Journée Scientifique BMBI 6ème édition

12 juillet 2017

 Centre Pierre Guillaumat 1, L103

Equipe organisatrice: Méqane Beldjilali-Labro - Elodie Colaço – Karim El Kirat – Doriane Vesperini

N.B. : Les résumés sont classés par ordre alphabétique du premier auteur.

Programme de la JS6 – BMBI - 2017 10h15 Accueil à PG1-L103

10h30 ZGHEIB Elias 10h45 CARRIOU Vincent 11h00 NURIN-BAITI Risa

*** PAUSE *** 11h45 LETOCART Adrien 12h00 LEPETIT Kevin 12h15 VESPERINI Doriane *** BUFFET *** 14h00 KHLAIFI Hajer 14h15 DUMONT Emmanuel 14h30 TANNOUS Halim 14h45 Délibération

Mot de la Directrice de BMBI, La journée très attendue aussi bien par les doctorants que les membres du laboratoire arrive enfin. Il faut que dire que les deux précédentes éditions furent une réussite, avec des présentations de très bonnes qualités. L’ exercice qui consiste à présenter ses activités devant un public par essence pluridisciplinaire et celles/ceux qui y réussissent y trouveront une très grande satisfaction et le public aussi.

Nous retiendrons aussi que les vainqueurs des deux précédentes éditions ont été par la suite lauréats des Prix de thèse Guy Deniélou… Cette journée est d’autant plus agréable que la présidente du jury Chantal Pérot ancienne directrice de l’Ecole Doctorale de l’UTC y met une coloration particulière, la 1ère édition ce fut la mode des toques blanches de chefs cuisiniers, puis la 2ème des coiffes de docteurs .. que nous réserve t- elle pour la 3ème édition ? Cette journée est encore une occasion pour découvrir, redécouvrir les activités de recherches de BMBI, sa richesse, sa ‘biodiversité’, et je termine en remerciant chaleureusement les organisateurs pour leur contribution à la réussite de cette journée. Bonne lecture, Marie-Christine HO BA THO Directrice BMBI

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CHARACTERIZATION OF THE NANO MECHANICAL PROPERTIES OF BIOLOGICAL LIPID MEMBRANES WITH THECIRCULAR MODE ATOMIC FORCE MICROSCOPY

Risa Nurin BAITI*, Karim EL KIRAT*and Pierre-Emmanuel MAZERAN**

*: UMR CNRS 7338 Bio mechanic and Bio-engineering, Université de Technologie de Compiègne

**: UMR CNRS 7337 Roberval, Universitéde Technologie de Compiègne

risa-nurin.baiti@utc.fr, karim.elkirat@utc.fr, pierre-emmanuel.mazeran@utc.fr

RESUME: In lipid-based drug delivery systems,the fusion mechanism between liposome (lipid-based nanocapsule) and cell envelope is significantly related to the mechanical properties of lipid membranes. Atomic Force Microscopy (AFM) has been widely used to assess the mechanical properties of biological samples on the nanoscale. We applied recently developed Circular Mode AFM for nanotribological measurements of biomembranes. AFM mode circular is able to quantify the both mechanical and viscous properties of lipid membranes simultaneously due to its constant and high sliding velocity.

1. INTRODUCTION

In drug delivery system, lipidic nanocapsules fuse with cell wall to release the drug [1]. This phenomenon is related to dynamic mechanical mechanism which is not well explained yet. Thus, understanding the mechanical properties of biomembranes can help to select lipid type and co-factor and to modulate the nanocapsule properties [2] for enhanced fusion/delivery process. Atomic Force Microscope (AFM) has opened new perspectives for investigations of phenomenological mechanisms at the nanoscale especially for biological materials [3]). Recently, Circular Mode AFM (CM-AFM) was developed and it offers new capabilities for surface investigations specially for measuring physical properties that requires high scanning velocities and/or continuous displacements without rest periods [4]. CM-AFM allows real-time measurement of mechanical response to normal and lateral force.. So, in this research, we measured the mechanical response of supported lipid bilayers (SLBs) under physiological condition to have a better insight about the organization of the membranes.

2. METHODES

Circular displacement is generated by a sinusoidal displacement in the X direction and a quadrature sinusoidal displacement in the Y direction. There are two parameters to control the sliding velocity: the voltage half amplitude and the frequency. Coupled with the force spectrum mode, it allows the measurement of friction load dependence. AFM images are acquired with a NanoScope III Multimode AFM (Veeco Metrology LLC, Santa Barbara, CA) at 21°C. It is equipped with a 125 μm× 125 μm× 5 μm J-scanner. We used oxide-sharpened micro fabricated Si3N4 cantilevers (Microlevers, Veeco Metrology LLC, Santa Barbara, CA). The spring constants of tips are ranging from 0.01 to 0.5 N/m. The calibration constant of lateral force are done by recently developed scratched method which is adopted from wedge technique [5]. Supported lipid bilayers (SLBs) are prepared by using the vesicle fusion method with final concentration of 1 mM in 10 mM TBS containing 150 mM alkali halide salts. After sonication, it was heated at 60oC for 1 h. Lipid bilayers are formed on glass solid support by successive adhesion, flattening and rupture of lipid vesicles.

3. RESULTATS

Our continuous DOPC SLBs present a thickness of 4.7 + 0.07 nm and a punch-through force of 2.52 + 0.01 nN. Both values are in agreement with the literature |6]. Continuous friction curves have been successfully acquired instead of the curves made of discrete points obtained by conventional AFM. Our curves followed the same pattern as published results on SLBs [7]. Measurement at sliding velocity ranged between 200 – 2200 µm/s shows the linear increase of friction force. Viscous friction coefficient is measured as slope of linear regression line. For DOPC, we acquired η = 72.85+8.86 nN.s/m. We varied the sliding velocity by modifying the diameter of circular displacement through amplitude changes; 1-10 Volts (the sliding distance) while the frequency was remained constant (175 Hz). Thus, we did a series of experiments with sliding velocities ranging from 214 to 2144 µm/s. Furthermore, the use of different alkali halide salts can alter the mechanical response of DOPC SLBs. While Li+ and Na+ brought relatively the same effect, K+ improves the compactness of viscosity of DOPC SLBs (see Figure 1.B).

4. DISCUSSION

The tribological profile shows the interaction between tip and membrane (see Figure 1.A). Thanks to its fast, constant, and continuous scanning velocity, CM-AFM allows us to assess nanotribological properties of biomembranes. A small jump can

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be observed at the beginning of the curve due to repulsive electrostatic interactions between DOPC headgroups and the AFM tip. A certain amount of force was needed to recover this repulsion so the tip can touch the membrane. We assumed that the friction force at this moment was the initial friction force of membrane and we followed its evolution as function of sliding velocity. Then, as the tip slides onto the membrane with increasing normal force, there was plateau formed. In fact, this plateau is not flat but it is slightly tilted as the tip crosses the hydrophilic region of the biomembrane. When the tip reached the threshold value of normal force, there was significant jump of friction force. We noted the normal force at this point as punch-through force of membrane. Then, the tip continued to slide on the solid support. Friction force of membrane is increasing linearly at higher sliding velocity. It followed the typical profile of viscous friction (8) showing that lipid membrane acts as a confined liquid.

The presence of different alkali halide salts has been proved improving the mechanical resistance of the membrane (7). Among the alkali cations, potassium shows the highest increase due to its optimal size-fit between efficient ionic size and distance between lipid headgroups.

5. CONCLUSIONS

CM-AFM successfully quantifies the punch-through force and friction force simultaneously in real-time with better time efficiency. The reliability of this method was showed by the punch-through force which was comparable with the values found in the literature. CM AFM allows direct measurement of membrane viscosity in nN.s/m. Thus, it gives a new possibility to study both mechanical and viscous properties of any lipidic system, including live cell membrane.

6. REMERCIEMENTS

This work was supported by doctoral fellowship from Labex MS2T Control of Technological Systems-of-Systems (LABEX-UTC) and Picardie Region (FrixOrg) grant with the European Funds for the Development of the Regions (FEDER).

