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NANOPHYSICAL ANALYSIS TO STUDY         EVOLUTION OF    VASCULAR AND ARTICULAR  INFLAMMATORY PATHOLOGIES  Doctorant: MIREA ...
Plan   General introduction     Inflammatory pathologies     Chosen strategies   Vascular pathologies     Atheroscler...
General Introduction                  Inflammatory Pathologies     Vascular                                               ...
VASCULAR PATHOLOGIES                              Objectives                ARTICULAR PATHOLOGIES         DEATH           ...
Strategy   1V        Measuring the affinity between antibodies                   and inflammation markers   1A    Measurin...
Strategy 2A       Studying the rheological and tribological properties                          of synovial lubricant   Fl...
Atomic Force Microscopy – Force SpectroscopyPrinciple                                            7
AFM functionalization techniques to      determine interaction forcesPrinciple: Generating chemical interactions between f...
AFM Force Spectroscopy to measure mechanical       properties of biological samples      Nanoindentation will be used here...
Analytical models used to calculate the       contact stress repartition                                                  ...
The Hertz model with respect to indenter’s               geometry   Data obtained in the experiments to determine the ela...
Fluorescence Recovery After   Photobleaching (FRAP)                              12
Tribological AnalysisThrough a collaboration with B.Munteanu (Lamcos, INSA, Lyon)      Normal load          Fluorescence  ...
Plan   General introduction     Inflammatory pathologies     Chosen strategies   Vascular pathologies     Atheroscler...
Vascular Pathologies                                                             Inflammatory marker           Atheroscle...
Vascular Pathologies    Detection of atherosclerosis through MRI using contrast agents     Collaboration with       Cardi...
AFM functionalization techniques to study interaction       between Fucoidans and P-Selectin                      Silanisa...
AFM study of Fucoidan’s affinity for different proteins                            Experimental Procedure   AFM cantilever...
Fucoidan Type       Adhesion Force (nN)   Adhesion percentage               CMA              0.05                 43.891st...
Vascular inflammation: Affinity of Fucoidan for               different proteins     Forces of adhesion between both Fucoi...
Study of mechanical properties for         vascular tissues AFM-FS was used to test the sample’s elasticityCollaboration w...
Study of mechanical properties of vascular                tissues         Pathological and healthy vascular tissue harvest...
Experimental Procedure                     AFM Microscope                    Park Systems XE 70Pyramidal                  ...
Study of mechanical properties of           vascular tissuesIndentation in tissue determined from the force distance curve...
Results            Elastic modulus (MPa)               Elastic modulus (MPa)             Pathological tissue              ...
Results and discussion    A higher elastic modulus is found for the healthy zones (in agreement with                      ...
Vascular: conclusions and perspectives        These results suggest that F7200 can successfully detect P-selectin         ...
Plan   General introduction     Inflammatory pathologies     Chosen strategies   Vascular pathologies     Atheroscler...
Articular pathologies  2 cmOsteoarthritis is the most common joint disease affecting especially people aged over 55. It in...
Synovial joints         Joint Implants                               2 cm                                       Remarkable...
The Synovial Fluid            Composition of the Synovial FluidPhysiologic serum        +               Molecular chain   ...
AFM methods to detect affinities betweenmolecular components of the synovial fluid          Substances of interest     1. ...
AFM functionalization techniques to study affinitybetween molecular components of the synovial fluid                      ...
Lipid bilayer deposition techniques                       DOPC                                Co-adsorption technique   2....
Experimental Procedure                  Affinity of the synovial fluid components for lipid bilayers                      ...
Results                Functionalized   Penetration    Adhesion    Medium Adhesion                  substance      percent...
Discussion The results are analyzed in terms of adhesion force between substances ofinterest and lipid bilayers with respe...
FRAP studies Diffusion of the synovial fluid’s main components incubated on lipid bilayers                     was studied...
FRAP studies                                              2.5μm                                     ROI5 nm               ...
