A study aimed at offering new physical methods capable of highlighting the inflammatory stages of vascular and articular pathologies.

1. 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

2. Plan
 General introduction
 Inflammatory pathologies
 Chosen strategies
 Vascular pathologies
 Atherosclerosis
 Results
 Articular pathologies
 Osteoarthritis
 Results
 General discussion
2

3. General Introduction
Inflammatory Pathologies
Vascular Articular
mortality 37% (2010) invalidity 33% (2010)
Causes ?
Hypertension Patient's sex
Smoking habits Only the risk factors are known Genetically factors
Age Hormonal factors
Presently medical treatments are available only
in advanced stages of pathologies
Surgery Antalgic medicines
Prothesis implant surgery
restore blood flow No synthetic synovial
tissue rupture risks fluid available
(efficient as biolubricant)
Need for
Detection of early inflammatory stage (vascular pathologies especially)
and follow up of subsequent stages of pathology evolution
3

4. VASCULAR PATHOLOGIES
Objectives ARTICULAR PATHOLOGIES
DEATH RISKS INVALIDITY
New physical approaches to study the evolution of vascular and articular
inflammatory pathologies
Objectives for Objectives for
Vascular Pathologies Articular Pathologies
Testing new mimetics of
1V antibodies to detect vascular Improving knowledge of
1A
inflammatory markers synovial fluid’s structure
Measuring mechanical/elastic
properties of Correlating synovial fluid
2V healthy and pathological 2A structure with rheological
vascular tissues and tribological properties
(what can improve surgical
gesture)
4

5. Strategy
1V Measuring the affinity between antibodies
and inflammation markers
1A Measuring the affinity between different molecular
components of the synovial fluid
ELISA test can not :
Atomic Force Microscopy:
detect weak adhesion forces
Force Spectroscopy
be used for lipids
Measuring changes in elasticity of
2V
healthy or pathological vascular tissues
Rheometric analysis:
requires large and not Atomic Force Microscopy:
ruguous samples Indentation analysis
5

6. Strategy
2A Studying the rheological and tribological properties
of synovial lubricant
Fluorescence Recovery Tribological analysis:
After Photobleaching (FRAP): used to determine friction
used to measure coefficient of coefficients between
diffusion of different different fluid components
molecules with respect to and lipids
other components (towards analysis of joint
(towards rheological properties) lubrication mechanism)
6

7. Atomic Force Microscopy – Force Spectroscopy
Principle
7

8. AFM functionalization techniques to
determine interaction forces
Principle: Generating chemical interactions between free radicals from the
substances of interest and the radicals from the cantilever binding
the substances to the cantilevers
Separator. Offers a higher freedom
degree to the linked molecules
8

9. AFM Force Spectroscopy to measure mechanical
properties of biological samples
Nanoindentation will be used here to determine the elastic
properties of vascular tissues
Calibration needed
9

10. Analytical models used to calculate the
contact stress repartition
Hertz model
Can be applied for very rigid materials for which elastic deformations are very
low or in the lack of adhesion
JKR model DMT model
10

11. 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

12. Fluorescence Recovery After
Photobleaching (FRAP)
12

13. Tribological Analysis
Through a collaboration with B.Munteanu (Lamcos, INSA, Lyon)
Normal load Fluorescence
(NL = 2.5N) Microscope
Glass
0.2 nm RMS Liquid
environments
Flurescent Lipid
Bilayers
Foucault sensor Hydrogel ~ few
nm RMS
Measurement of T Flexible lames
x
Moving table (v = 0.6 mm/s)
5mm
13

14. Plan
 General introduction
 Inflammatory pathologies
 Chosen strategies
 Vascular pathologies
 Atherosclerosis
 Results
 Articular pathologies
 Osteoarthritis
 Results
 General discussion
14

15. Vascular Pathologies
Inflammatory marker
 Atherosclerosis
I II III IV V VI 5 mm
Inflammatory stage Plaque formation
P-Selectin
MRI Detection
Surgical Treatment
Targeting contrast agents to improve MRI detection 15