(A) (B) Figure 1 – (A) Curve of normal and friction force as function of tip-membrane separation. The curve can be divided into four zones: (a) tip is relatively far from membrane , (b) tip touch the membranes’ surface, (c) tip breaks the membrane, and (d) tip is in contact with solid surface. (B) Punchthrough force and friction viscous coefficient for different cations.

7. REFERENCES

[1] Felice B, Prabhakaran MP, Rodriguez AP, Ramakrishna S. Drug delivery vehicles on a nano-engineering perspective. Mat Scie and Eng C 2014; 41: 178-195

[2] Garcia-Manyes S, Sanz F. Nanomechanics of lipid bilayers by force spectroscopy with AFM : A perspective. BiochimetBiophys Act 2010; 1798: 741-749.

[3] Morandat S, El Kirat K. Exploring the properties and interactions of supported lipid bilayers on the nanoscale by atomic force microscopy. Microscopy 2010;1925-1939

[4] Nasrallah H, Mazeran PE, Noel O. Circular mode: A new scanning probe microscopy method for investigating surface properties at constant and continuous scanning velocities. Rev of SciInstrum 2011; 82: 113703Pelling and Horton, 2008

[5] Ogletree DF, Carpick RW, Salmeron M, Calibration of frictional forces in atomic force microscopy. Rev SciInstrum 1996; 67: 3298-3306

[6] Garcia-Manyes S, Redondo-Morata L, Oncins G. Nanomechanics of Lipid Bilayers: heads or tails ?. JACS 2010;14:12874-12886

[7] Oncins G, Garcia-Manyes S, Sanz F. Study of frictional properties of a phospholipid bilayer in a liquid environment with lateral force microscopy as a function of NaCl concentration. Langmuir Vol.21 2005; 16: 7373-7379

[8] Mougin K, Hamidou H. Nanoscale friction of self-assembled monolayers. In “Fundamentals of friction and wear on the nanoscale”, Gnecco E, Meyer E.Springer (eds), London, 2015

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MULTI-SCALE ANALYSIS OF MULTI-LAYERED TISSUES CONSTRUCTS AND COMPARISON WITH NATIVE TISSUE CHARACTERISTICS: INVESTIGATION OF THE MYOTENDINOUS JUNCTION

Megane Beldjilali Labro*,** , Murielle Dufresne* Jean Francois Grosset* , Cecile Legallais*

*: UMR CNRS 7338 Biomechanics and Bioengineering (BMBI) ***: LABEX MS2T

megane.beldjilali-labro@utc.fr ; cecile.legallais@utc.fr ; jean-françois.grosset@utc.fr ; murielle.dufresne@utc.fr RESUME: The project is to develop a biohybrid substitute for the reconstruction of the continuum tendon-muscle ensuring cohesive interfaces during and beyond the cell culture process, using tissue engineering approaches based on a deep knowledge of in vivo native structures. After a bibliographical analysis of native and reconstructed structures at and near the muscle-tendon junction, a biomimetic approach will guide tissue reconstruction from the characteristics of native one. Tendon and muscle will be reconstructed using electrospun materials mimicking the collagen structure and the muscle fibers, seeded with fibroblasts and myoblasts (muscular cells). The interface between muscle and tendon will be simulated, with close analysis of the morphological and biochemical interactions between collagen microfibrills and transmembrane proteins of the myoblasts.

1. INTRODUCTION

The myotendinous junction is a highly specialized region in the muscle-tendon unit. In this area, the tension generated by muscle fibres is transmitted to the collagen fiber of the tendon. Morphological studies have demonstrated that, at the myotendinous junction, collagen fibrils insert into deep recesses, which are formed between the finger-like ends of the muscle cells(1). It is thus interesting to study the interaction between chemical and mechanical inter-tissue signaling in cell adhesion, tissue morphogenesis and differentiation, that leads to a functional MTJ(2). In case of injury, traditional surgery methods such as autograft or allograft have limitations due to donor site unavailability or immunogenicity problems (3). Tissue engineering, that consists in cultivating cells in a polymeric artificial or natural scaffold, usually using external stimuli such as biochemical factors, mechanical or electrical constraints in order to obtain a final construct mimicking the native organ or tissue, could overcome those limits (4). A biohybrid tissue can also be used to study alterations at the MTJ place. The aim of this thesis is to develop a scaffold with specific regions that exhibit mechanical property differences mimicking the trends observed in native MTJ.

2. METHODS

Electrospinning: For muscle tissue constructs we propose to elaborate 3D scaffolds with electrospun poly-ε-caprolactone because of its known biocompatibility, biodegradability, at 8 ;10, 12,5 or 15% wt/V in 80/20 DCM/DMF (V/V).

Materials’ analyses: All materials were observed by SEM (Scanning Electron Microscopy) after a metallization of the scaffolds. They will be characterized for the mechanical properties on Bose Electroforce device (Equipex FIGURES).

Cell culture: Skeletal muscle cells (C2C12 mouse muscle myoblasts, American Type Culture Collection) were maintained at 37°C in a 5% CO2 atmosphere in growth medium. The C2C12 growth medium consisted of Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. The C2C12 differentiation medium consisted of DMEM with 2% horse serum. To determine if the scaffolds were capable of allowing cell attachment and differentiation of myoblasts into myotubes, a series of cell seeding experiments were performed using C2C12 myoblasts seeded under static conditions at a density of 5.103 cells/cm2 with or without laminin coating (2µg/cm2).

3. RESULTS

The electrospun PCL 10%wt/V was characterized using a scanning electron microscopy [Figure (1)]. The mean fiber diameter was evaluated to be of 1.2µm +/- 10%. C2C12 myoblasts were cultivated under static condition, first in culture flask to optimize their differentiation into myotubes, then on the scaffold (PCL 10%). The cells were incubated in proliferative media for 5 days, then switched to differentiation media. The cell cycle stopped and the cells started to fuse together to form randomly oriented multinucleated myotubes. The unaligned myofibers did not fully matured and did not all gain full contractile function. Murine C2C12 myoblasts were cultured on culture surfaces and scaffold uncoated or coated with Laminin (2ug/ml) The levels of C2C12 cell proliferation observed on coated surfaces were significantly greater than those observed for cells grown on uncoated scaffold and culture plastic.

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4. DISCUSSION

Skeletal myogenesis is a highly organized terminal differentiation process in which the proliferating mono nucleated myoblasts differentiate and fuse to form multi nucleated myotubes. Controlling the alignment of cells is critical for any tissue engineered graft. In vivo, many cellular tissues have a very high degree of alignment which in turn enhances their functionality. In particular, for skeletal muscles, alignment of cells is extremely important in order to maximize the contractile power of the tissue. The future optimization focuses on strengthening the initial tissue’s mechanical integrity by co-culture with fibroblast cells, which contribute to the structural integrity of skeletal muscle tissue in vivo. These cells may strengthen the early tissue by depositing extracellular matrix as the tissue self-assembles. Further development of the device itself may focus on incorporation of mechanical or electrical stimulation of the tissue, as well as the ability to accurately produced by electrospinning a well aligned scaffold.

5. CONCLUSIONS

In this first step of the project, we managed to culture a myoblast cell line on electrospun PCL. Our first results strongly suggest to functionalize the support so as to favor myotube formation. The next steps will consist in submitting the biohybrid constructs to mechanical or electrical solicitations, and on developing the muscle-tendon junction itself.

6. ACKNOWLEDGEMENTS

This project is funded by LABEX MSST, in the framework of the Challenge Interfaces.

Figure 1- Electrospun scaffolds. left : unaligned fiber PCL 10% wt/v in 80/20 DCM/DMF; right : Live&Dead cell viability assays, (calcein and propidium iodide) visualization by epifluorescence microscope after 3 days of culture on the scaffold.