Results               Incubated            Number of      Diffusion        Immobile              substance of         meas...
Discussion  Over the two series no significant difference in the diffusion coefficient values   depending on lipid compoun...
Tribological Analysis        Through a collaboration with B.Munteanu (Lamcos, INSA, Lyon)    Normal load           Fluores...
Tribological results Lubricant           Fluorescence    Friction        Velocity                      microscopy    coeff...
Articular Pathologies: conclusions and perspectives               VOLUME                                                  ...
Plan   General introduction     Inflammatory pathologies     Chosen strategies   Vascular pathologies     Atheroscler...
General Discussion, Conclusions and              PerspectivesVascular pathologies    Collaboration with Cardiovascular Bi...
General Discussion, Conclusions and              PerspectivesArticular pathologies          AFM - Measuring               ...
I would like to give thanks to all everybody from LPMCN and UPB, you might not notice it but your       help was decisive ...
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NANOPHYSICAL ANALYSIS TO STUDY EVOLUTION OF

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

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  1. 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. 2. Plan General introduction  Inflammatory pathologies  Chosen strategies Vascular pathologies  Atherosclerosis  Results Articular pathologies  Osteoarthritis  Results General discussion 2
  3. 3. General Introduction Inflammatory Pathologies Vascular Articularmortality 37% (2010) invalidity 33% (2010) Causes ?Hypertension Patients sexSmoking habits Only the risk factors are known Genetically factorsAge Hormonal factors Presently medical treatments are available only in advanced stages of pathologies Surgery Antalgic medicines Prothesis implant surgeryrestore blood flow No synthetic synovialtissue 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. 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. 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 tissuesRheometric analysis:requires large and not Atomic Force Microscopy:ruguous samples Indentation analysis 5
  6. 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. 7. Atomic Force Microscopy – Force SpectroscopyPrinciple 7
  8. 8. AFM functionalization techniques to determine interaction forcesPrinciple: 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. 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. 10. Analytical models used to calculate the contact stress repartition Hertz modelCan 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. 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. 12. Fluorescence Recovery After Photobleaching (FRAP) 12
  13. 13. Tribological AnalysisThrough 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. 14. Plan General introduction  Inflammatory pathologies  Chosen strategies Vascular pathologies  Atherosclerosis  Results Articular pathologies  Osteoarthritis  Results General discussion 14
  15. 15. Vascular Pathologies Inflammatory marker  Atherosclerosis I II III IV V VI 5 mm Inflammatory stage Plaque formation P-Selectin MRI Detection Surgical TreatmentTargeting contrast agents to improve MRI detection 15
  16. 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 ofPSGl-1 ligand of P-Selectin F7200 and F50500 with different molecular weights 16
  17. 17. AFM functionalization techniques to study interaction between Fucoidans and P-Selectin Silanisation with APTESAFM cantilever Glass substrate Glass substrate CMA separator molecule used Fucoidans F7200 F50500 P-Selectin and Fucoidan BSA Proteins 17
  18. 18. AFM study of Fucoidan’s affinity for different proteins Experimental Procedure AFM cantilever No adhesion AdhesionF7200 and Adhesion PeakF50500 Glass substrate Glass substrate P-Selectin and BSA CMA control test Microscope Veeco Multimode 18
  19. 19. Fucoidan Type Adhesion Force (nN) Adhesion percentage CMA 0.05 43.891st series BSA 0.08 61.33F7200 P-Selectin 0.05 99.01 CMA 0.07 91.981stseries BSA 0.06 74.58F50500 P-Selectin 0.08 97.052nd series BSA 0.11 32.67F7200 P-Selectin 0.06 44.49 CMA 0.29 1002ndseries BSA 0.31 100F50500 P-Selectin 0.33 100 CMA 0.22 24.793rd series BSA 0.28 47.22F7200 P-Selectin 0.47 95.66 CMA 0.21 23.363rdseriesF50500 BSA 0.05 43.89 P-Selectin 0.08 61.33 19
