal Medicine

Journal 2002

INTERFACE

Lubricin- adhesion and

COF on lipid- adhesion on cartilage (Rhee D.K.,

2005)

Lubricin fixes the

lipid layers on

the cartilage

Articular Pathologies: conclusions and perspectives

Trunfio-Sfarghiu A.M, and all. BiomMedD'2008





General discussion

45

46

General Discussion, Conclusions and Perspectives

Vascular pathologies

Fucoidan F7200

Fucoidan F50500

Positive results for F7200 to detect P-Selectin inflammatory marker

Testing new antibody mimetics to detectP-Selectin inflammatory marker

Collaboration with Cardiovascular Bioengineering Laboratory (Inserm, U698), University Paris 7, F-75877, France

Measuring elasticity for pathological and healthy vascular tissues

Collaboration with Department of Vascular and Endocrine Surgery from Hospital Henri Mondor, Rennes & Lamcos, INSA, Lyon, France

This study may help reduce the risk of vascular tissue rupture during angioplasty surgery

Noticeable differences for elastic modulus values between healthy and pathological tissue samples

Further tests repeated about ability of F7200 to detect P-, E-,

L-Selectin proteins

47

General Discussion, Conclusions and Perspectives

Articular pathologies

AFM - Measuring molecular

affinities between synovial fluid’s

components

Identification of possible key components for the synovial structure

FRAP – Studying the diffusion of different components incubated

on lipid substrates(towards rheological properties)

Tribological test performed to understand the role of these different components in joint lubrication

Towards optimization of artificial synovial fluids

3D model proposed for synovial fluid’s volume structure

Thank you for your attention!

I would like to give thanks to all everybody from LPMCN and UPB, you might not notice it but your

help was decisive for me, Thank-You !

Also I have reserved special thanks to my colleagues and everybody else, especially Ana-Maria et Mr. Berthier from Lamcos, INSA, Lyon and also to the team from Laboratoire Inserm, Paris, Thank-You!

And last but not least, to all of my friends here, Bogdan, Ionut, Livia, Ana, Liliana, Mihai, Antonio

andTo all of my coleagues Jose, Arnaud, Samuel, Lucas,

Clement, Guido, Dimitri, Lauri, Alejandro, Tomita, Marilena, Na et Simon

Thank-You !

Health & Medicine

NANOPHYSICAL ANALYSIS TO STUDY EVOLUTION OF