7. REFERENCES

[1] B.Charvet, F.Ruggiero, D. Le Guellec. The development of the myotendinous junction. A review. Muscles, Ligaments and Tendons Journal 2012; 2 (2): 53-63 [2] Arul Subramanian and Thomas F. Schilling. Tendon development and musculoskeletal assembly: emerging roles for the extracellular matrix. Development (2015) 142, 4191-4204 [3] Murugan R, Ramakrishna S. Development of nanocomposites for bone grafting. Composites SciencesTechnologies 2005; 65:2385–406. [4] Mitchell R. Ladd et al. Co-electrospun dual scaffolding system with potential for muscleetendon junction tissue engineering. Biomaterials (32) 2011:1549-1559

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CRYOGEL-BASED HEPATIC CELL CULTURE DEVICES FOR LIVER TISSUE ENGINEERING

Lilandra BOULAIS, Sidi BENCHERIF and Cécile LEGALLAIS

Université de Technologie de Compiègne UMR CNRS 7338

lilandra.boulais@utc.fr, sidi.bencherif@utc.fr , cecile.legallais@utc.fr

RESUME: The aim of this project is to improve the microfluidic liver on chip already developed in our laboratory so as to offer the cells a surrounding niche mimicking in vivo situations. Such model should allow co-cultures of native and pathological cells, for cancer research for instance. It is therefore proposed to fill biochips with cryogels presenting an interconnected macro-porosity, so as to promote the formation of clusters of different cell types.

1. INTRODUCTION

Cell environment is essential for biological responses and it has been shown that cells cultivated in 3D structures have a better metabolic functions, getting closer to the in vivo reality. In liver tissue engineering, cell aggregation and culture in dynamic conditions have been shown to enhance the viability and the functions of hepatocytes. The cell culture in dynamic conditions as already performed in the lab thanks to biochips coated with fibronectin [1,2]. To improve the current device, an alginate macroporous hydrogel is created inside the microchip to protect the cells from the shear stress and to provide them a better 3D environment. The alginate macroporous hydrogel inside the microchip will be characterized. To test the feasibility of this new microchip HepG2C3a will first be used, followed by HepaRG as a standard of liver cells. Finally co-cultures of healthy and pathological cells will be investigated.

2. METHODS

Microchip. The microchips are made of two polydimethylsilxane (PDMS) layers. Each layer has a specific microstructure with microchambers and microchannels. The two layers are bound thanks to a plasma surface treatment. The total volume of the resulting microchip is about 40µL and the surface area about 2cm². Alginate hydrogel. Purified alginate is dissolved at 1% in MES buffer and injected inside the microchip. The alginate is then cryopolymerized at -20°C overnight. After thawing, the microchip is washed with water. Cell culture. The human liver cancer cell line HepG2 C3a were used with the adapted cell medium culture. After sterilization of the alginate based microchip using an autoclave, the cells were inoculated inside the microchip at 0.3x106 cells/cm². Hydrogel characterization. The alginate hydrogels were observed at the confocal micr oscope after overnight incubation with rhodamine at 10µL/mL. Biochip study. Cells were stained with Hoescht. After 24h in static condition, a live/dead assay was performed under epifluorescent microscope.

3. RESULTS

Hydrogel characterization. The alginate hydrogel structure is homogeneous inside the microchip. A network of alginate can be observed under confocal microscopy (Fig. 1). Pressure drop measurements indicated that the biochip can be correctly perfused. Biochip study. After 24h in static condition inside the alginate based microchip, the cells create aggregates. The live/dead assay shows that most of the cells are still alive 24h after the inoculation.

4. DISCUSSION

The 3D structure of the alginate hydrogel is composed of random pores due to the freeze drying process to create them. The size of the pores is not easy to distinguish and other observation tools will be used to characterize them. Further studies should be done on the hydrogel including possible preferential paths inside the microchip. The macroporous structure of the hydrogel trap the cells inside the microchip which then form aggregates. Further studies should be done to investigate on the viability of these aggregates in static and dynamic conditions.

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5. CONCLUSIONS

The alginate hydrogel is well formed inside the microchips and random pores can be observed. The cells show a good viability after 24h inside the microchip containing the alginate hydrogel. This first step is very encouraging, and will be followed by the implementation of alginate functionalized by peptides to promote cell adhesion, and by further characterization of the biological properties of the seeded hepatic cells.

6. ACKNOWLEDGEMENTS

This project is funded by a Ministerial Fellowship, by a support of the Comité Départemental de la Ligue contre le cancer and by the PIA RHU Ilite (ANR16-RHUS-0005).

Figure 1 – Microchips. left : the microchip design ; center : alginate hydrogel with rhodamine observed with confocal microscope ; right: HepG2 C3a organization inside the alginate hydrogel after 24h in static condition.

7. REFERENCES

[1] Baudoin R, Leclerc E. Behaviour of HepG2 C3a cell cultures inside a microfluidic bioreactor. Ann Biochem Eng. 2011;53:172-181. [2] Prot JM, Leclerc E. The current status of alternatives to animal testing and predictive toxicology methods using liver microfluidic biochips. Ann Biomed Eng. 2012;40(6):1228-43.

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MULTISCALE, MULTIPHYSIC MODELING OF THE SKELETAL MUSCLE DURING ISOMETRIC CONTRACTION

Vincent CARRIOU*, Sofiane BOUDAOUD* et Jérémy LAFORET*

*: Sorbonne Université, Université de Technologie de Compiègne, CNRS UMR 7338 BMBI, 60200 Compiègne

vincent.carriou@utc.fr; sofiane.boudaoud@utc.fr; jeremy.laforet@utc.fr

RESUME: This thesis is interested in the modeling of the physical phenomena performed during a muscular isometric contraction of the skeletal muscles. The skeletal muscles can be seen as a complex System of Systems. Thus, we divided this complex system into three independents models: a neural model describing the Motor Units (MUs) recruitment, a model generating the electrical activity at the skin surface and a mechanical model describing the muscle mechanical behavior.

1. INTRODUCTION

The neuromuscular and musculoskeletal systems are complex System of Systems (SoS) that mutually interact during motion genesis. In fact, the human motion is managed by the Central Nervous System (CNS) through activation of skeletal muscle fibers. The muscle activation produces two types of contractile responses; mechanical and electrical. These two activities have different properties, nevertheless one cannot occur without the other. The mechanical outcome of skeletal muscle contraction manifests by a force production and the deformation of the muscle. This electrical response is called the Electromyogram (EMG) and can be measured in a non-invasive manner at the skin surface using surface electrodes. Considering the complex underlying interactions arising during the muscle contraction these disruptions are hardly diagnosed. For these reasons, bioreliable modeling of the skeletal muscle during contraction is one of the leading challenges in biomechanics and motor control. Bioreliable models can accurately describe the mechanisms controlling the muscle activation. In this thesis, we propose to develop a multiscale, multiphysic model of the skeletal muscle during isometric, isotonic and anisotonic non-fatiguing contraction. For this purpose, the model computes mechanical and electrical activities of the muscle considering the muscle deformation during contraction.

2. METHODS

The proposed quasi-static electro-mechanical skeletal muscle model is described on Fig. 1. This model is driven by a Motor Unit (MU) recruitment scheme describing the firing times of each MU composing the muscle. In this work, the limb is considered as a multi-layered cylinder where the muscle area can be defined using MRI technique [1].

The MU recruitment scheme considers several physiological mechanisms observed during experiences of isometric contraction of the muscle. Thus, the size principle recruitment, the rate coding and the onion skin recruitment are considered in this model [2].

From this MU recruitment, a mechanical multiscale modeling of the skeletal muscle computes the corresponding force, stiffness and deformation. This model decomposes the muscle at the MU scale where each is driven by its firing times. Once the motoneuron composing the MU is recruited, a different calcium dynamic is induced within the fiber composing the MU according to the MU type. Based on this calcium dynamic, we determine the contraction state of the MU: in contraction, in relaxation or relaxed. From this definition, this model is one of the first to be able to give a physiological meaning to the activation parameter usually used in the classical Hill type models. Then, using the moment distribution method proposed by Zahalak [3] and the assumption that all the sarcomeres are placed in serie within the fiber, we can determine the mechanical contribution of a fiber. Then, the mechanical participation of the MUs is determined by assuming that all the fibers within the MUs are placed in parallel. And finally, the muscle mechanical outcomes are computed assuming that all the MUs are placed in parallel into the muscle.