16. Vascular Pathologies
 Detection of atherosclerosis through MRI using contrast agents
Collaboration with
Cardiovascular
Magnetic core
Bioengineering Laboratory
(Inserm, U698), University
Paris 7, F-75877, France
Antibody Polymeric cover
Antibody tested:
Fucoidan - Mimetic of
PSGl-1 ligand of P-Selectin
F7200 and F50500 with
different molecular weights 16

17. AFM functionalization techniques to study interaction
between Fucoidans and P-Selectin
Silanisation with APTES
AFM cantilever
Glass substrate
Glass substrate
CMA separator
molecule used
Fucoidans
F7200
F50500
P-Selectin
and
Fucoidan BSA Proteins 17

18. AFM study of Fucoidan’s affinity for different proteins
Experimental Procedure
AFM cantilever
No adhesion Adhesion
F7200
and Adhesion Peak
F50500
Glass substrate
Glass substrate
P-Selectin
and
BSA
CMA control test Microscope Veeco Multimode 18

19. Fucoidan Type Adhesion Force (nN) Adhesion percentage
CMA 0.05 43.89
1st series
BSA 0.08 61.33
F7200
P-Selectin 0.05 99.01
CMA 0.07 91.98
1stseries
BSA 0.06 74.58
F50500
P-Selectin 0.08 97.05
2nd series BSA 0.11 32.67
F7200 P-Selectin 0.06 44.49
CMA 0.29 100
2ndseries
BSA 0.31 100
F50500
P-Selectin 0.33 100
CMA 0.22 24.79
3rd series
BSA 0.28 47.22
F7200
P-Selectin 0.47 95.66
CMA 0.21 23.36
3rdseries
F50500
BSA 0.05 43.89
P-Selectin 0.08 61.33 19

20. 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
•First andone hand series of rd series of results on the other hand
•Third series of experiments
Second and the 3experiments
The results may beadhesion forceserratic duetest arevalues in medium
Medium considered as in control to low weak
The medium adhesion force values are respect to CMA and BSA to
adhesion force for P-Selectin with lower in BSA with respect
P-Selectin protein test
Failure of purification process of Fucoidan?
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
20

21. Study of mechanical properties for
vascular tissues
AFM-FS was used to test the sample’s elasticity
Collaboration with the Department of Vascular and
Endocrine Surgery, Hospital Henri Mondor, Rennes
Healthy and Pathological
samples of human aorta
affected by atherosclerosis
were obtained
Stored at -80 C
immersed in DMSO 10%
21

22. Study of mechanical properties of vascular
tissues
Pathological and healthy vascular tissue harvesting
5 mm
2 cm
2 cm 22

23. Experimental Procedure
AFM Microscope
Park Systems XE 70
Pyramidal Spherical
Indenter Indenter
23

24. Study of mechanical properties of
vascular tissues
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,
24

25. Results
Elastic modulus (MPa) Elastic modulus (MPa)
Pathological tissue Healthy tissue
0.9
Spherical 3.7
Indenter 0.55
0.11 Spherical
4.1
2.7 Indenter
Pyramidal 1.7
Indenter 3.4
1.1
25

26. Results and discussion
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
26

27. Vascular: conclusions and perspectives
These results suggest that F7200 can successfully detect P-selectin
while 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
27

28. Plan
 General introduction
 Inflammatory pathologies
 Chosen strategies
 Vascular pathologies
 Atherosclerosis
 Results
 Articular pathologies
 Osteoarthritis
 Results
 General discussion
28

29. Articular pathologies
2 cm
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 chronic
inflammation of synovial joints.
29

30. Synovial joints
Joint Implants
2 cm
Remarkable tribological performances:
Synovial fluid
Life expectancy over 80 years!
Many of the current treatments Current research is focused on
available are based on the partial determination of synovial fluid’s
or total replacement of the synovial structure to obtain a more
joint by an artificial one efficient artificial lubricant
30