  20. 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. 21. Study of mechanical properties for vascular tissues AFM-FS was used to test the sample’s elasticityCollaboration with the Department of Vascular andEndocrine 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. 22. Study of mechanical properties of vascular tissues Pathological and healthy vascular tissue harvesting 5 mm 2 cm 2 cm 22
  23. 23. Experimental Procedure AFM Microscope Park Systems XE 70Pyramidal Spherical Indenter Indenter 23
  24. 24. Study of mechanical properties of vascular tissuesIndentation 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. 25. Results Elastic modulus (MPa) Elastic modulus (MPa) Pathological tissue Healthy tissue 0.9Spherical 3.7Indenter 0.55 0.11 Spherical 4.1 2.7 IndenterPyramidal 1.7Indenter 3.4 1.1 25
  26. 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. 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. 28. Plan General introduction  Inflammatory pathologies  Chosen strategies Vascular pathologies  Atherosclerosis  Results Articular pathologies  Osteoarthritis  Results General discussion 28
  29. 29. Articular pathologies 2 cmOsteoarthritis 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. 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’sor total replacement of the synovial structure to obtain a more joint by an artificial one efficient artificial lubricant 30
  31. 31. The Synovial Fluid Composition of the Synovial FluidPhysiologic 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. 32. AFM methods to detect affinities betweenmolecular 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. 33. AFM functionalization techniques to study affinitybetween molecular components of the synovial fluid Silanisation with APTESAFM cantilever CMA separator molecule used Mucin III, Proteo- Glycan 4, BSA and Hyaluronic Acid γ-Globulin Proteins 33
  34. 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. 35. Experimental Procedure Affinity of the synovial fluid components for lipid bilayers measured from AFM force distance curves Microscope Veeco Multimode AFM cantileverMeasurement of intermolecular affinity 5 nm Lipid bilayer Specific force versus distance curves recorded 35
  36. 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% 0.18Second 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. 37. Discussion The results are analyzed in terms of adhesion force between substances ofinterest 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. 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 bothLangmuir-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. 39. FRAP studies 2.5μm ROI5 nm 50μm Between 2 and 45 ROIs + 1 Reference non-bleached Displaced exponential law Half-Life time Diffusion coefficient 39 39
  40. 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 Acidseries 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 0.52Second 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. 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 byAFM-Force Spectroscopy also exhibit here significantly lower diffusion coefficients 41
  42. 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 solutionFlurescent Lipid 200 µg/ml Bilayers 3. Glycoproteic gel: solutionFoucault sensor Hydrogel ~ HA few nm FlexibleMeasurement 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. 43. Tribological results Lubricant Fluorescence Friction Velocity microscopy coefficient accommodation Physiological salt 0.008 solution Lubricin solution 0.035 80μm Glycoproteic gel 0.1Lipid vesicles 0.008 43
  44. 44. Articular Pathologies: conclusions and perspectives VOLUME INTERFACE Trunfio-Sfarghiu A.M, and all. BiomMedD2008 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 includedHyaluronic acid + seric proteins Cartilage Lipid layers Lubricin 44
  45. 45. Plan General introduction  Inflammatory pathologies  Chosen strategies Vascular pathologies  Atherosclerosis  Results Articular pathologies  Osteoarthritis  Results General discussion 45
  46. 46. General Discussion, Conclusions and PerspectivesVascular 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. 47. General Discussion, Conclusions and PerspectivesArticular 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. 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 myThank you for yourcolleagues and everybody else, especially Ana-Mariaet Mr. Berthier from Lamcos, INSA, Lyon and also tothe 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 andTo all of my coleagues Jose, Arnaud, Samuel, Lucas, Clement, Guido, Dimitri, Lauri, Alejandro, Tomita, Marilena, Na et Simon Thank-You !
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