From the deformation computed using the mechanical model, the electrical model describing the muscle as a three layered (muscle, adipose and skin tissues) cylinder is updated assuming isovolumic and incompressible volume of the muscle. Thus, during isometric contraction the muscle length shortens while its thickness swells. Once this update considered, the electrical classically computes the electrical activity at the skin surface. This electrical activity is computed from the Poisson equation with quasi-stationnary condition. As for the mechanical, once the MU is recruited, all the fibers composing will generate an action potential that is generated at the neuromuscular junction and propagated along the fiber. Then, these action potentials are propagating to the skin surface undergoing the different layer filtering. Due to the complex computation, all the computation is performed in Fourier domain. Finally, the surface EMG (sEMG) signal recorded by the electrode is determined from a numerical surface integration of the electrical values under the defined electrode area.

3. RESULTS

Simulation results for both models exhibit comparable force and sEMG with experimental recording. Mechanical model has been validated with experimental force profile recorded on human paraplegic subject implanted with an electrical stimulation device [4]. On the other hand, electrical model simulates similar statistics trend on the generated sEMG signals compared to experimental sEMG signal recording [1]. Nevertheless, comparison between the static electrical model and quasi-static electro-

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mechanical model clearly exhibits the effect of the muscle deformation on the generated signals. The computed statistic trends remain the same, yet amplitude values computed on the statistics change. This comparison clearly outlines the necessity to consider the muscle deformation for simulation of sEMG signals during isometric contraction in order to provide more accurate and bioreliable simulated signals. Moreover, thanks to the modular programming used in this thesis, model personalization is quickly feasible by providing the desired parameter value in the input file. In total, the quasi-static electro-mechanical model considers 80 physiological parameters describing the skeletal muscle.

4. DISCUSSION

Even if the electro-mechanical model accurately describes the physiological phenomena arising during the skeletal muscle isometric contraction. Some physiological effects during the contraction of the skeletal muscle are not considered such as the muscle fatigue phenomenon or the muscle fibers pennation angle. Yet, thanks to the modular program developed during this thesis, upgrading the models is an easy and fast task to perform.

5. CONCLUSION

We believe this quasi-static electro-mechanical model will help researchers as well as clinicians to provide preliminary investigations that will help them to better understand and investigate muscle diseases. Moreover, a particular effort was brought on the computation time of the models. Thanks to parallel programming and optimization included in the models, global sensitivity analysis or inverse problem studies are possible. These studies can bring some new knowledge to the scientific community since some experimental investigations are genuinely hard to investigate due to the invasive procedure and the inter-variability between subjects. With this model, investigations of effect impossible to study in experimental conditions are feasible [1, 5].

Figure 1 – Quasi-static electro-mechanical diagram. From a MU recruitment pattern, the multiscale mechanical muscle model computes the mechanical muscle outcomes (force, stiffness and deformation). This deformation is used in the

electrical model to update the muscle anatomy with incompressible assumption and then, usually computes the electrical activity at the skin surface.

6. REFERENCES

[1] M. Al Harrach, V. Carriou, S. Boudaoud, J. Laforet and F. Marin. Analysis of the sEMG/Force Relationship using HD-sEMG Technique and Data Fusion: A simulation study. Comp Biol Med 2017; 83: 34-47. [2] V. Carriou, S. Boudaoud, J. Laforet and F.S. Ayachi. Fast generation model of high density surface EMG signals in a cylindrical conductor volume. Comp Biol Med 2016, 74: 54–68 [3] G. I. Zahalak. A distribution-moment approximation for kinetic theories of muscular contraction. Math Biosci 1981, 55: 89-114. [4] Mourad Benoussaad, Philippe Poignet, Mitsuhiro Hayashibe, Christine Azevedo-Coste, Charles Fattal, and David Guiraud. Experimental parameter identification of a multi-scale musculoskeletal model controlled by electrical stimulation: application to patients with spinal cord injury. Med & Biol Eng & Comp 2013, 51:617–631. [5] V. Carriou, J. Laforet, S. Boudaoud, and M. Al Harrach. Sensitivity analysis of HD-sEMG amplitude descriptors relative to grid parameter variations of a cylindrical multilayered muscle model. Biom Phy & Eng Exp 2016, 2(6).

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FACTORS INFLUENCING THE INTERACTION OF COLLAGEN WITH HYDROXYAPATITE NANOPARTICLES

Elodie COLACOa, Karim EL KIRATa, Jessem LANDOULSIa,b,c

a: Laboratoire de Biomécanique & Bioingénierie, CNRS, UMR 7338, Université de Technologie de Compiègne, BP 20529, F-60205 Compiègne Cedex, France

b:Sorbonne Universités, UPMC Univ Paris 06, F-75005 Paris, France c: CNRS, UMR 7197, Laboratoire de Réactivité de Surface, F-75005 Paris, France

elodie.colaco@utc.fr

RESUME: We investigate the mechanism by which collagen interact with hydroxyapatite nanoparticles in aqueous solutions with biological interest with the aim to designing biomimetic material with tailored properties and functions.

1. INTRODUCTION

The design of biocomposites based on collagen and hydroxyapatite has attracted much interest for different purposes such as bone tissues engineering, bone regeneration or even in drug delivery systems.1,2 Different strategies have been used to synthesize HAp-Col composites including mineralization3, PILP process4, layer-by-layer method5 and assembly with preformed nanoparticles6. The latter method is particularly investigated in this work since the studies on collagen and hydroxyapatite nanoparticles remains poorly documented. The advantages of using preformed hydroxyapatite nanoparticles are their well-defined dimensions and their high stability in a wide range of pH. Thus, this requires a better understanding of the mechanism of the interaction between collagen molecules and HAp nanoparticles in aqueous solution. While protein-nanoparticle interaction has been the subject of a vast literature,7 In this work, we study the interaction of collagen HAp crystals in solution and in adsorbed phase.

2. METHODS

The interaction between collagen and HAp nanoparticles was investigated (i) in solution (bulk) or (ii) in adsorbed phase. (i) In solution, the preparations were carried out at room temperature by mixing in ultra-pure water 1 mg/mL of HAp suspension (5-10% w/v, Alfa Aesar, Germany) and 0.1 mg/mL of bovine collagen solution (Type I 99.9%, 3.1 mg/mL) from Advanced BioMatrix (USA). The pH was adjusted by addition of HCl 0.5 M or NaOH 0.1 M. These samples were characterized by using Turbiscan technique which measures the infrared light transmitted or backscattered by the particles in solution over the whole height of the tube. To define and compare the destabilization kinetics of the different samples, we used a parameter called Turbiscan Stability Index (TSI) which is obtained from the raw data. The higher is the TSI value, the higher is the destabilization of the colloidal solution. (ii) In the adsorption process, the interaction was investigated by means of quartz crystal microbalance with dissipation monitoring (QCM-D). After injecting water to define the baseline, 0.1 mg/mL of collagen solution was injected for 2 h. Rinsing was then carried out using water for 20 min followed by the injection of 0.1 mg/mL of HAp suspension for 2 h. The samples were imaged using atomic force microscopy (AFM) on glass substrates and the chemical composition of the film was determined by X-ray photoelectron spectroscopy (XPS) by calculated the Ca/N ratio. All procedures were conducted at pH 4 and 7.4 in ultra-pure water and in NaCl 150 mM. The pH values were chosen according to the measurement of surface particles charges by using electrophoretic mobility (EPM) technique.