31. The Synovial Fluid
Composition of the Synovial Fluid
Physiologic serum
+ Molecular chain
Hyaluronic
L ~ 12 000 nm acid
Glucides:
Hyaluronic Acid 3g/l
+
Proteines: 3 nm
8 nm
Albumin 18 g/l Albumin
Globulin 2 g/l Glycoproteic gel
Globular protein
Oates K.M.N. et all, 2005
+
0,5 nm
Lipids 3 g/l
2,5 nm
Lipid bilayer
31

32. AFM methods to detect affinities between
molecular components of the synovial fluid
Substances of interest
1. Hyaluronic acid AFM cantilever
CMA – a “separator”
in order to keep the
Lubricin molecular
configuration in
solution
2. Globular proteins (BSA,  globulin ) Measurement of
intermolecular
3 nm affinity
Polyelectrolyte
8 nm 5 nm
Mimicked here by Mucin III or
3. Lubricin
Proteo-Glycan 4
Lipid bilayer
32

33. AFM functionalization techniques to study affinity
between molecular components of the synovial fluid
Silanisation with APTES
AFM cantilever
CMA separator
molecule used
Mucin III, Proteo-
Glycan 4,
BSA and
Hyaluronic Acid γ-Globulin Proteins 33

34. Lipid bilayer deposition techniques
DOPC
Co-adsorption technique
2.5 nm 0.5 nm
Langmuir-Blodgett technique
Vesicle burst technique
DLPC
2 nm 0.5 nm
5 nm
Te
34

35. Experimental Procedure
Affinity of the synovial fluid components for lipid bilayers
measured from AFM force distance curves
Microscope Veeco Multimode
AFM cantilever
Measurement of
intermolecular
affinity
5 nm
Lipid bilayer
Specific force versus
distance curves recorded 35

36. Results
Functionalized Penetration Adhesion Medium Adhesion
substance percentage percentage Force (nN)
CMA 0% 9.14% 0.21
First Series
BSA 0% 25.05% 0.31
γ-Globulin 0% 23.67% 0.32
Hyaluronic 21.54% 61.15% 1.45
Acid
Mucin III 0% 82.85% 0.58
CMA 0% 6.1% 0.18
Second Series
BSA 0% 19.8% 0.36
γ-Globulin 0% 25.3% 0.23
Hyaluronic 15.4% 67.62% 1.06
Acid
PG 4 0% 65.1% 0.56 36
36

37. Discussion
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
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 37

38. FRAP studies
Diffusion of the synovial fluid’s main components incubated on lipid bilayers
was studied using FRAP techniques
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% NBD
fluorescent molecules in the initial lipid solution
1mg/ml in PBS
0.25mg/ml
Leica Confocal TSP3 microscope equipped with a
488nm line of the argon laser for photobleaching was used
38

39. FRAP studies
2.5μm
ROI
5 nm 50μm
Between 2 and 45 ROIs
+ 1 Reference non-bleached
Displaced exponential law
Half-Life time
Diffusion coefficient
39
39

40. Results
Incubated Number of Diffusion Immobile
substance of measurements coefficient fraction
-9
interest (cm2/s) •10
DLPC 6 0.52
5.93
DLPC + Mucin III 89 0.58
1.51
First DLPC + Hyaluronic Acid
series
30 1.41 0.51
DLPC + γ-Globulin 58 0.54
4.94
DLPC + BSA 55 0.59
4.56
DLPC 51 0.53
8.12
DOPC 48 0.55
7.86
DOPC + Mucin III 89 0.52
Second 2.77
series DOPC + Hyaluronic Acid 20 0.54
5.41
DOPC + γ-Globulin 52 0.51
7.41
DOPC + BSA 23 0.59
6.74 40
40