3. RESULTS

EPM measurements indicated that HAp nanoparticles are negatively charged from pH 3 to 12 (i.e. -0.42 µmcm/Vs at pH 3 to -3.08 µmcm/Vs at pH 12). The same trend was observed in the presence of NaCl. By contrast, collagen was shown to be negatively charged over pH values ranging from 6 to 12 and positively charged at pH values below 5. This suggests an apparent isoelectric point (iep) of the molecule between 5 and 6. In presence of NaCl, EPM results showed less negative values at pH above 6 and iep shifted to a value between 4 and 5. These measurements suggested favourable electrostatic interactions at pH 4. The system at pH 7.4 was also investigated owing to its biological interest. Turbiscan results exhibited higher TSI values at pH 4 than at pH 7.4 in presence of NaCl or not. This suggests that the colloidal system is more stable at pH 7.4 than at pH 4, probably to a higher aggregation/agglomeration in the latter pH. In the adsorbed phase, QCM-D graphs showed a significant shift of the resonance frequency (Δf) and of dissipation (ΔD) when adsorbing collagen molecules onto the surface in ultra-pure water (Figure 1A and B). This suggests a deposition of a soft and viscoelastic layer. The addition of hydroxyapatite nanoparticles led to a shift of Δf with a strong decrease of ΔD at pH 4 (Figure A) and an increase of ΔD at pH 7.4 (Figure B). At pH 4, the significant decrease of the dissipation (see red curve at about 140 min, Figure A) may be due to a reorganization of the layer and/or a loss of adsorbed compounds from the surface. In presence of NaCl, a similar trend was observed.

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The AFM image showed a surface essentially covered by collagen after adsorption of hydroxyapatite solution at pH 4 (Figure C). At pH 7.4, the formation of dense hydroxyapatite crystals deposited, specifically, onto collagen fibrils was noticed (Figure D). This findings are in agreement with XPS results which showed a Ca/N three times lower at pH 4 (Ca/N = 0.006) than at pH 7.4 (Ca/N = 0.019).

4. DISCUSSION

The above findings suggest a strong interaction of collagen with hydroxyapatite at pH 4 in solution. This leads to the formation of aggregates/agglomerates which sediment. In the adsorbed phase this strong interaction leads to the formation of a soluble Col-HAp complex which desorb from the surface. By contrast, at physiological pH, hydroxyapatite nanoparticles interact specifically with adsorbed collagen fibrils.

5. CONCLUSIONS AND PERSPECTIVES

The interaction of collagen with hydroxyapatite is significantly impacted by the pH. The effect on nanoparticles size can be also investigated. Our results provide practical information and allow the assembly of Col and HAp to be carried out under optimised conditions. This is particularly important in applications such as drug loading or cell culture. .

Figure 1 – QCM-D measurements showing frequency changes with the corresponding dissipation curve of HAp/Col system in ultra-pure water at pH 4 (A) and at pH 7.4 (B). AFM images corresponding to HAp/Col system after adsorption

process in ultra-pure water at pH 4 (C) and at pH 7.4 (D)

6. REFERENCES

[1] Lee, C. H.; Singla, A.; Lee, Y. Int. J. Pharm. 2001, 221 (1), 1–22. [2] Cui, F.-Z.; Li, Y.; Ge, J. Mater. Sci. Eng. R Rep. 2007, 57 (1–6), 1–27 [3] Zhang, W.; Liao, S. S.; Cui, F. Z. Chem. Mater. 2003, 15 (16), 3221–3226. [4] Hu, C.; Zhang, L.; Wei, M. ACS Biomater. Sci. Eng. 2015, 1 (8), 669–676 [5] Ficai, A.; Andronescu, E.; Voicu, G.; Manzu, D.; Ficai, M. Mater. Sci. Eng. C 2009, 29 (7), 2217–2220 [6] Tampieri, A.; Celotti, G.; Landi, E.; Sandri, M.; Roveri, N.; Falini, G. J. Biomed. Mater. Res. A 2003, 67 (2), 618–625 [7] Mahmoudi, M.; Lynch, I.; Ejtehadi, M. R.; Monopoli, M. P.; Bombelli, F. B.; Laurent, S. Chem. Rev. 2011, 111 (9), 5610–

5637.

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MULTI-SCALE ANALYSIS OF MULTI-LAYARED TISSUES CONSTRUCTS: INTERFACES IN THE MUSCULO-SKELETAL SYSTEM BASED ON TISSUE ENGINEERED MYOTENDINOUS AND OSTEOTENDINOUS

JUNCTIONS

Alejandro GARCIA GARCIA*,***, Fahmi BEDOUI** et Cécile Legallais*

*: UMR CNRS 7338 Biomechanics and Bioengineering **: UMR CNRS 7337 Laboratoire Roberval ***: LABEX MS2T alejandro.garcia-garcia@utc.fr ; fahmi.bedoui@utc.fr ; cecile.legallais@utc.fr

ABSTRACT: The present PhD thesis aims at simultaneously reconstructing two adjacent tissues (ex. tendon-bone) by electrospinning, ensuring cohesive interfaces during and beyond the cell culture process. For bone tissue engineering, constructs were built after designing and synthetizing materials with a honeycomb-like structure, mimicking the bone native topography. For tendon tissue engineering, materials were realised electrospinning polycaprolactone to mimic the size of collagen fibers in the native tissue.

1. INTRODUCTION

The osteotendinous/osteoligamentous junction or enthesis is designed to allow smooth transmission of strain between tendon or ligament and bone. In case of damages, traditional surgery methods such as autograft or allograft have limitations due to donor site unavailability or immunogenicity problems (1). The present PhD thesis aims at simultaneously reconstructing two adjacent tissues (ex. tendon-bone) ensuring cohesive interfaces during and beyond the cell culture process. After preliminary promising results from the first year of thesis, the tendon is reconstructed based on a polycaprolactone electrospun material mimicking the collagen structure (microscale level), seeded with mesenchymal stem cells isolated from Sprague Dawley femur. Mechanical stress field will be used as differentiation stimulator during the cell culture phase. For bone tissue engineering, trials of reconstruction of a critical size calvarial defect in an experimental model with rats have been carried out in cooperation with the CHU Amiens team (Prof B. Devauchelle, Service de Chirurgie Maxillo-faciale) by a post-doc Dr Marie Naudot. The polycaprolactone honeycomb scaffolds (2) developed during the first year with ICEPEES (Strasbourg) were inserted into a critical size calvarial defect for 1 month. RMN, Histology and Nano dentation are currently carried out to characterise the implant behaviour.

2. METHODS

Electrospinning: For bone tissue constructs we propose to elaborate 3D scaffolds showing a honeycomb like morphology with electrospun poly-ε-caprolactone (PCL) nanofibers and electrosprayed hydroxyapatite (HA) nanoparticles to mimic native tissue. The scaffold was made by alternative deposition of electrospun PCL layers (15wt% PCL (80KDa) in 60/40 V/V DCM/DMF) and electrosprayed HA (6% HA/ethanol; HA nanopowder <200nm) layers over a microstructured collector. These constructs were built in collaboration with ICEPEES (Dr. G. Schlatter) Tendon scaffolds were made by electrospinning PCL at 10% wt/V in 80/20 DCM/DMF (V/V). Material analyses: Traction tests have been realized on Bose Electroforce or Bose Biodynamic systems (Equipex FIGURES) in order to observe their mechanical properties both with or without cells. Cell culture: Bone Marrow Stromal Cells from 5 weeks Sprague Dawely rats were cultured over the electrospun materials at a cellular density of 3x105 cells/cm2 for 7 days (static or dynamic). Dynamic culture was performed after 2 days of static culture. Stretching conditions were imposed cyclically with T6 CellScale Bioreactor (CellScalle) (Fig.1A). Biological analyses: Cell morphology was assessed using Rhodamine Phalloidin (Invitrogen, USA) to selectively stain the F-actin (Red). Hoechst 33342 was added on every experiment to stain the nuclei (Bleu). Matrix deposition was followed by staining the Type I Collagen (Green).

3. RESULTS

Tendon tissue engineering: The second year of the PhD thesis was dedicated to establish the mechanical behaviour of the 10% polycaprolactone (PCL) electrospun scaffold synthetized the first year, with or without cells, to mimic tendon tissue. The objective is the implementation of a protocol that allows a monitoring of the changes of viscoelasticity in the material due to cellular behaviour. In a first time, C3H10 cell line (used in the 1st year for feasibility study) was cultured on the scaffolds under both static and dynamic conditions for up to 3 weeks. Because the very low matrix’s synthesis, it was thus decided to switch to Bone Marrow Stromal cells from Spragle Dawly rats and to follow-up the culture, so as to favour the neo-synthesis of an extracellular matrix. Cell culture over

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dynamic stretch shows an alignment of cells following the axis of deformation of the material (Fig.1B), while in static conditions cells remains randomly organized (1C). This aligned morphology reveals that BMSCs are responding to the stress and adapting their phenotype to the mechanical stimuli. In addition, collagen of type I (predominant in native tendons) seem to be aligned, mimicking the native structure of tendons.

4. DISCUSSION

Regarding the mechanical stimuli, the next step is to collect as many data as possible to validate the scientific approach. Increasing the duration of cultivation will potentially have more impact on the viscoelasticity of the material. From a biological point of culture, the proliferation, quantification of proteins and expression levels of genes of interest will be carried out to determine the most appropriate mechanical stimulation. Further experiments with the CHU Amiens are also planned to characterize the reconstruction of the bone with the polycaprolactone honeycombs implants. If the results with the PCL scaffold are positive, we will then proceed to the design of the bi-layer scaffold, in link with a post-doc to be recruited in Roberval team.

5. CONCLUSIONS

We showed that, by controlling the level of stretching in culture, we can modulate cell behaviour. We are going to prolong this study in order to investigate how stretch could modify cell's activities. The next step is to mimic the physiological range of stretch in the musculoskeletal system to better understand how stretch could influence the cell behaviour and to maximize the tissue engineered constructs.

6. ACKNOWLEDGEMENTS

This project is funded by LABEX MSST, in the framework of the Challenge Interfaces, and by the Région Picardie (Projet structurant INTIM). Universities of Strasbourg (ICEPEES), Pierre et Marie Curie (IBPS) and Leibnitz (Hannover) participated into this project.

Figure 1 – Cell culture under mechanical stimuli. A. Bioreactor T6 CellScale with 6 electrospun scaffold cultured with BMSCs. B. BMSCs in static conditions. C. BMSCs under dynamic stretching.

7. REFERENCES

[1] Murugan R, Ramakrishna S. Development of nanocomposites for bone grafting. Compos Sci Technol 2005;65:2385–406. [2] Nedjari S. et al. Electrospun Honeycomb as Nest for Controlled Osteoblast Spatial Organization. Macromol. Biosci. 2014, 14, 1580–1589.

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SYNTHESE ET CARACTERISATION D’HYDROGELS A BASE DE FIBRINE ET DE POLYETHYLENE GLYCOL POUR LE DEVELOPPEMENT DE DERMES EQUIVALENTS

Olfat GSIB*, Christophe Egles*, Sidi A. Bencherif*

*: Sorbonne Universités, Université de Technologie de Compiègne, BMBI UMR CNRS 7388, France

olfat.gsib@utc.fr

RESUME: l’objectif de cette thèse a été la mise au point ainsi que la caractérisation de réseaux interpénétrés de polymères pour l'ingénierie tissulaire cutanée associant un réseau de polymère naturel, la fibrine avec un réseau de polymère synthétique, le polyéthylène glycol. Cette thèse a permis de valider en trois étapes (in vitro, ex vivo puis in vivo) la biocompatibilité de ces nouvelles matrices, destinées à être utilisées comme support de culture 2D et pour l’augmentation de tissus mous. Cette thèse a également permis l’élaboration de nouvelles matrices macroporeuses optimisées pour la culture de cellules en 3D.

1. INTRODUCTION

Dans le domaine de la médecine régénérative appliquée à la peau, le principal challenge reste celui de développer un substitut cutané sans propriétés antigéniques, facilement manipulable, donnant des résultats cicatriciels satisfaisants tant du point de vue esthétique que fonctionnel et présentant un faible coût de production. Face à ces besoins, les hydrogels à base de fibrine constituent des candidats prometteurs : la fibrine joue un rôle central dans le processus de cicatrisation, ses précurseurs facilement isolables à partir du sang du patient en font une source autologue aisément disponible. Le principal inconvénient est qu’à concentration physiologique, les hydrogels de fibrine présentent de faibles propriétés mécaniques qui limitent leur utilisation en ingénierie tissulaire [1]. Ma thèse, a eu pour objectif de mettre au point ainsi que de caractériser de nouveaux hydrogels à base de fibrine pour le développement de dermes équivalents. Ils associent un réseau de fibrine avec un réseau de polyéthylène glycol (PEG) dans une architecture de réseaux interpénétrés de polymères (RIP). Les RIP sont une combinaison d’au moins deux réseaux polymériques réticulés simultanément. Les réseaux résultants sont alors enchevêtrés (d’où le terme « d’interpénétrés »), ce qui force la miscibilité des composants initiaux et octroie une stabilité au matériau final. Ces biomatériaux composites sont destinés à être utilisés comme aide à la cicatrisation des plaies aiguës et chroniques, la fibrine servant de support biologiquement actif provisoire aux cellules pour la formation d’une nouvelle matrice extracellulaire alors que le PEG assure le maintien mécanique de la matrice de fibrine. Cette thèse s’inscrit dans le cadre du projet ANR FibriDerm.

2. METHODES

Des RIP, associant un réseau de fibrine synthétisé par voie enzymatique avec un réseau de PEG formé par photo-polymérisation radicalaire (UV) ont été élaborés [2] et proposés par nos partenaires des laboratoires LPPI et ERRMECE de l’université de Cergy-pontoise. ] La cytotoxicité des RIP a été évaluée in vitro après mise en contact de cellules L929 avec les éluants de nos matrices (milieu complet dans lequel les matériaux ont été agités pendant 24 heures). 48 heures après ensemencement, un test MTS a été réalisé. La biocompatibilité des RIP a été par la suite évaluée ex vivo en utilisant de la culture organotypique. Des RIP ont été déposés sur différents fragments d’organes (dont la peau) isolés à partir d’embryons de poulet. Après 7 jours de culture, 3 paramètres caractérisant le comportement cellulaire ont été déterminés: prolifération, adhésion et migration cellulaires. La biocompatibilité a également été évaluée in vivo après implantation sous-cutanée des matrices acellulaires sur des souris nude. Dans une seconde étape, des fibroblastes de derme humain ont été cultivés au sein des RIP. Afin de déterminer l’impact des UV sur les cellules, celles-ci ont également été cultivées en 2D après avoir été ou non exposées aux UV dans les mêmes conditions que celles utilisées pour la polymérisation des RIP (rayonnement UV de 365 nm d’une puissance de 1,16 mW/cm2 pendant 1 heure). A différents temps de culture, la viabilité cellulaire ainsi que la morphologie cellulaire ont été analysées. Dans une troisième étape; un nouveau système macroporeux optimisé, s’affranchissant notamment de l’utilisation néfaste des UV a été mis au point au sein de notre laboratoire. Il s’agit de RIP synthétisés de manière séquentielle : un co-réseau de PEG associé à de l’albumine sérique a été synthétisé. L’albumine sérique a été ajoutée afin de promouvoir la biodégrabilité du réseau de PEG. Plusieurs lyophilisation-congélation ont ensuite abouti à la formation d’un système macroporeux. Ce système macroporeux a été finalement réhydraté par l’ajout des précurseurs de la fibrine. Différents tests de caractérisation physique (ESEM, marquage des réseaux, degré de gonflement etc.) et biologique (analyses in vitro de la viabilité, de la prolifération, de l’activité métabolique, de la morphologie cellulaire ainsi qu’in vivo après implantation des matrices sur des souris nude).