41. Discussion
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
No dependence of diffusion coefficients on ROI dimension
The substances that exhibit high affinity for the lipid bilayers as measured by
AFM-Force Spectroscopy also exhibit here significantly lower diffusion coefficients
41

42. Tribological Analysis
Through a collaboration with B.Munteanu (Lamcos, INSA, Lyon)
Normal load Fluorescence
(NL = 2.5N) Microscope
1. Physiological serum salt
Glass
0.2 nm RMS
2. Lubricin solution
Flurescent Lipid 200 µg/ml
Bilayers
3. Glycoproteic gel: solution
Foucault sensor Hydrogel ~ HA
few nm Flexible
Measurement of RMS 3mg/ml + BSA 18mg/ml +
lames
T
Globulin 2mg/ml
x
Moving table (v = 0.6 mm/s) 4. Lipid vesicles
containing
Friction coefficient (f) = T/N glycoproteic gel
Normal pressure: 0.3 – 1 MPa (similar to knee)
Speed : 0.1 – 1 mm/s (no hydrodynamic phenomena)
42

43. Tribological results
Lubricant Fluorescence Friction Velocity
microscopy coefficient accommodation
Physiological
salt 0.008
solution
Lubricin
solution 0.035
80μm
Glycoproteic
gel 0.1
Lipid vesicles
0.008
43

44. Articular Pathologies: conclusions and perspectives
VOLUME INTERFACE
Trunfio-Sfarghiu A.M, and all.
BiomMedD'2008
lipid multilamellar Presence of lipid Hills
A.B., Internal
vesicles multilamellar Medicine
layers Journal 2002
0.1µm
Lubricin
fixes the Lubricin
Hyaluronic acid (HA) HA and seric lipid layers -  adhesion and 
High affinity for lipid proteins remain
Seric proteins – low adhesion inside the
on the COF on lipid
lipid and reticulation with HA vesicles
cartilage
-  adhesion on
 glycoproteic gel cartilage (Rhee D.K., 2005)
COF non included glycoproteic gel
 COF glycoproteic gel included
Hyaluronic acid +
seric proteins
Cartilage
Lipid layers Lubricin
44

45. Plan
 General introduction
 Inflammatory pathologies
 Chosen strategies
 Vascular pathologies
 Atherosclerosis
 Results
 Articular pathologies
 Osteoarthritis
 Results
 General discussion
45

46. General Discussion, Conclusions and
Perspectives
Vascular pathologies
Collaboration with Cardiovascular Bioengineering Laboratory (Inserm, U698),
University Paris 7, F-75877, France
Fucoidan F7200
Testing new antibody mimetics to detect
P-Selectin inflammatory marker
Fucoidan F50500
Positive results for F7200 to detect P-Selectin inflammatory marker
Further tests repeated about ability of F7200 to detect P-, E-,
L-Selectin proteins
Collaboration with Department of Vascular and Endocrine Surgery from
Hospital Henri Mondor, Rennes & Lamcos, INSA, Lyon, France
Measuring elasticity for pathological and healthy vascular tissues
Noticeable differences for elastic modulus values
between healthy and pathological tissue samples
This study may help reduce the risk of vascular tissue rupture
during angioplasty surgery 46

47. General Discussion, Conclusions and
Perspectives
Articular pathologies
AFM - Measuring
molecular FRAP – Studying the diffusion of
affinities between different components incubated
synovial fluid’s on lipid substrates
components (towards rheological properties)
Identification of possible key components for the synovial structure
Tribological test performed to understand the role of these different
components in joint lubrication
3D model proposed for Towards optimization of
synovial fluid’s volume structure artificial synovial fluids
47

48. 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
Thank you for your
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!
attention!
And last but not least, to all of my friends here,
Bogdan, Ionut, Livia, Ana, Liliana, Mihai, Antonio
and
To all of my coleagues Jose, Arnaud, Samuel, Lucas,
Clement, Guido, Dimitri, Lauri, Alejandro, Tomita,
Marilena, Na et Simon
Thank-You !