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3. RESULTATS ET DISCUSSION

Les résultats des tests MTS ont indiqué que la viabilité des cellules L929 cultivées au contact des éluants des RIP de fibrine est supérieure à 70%, seuil à partir duquel la norme ISO 10993-5 considère un matériau comme non-cytotoxique. La biocompatibilité a été confirmée ex vivo par culture organotypique. Des différences significatives de comportement cellulaire ont été observées tant en termes de migration, d’adhésion que de prolifération cellulaires. Concernant la peau, les résultats de l’étude organotypique ont montré que les cellules migraient et adhéraient davantage sur les RIP comparés aux matériaux contrôles (lamelles traitées pour la culture cellulaire). Ces résultats peuvent être corrélés aux propriétés biologiques du réseau de fibrine qui est connu pour promouvoir la migration et l’adhésion des cellules durant la cicatrisation [3]. Les résultats d’implantations des matrices acellulaires ont montré la capacité de ces matrices à conserver leur forme et leur volume même après 3 mois d’implantation. Aucun phénomène inflammatoire majeur n’a été observé après implantation. En revanche, les cellules n’ont pas colonisé les matériaux in vivo. La culture de fibroblastes au sein des matrices a montré que ces derniers sont restés ronds et n’ont pas proliférer. La durée d’exposition, combinée à une concentration d’initiateur très élevée et à la chaleur générée par la lampe ont été très certainement responsables de cet effet délétère observé sur les cellules. De plus, il est possible que les cellules aient été emprisonnées dans les RIP après réticulation, ce qui expliquerait également le fait qu’elles soient restées rondes alors que les mêmes cellules cultivées dans des gels de collagène (non exposés aux UV) sont déjà étalées après 24 heures de culture. Pour s’affranchir, entre autres, de l’utilisation néfaste des UV, de nouveaux RIP macroporeux synthétisés sans recourir à la photo-polymérisation et optimisés pour la culture de cellules en 3D ont été élaborés au sein du laboratoire. Les analyses ESEM ainsi que confocales après marquage des réseaux de fibrine et de PEG ont démontré la coexistence des deux réseaux et la création d’une macroporosité après lyophilisation (> 50 µm). La viabilité cellulaire a été montrée comme supérieure à 90 % après 48 heures de culture. La culture de fibroblastes de derme humain au sein de ces RIP séquentiels a montré que les cellules étaient capables d’adhérer, d’adopter leur morphologie caractéristique fusiforme et de proliférer au sein des matrices. L’implantation de ces matrices a également permis de démontrer qu’elles étaient bien tolérées in vivo, que les cellules étaient capables de les coloniser. La synthèse de novo de collagène et l’apparition d’une néo-vascularisation au sein des matrices implantées ont été également observées.

4. CONCLUSIONS

La biocompatibilité des RIP de Fibrine/PEG a été démontrée in vitro, ex vivo et in vivo indiquant qu’ils constituent des biomatériaux prometteurs pour l’ingénierie cutanée mais également pour d’autres applications potentielles. Toutefois, il semb le qu’ils soient davantage utilisables en tant que support 2D de culture et pour l’augmentation de tissus mous qu’en tant que scaffold pour la culture en 3D. En effet, les résultats de culture de fibroblastes de derme humain au sein des RIP étant peu concluants suggèrent que des conditions plus favorables aux cellules sont à prévoir. Un nouveau système macroporeux optimisé a été mis au point au sein du laboratoire pour s’affranchir notamment de l’utilisation délétère des UV et pour offrir un environnement propice à la culture de cellules. Nos résultats indiquent que ces nouvelles matrices sont biocompatibles et sont des supports colonisables par des fibroblastes de derme humain aussi bien in vitro qu’in vivo. Ensemble, ces résultats tendent à montrer que ces RIP séquentiels constituent des candidats prometteurs pour le développement de dermes équivalents.

5. REMERCIEMENTS

Ces travaux de thèse sont sous la direction du P. Christophe Egles et du Dr. Sidi A. Bencherif. Ils ont bénéficiés d’un financement ANR TECSAN FibriDerm et d’un soutien de la région de Picardie.

6. REFERENCES

[1] Janmey, P. A., Winer, J. P., & Weisel, J. W. Fibrin gels and their clinical and bioengineering applications. Journal of the Royal Society Interface 2009; 6:1-10. [2] Akpalo, E., Bidault, L., Boissiere, M., Vancaeyzeele, C., Fichet, O., & Larreta-Garde, V. Fibrin-polyethylene oxide interpenetrating polymer networks: new self-supported biomaterials combining the properties of both protein gel and synthetic polymer. Acta biomaterialia 2011; 7: 2418-2427. [3] Ahmed TA, Dare EV, Hincke M, Fibrin: a versatile scaffold for tissue engineering applications, Tissue Engineering Part B Reviews 2008; 14:199-215. Berger K, Sauvage LR. Late fiber deterioration in Dacron arterial grafts. Ann Surg 1981; 193: 477-491.

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TRAITEMENT TEMPS RÉEL DES SIGNAUX ACQUIS À DOMICILE

Hajer Khlaifi*, Dan Istrate* Jacques Demongeot**, Jérôme Boudy*** et Dhafer Malouche****

*: UTC

**: Université Grenoble Alpes, Grenoble

***: Télécom SudParis

****: Ecole Supérieur de la Statistique et Analyse de l’Information, Tunisie

Hajer.khlaifi@utc.fr

RESUME: Touchant environ 130 000 nouveaux patients chaque année. Toutes les 4 minutes en France, l’hospitalisation des victimes de l’AVC coûte très cher pour la sécurité sociale car l’AVC n’est pris en charge à 100% par la sécurité sociale qu’à partir du moment où il est considéré invalidant [1]. Les suites des AVC demandent de la rééducation fonctionnelle qui doit se poursuivre même après la fin de l’hospitalisation. Cela implique un suivi multidisciplinaire à l’hôpital et aussi à domicile. La possibilité de suivre le patient en dehors des séances de rééducation pourrait réduire le temps nécessaire et améliorer le taux de récupération. Dans ce contexte le projet ESwallHome se propose de développer des outils depuis les soins à l’hôpital jusqu’au retour à domicile du patient. Étant donné le nombre important des personnes dysphagiques parmi la population victime de l’AVC allant jusqu’à 76% (Zhou Z. 2009), ce sujet de thèse du projet ESwallHome participe au suivi des patients ayant souffert d’un AVC en proposant des méthodes automatiques de surveillance et d’évaluation de la rééducation fonctionnelle des troubles de la deglutition.

1. INTRODUCTION

L’objectif de la prise en charge des troubles de la déglutition est d’assurer l’alimentation sécurisée du patient en respectant au maximum la qualité de vie de la personne. C’est dans ce contexte qu’on traite des signaux acquis via des capteurs ambulatoires et facile à portés par la personne tels que le microphone pour des signaux sonores de la déglutition, le gilet Visuresp pour les signaux respiratoires pendant la prise alimentaire et l’électroglottographe pour les mesures de l’activité des cordes vocales . Le but de traitement des signaux est de pouvoir surveiller la personne à distance tout en assurant sa sécurité alimentaire et son autonomie. Dans cette première partie de thèse, on a travaillé, dans un premier temps, sur la détection automatique des signaux utiles à partir d’un signal sonore continu et dans un second temps, on a travaillé sur la classification par le biais des modèles de mélange gaussiens (GMM).

2. METHODES

L’enregistrement comprend des cycles de ventilation, des déglutitions spontanées et provoquées en ingérant différents volumes de textures homogènes différentes (salive, eau et compote), des séquences de lecture à haute voix sont aussi enregistrés suivi par des toux volontaires. Pour la détection automatique, les segments de signal utile est basé sur le calcul de l’énergie par une fenêtre glissante de 100 ms le long du signal ainsi le calcul d’un seuil pour chaque position de la fenêtre est effectué. Ainsi, tous énergie du signa l dépassant le seuil est détecté. Par la suite, un offset de 250 ms est rajouté pour chaque segment détecté à cause de la petite durée des segments de la déglutition et une phase de fusion est par la suite faite afin d’éliminer le maximum des détections partielles par rapport aux évènements de référence. Pour la classification, on utilise les modèles de mélange gaussiens pour différencier entre les sons de la déglutition et autres sons et qui a donné des résultats prometteurs. Cette partie de classification doit être validé par la première phase qui est la phase de détection automatique.

3. RESULTATS

Le taux de bonne reconnaissance pour la détection automatique est de l’ordre de 87.31 % dans les quelles 90.11 % taux de bonne reconnaissance pour la déglutition, 80 % pour la parole et 100 % pour la toux. Alors que pour la classification des

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évènements manuellement extraite a donné un taux de bonne reconnaissance de 84.57 % pour les segments de la déglutition Alors que pour les segments automatiquement détectés les résultats sont moins bons (8.33 %) et ça repose la question sur les conditions de validation des segments détectés. Le tableau suivant montre les résultats de classification avec les segments manuellement extraits :

Sounds Speech Swallowing Sounds 70.57 % 14.46 % 14.96 % Speech 25 % 75 % 0 %

Swallowing 11.17 % 4.26 % 84.57 %

4. DISCUSSION

L’algorithme de classification repose sur essentiellement sur les résultats de détection automatiques qui détecte la plupart des segments pré annotés mais toutes en des détections partielles. Mais en plus il y a détection du bruit.

5. CONCLUSIONS

Les résultats de la détection automatiques sont moins prometteurs par rapport aux résultats de la classification. Comme perspectives, on compte rentrer le rapport signal sur bruit dans l’algorithme de la détection automatique afin de réduire le nombre des fausses alarmes. Pour les GMM, on compte rajouter une étape d’adaptation qui sert à dériver le modèle des sons de déglutition en mettant à jour les paramètres bien formés lors de l’apprentissage du modèle du monde par la méthode MAP (Maximum a Posteriori)

6. REMERCIEMENTS

Je remercie toute l’équipe avec laquelle je travaille et spécifiquement Dan Istrate et Jacques Demongeot.

Figure 1 – Résultat de la détection automatique (rouge : évènement de référence, vert : détection validé, gris : détection partielle)

7. REFERENCES

[1]: https://www.news-assurances.com/fiche-pratique/comment-suis-je-pris-encharge-en-cas-davc-invalidant/016764094 Zhou Z., Accidents Vasculaires cérébraux (AVC) : Conséquences fonctionnelles et Dysphagie Associée, Université de Limoges, 22 juin 2009

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QUANTIFICATION DU TEST DE LEVER DE CHAISE POUR LA PREVENTION DES RISQUES DE CHUTE

Kevin LEPETIT*, Khalil BEN MANSOUR*, Sofiane BOUDAOUD*, Kiyoka KINUGAWA-BOURRON** et Frédéric MARIN*

*: Sorbonne Universités, Université de Technologie de Compiègne, UMR CNRS 7338, Compiègne, France

**: Hôpital Pitié-Salpêtrière – Charles Foix (AP-HP), France

kevin.lepetit@live.fr ; khalil.ben-mansour@utc.fr ; sofiane.boudaoud@utc.fr ; kiyoka.kinugawa@aphp.fr ; frederic.marin@utc.fr

RÉSUMÉ : Le sujet de ma thèse concerne l’évaluation de l’énergie mécanique au moyen de centrales inertielles. Dans ce contexte, l’estimation de l’énergie lors du test de lever de chaise s’inscrit en tant qu’application concrète. Un protocole d’évaluation de l’énergie cinétique du buste lors d’un test de lever de chaise avec une centrale inertielle placée sur le sternum a été développé. L’un des enjeux a été d’estimer la vitesse de translation du buste. Par la suite, les résultats quantifiés lors du test de lever de chaise de populations jeunes saines et âgées saines et pathologiques ont été comparées.

1. INTRODUCTION

Deux des problématiques de santé majeurs que l’Organisation Mondiale de la Santé (OMS) et l’Union Européenne (UE) ont identifiées dans le cadre de l’allongement de l’espérance de vie, notamment à travers le programme « More Years, Better Lives », sont le bien-être et le bien-vieillir. En gériatrie et plus généralement chez les personnes à risques, l’une des réponses est la prévention des chutes qui touchent 10 à 25% des personnes âgées de plus de 65 ans et dont les conséquences peuvent favoriser la perte d’indépendance et d’autonomie [1]. À l’heure actuelle, le test de timed-up and go et de lever de chaise sont souvent utilisés en clinique afin de déterminer les risques de chutes [2]. Le diagnostic s’appuie sur les observations qualitatives des cliniciens et la durée de test, ce qui n’est pas suffisant pour évaluer précisément les problèmes de stabilité. De plus, des études montrent que l’énergie cinétique, tenant compte de la morphologie de l’individu, décroit lors du test de lever de chaise lorsque les risques de chutes augmentent [3]. Ce paramètre énergétique se détermine par l’estimation de données cinématiques et de données inertiels. Alors que ces dernières peuvent être évaluées via des tables anthropométriques, les premières nécessitent une capture et une analyse quantifiée du mouvement. Actuellement, la capture du mouvement est très bien maitrisée grâce aux systèmes optoélectroniques. Malgré une utilisation rependue, le coût, la mise en place et l’encombrement de ce type de technologie la rend inutilisable pour des applications cliniques de routine. D’autre part, les capteurs magnéto -inertiels se sont largement démocratisés. La miniaturisation a notamment permis l’apparition de centrales inertielles permettant la mesure de l’accélération, de la vitesse angulaire, du champ magnétique et de l’orientation dans l’espace. Cette technologie légère et bon marché apparait comme une alternative possible pour des applications écologiques. L’objectif de cette étude a ainsi été, dans un premier temps, de proposer un protocole d’évaluation de l’énergie cinétique lors du test de lever de chaise à l’aide d’une centrale inertielle. Dans un second temps, des populations composées de jeunes sains et de de séniors sains et pathologiques ont réalisé le test de lever de chaise afin d’observer l’évolution de différents paramètres spatiotemporels et énergétiques.

2. METHODES

2.1. Évaluation de l’énergie cinétique Afin de proposer un protocole rapide à mettre en place pour le clinicien, le choix a été fait de positionner une seule centrale inertielle sur le buste qui est le segment le plus massif du haut du corps. Après avoir montré que la composante rotationnelle était négligeable, seule l’énergie cinétique de translation a été calculée lors du test de lever de chaise. La masse du buste a été obtenue grâce au modèle anthropométrique proposé par Dumas et al [4]. La vitesse de translation du centre de masse du buste a été calculée par intégration directe de l’accélération mesurée par la centrale. Pour limiter la dérive, une correction linéaire a été appliquée. De plus, l’intégration est limitée à la phase de mouvement grâce à un algorithme de seuillage inspirée du concept de zero-velocity update [5]. Pour quantifier la précision de cette méthode, un système optoélectronique composé de 18 caméras (T160, Vicon motion systems Inc., Oxford, UK) avec des marqueurs passifs positionnés sur la centrale a été utilisé simultanément et considéré comme référence. Vingt-six sujets (14 hommes (29±4 ans, 74±12kg, 175±7cm) et 12 femmes (23±3 ans, 58±8kg, 165±7cm)) ont réalisé des tests de lever de chaise à trois différentes vitesses (rapide, naturelle, lente). L’erreur quadratique moyenne normalisée (nRMSE) et le coefficient de corrélation de la méthode proposée comparée à la référence ont été calculés. 2.2. Influence de l’âge et de pathologies diverses sur les paramètres quantifiés du test de lever de chaise Trois populations d’hommes ont réalisé des tests de lever de chaise : 19 jeunes sains (25±3 ans, 75±11kg, 180±9cm), 34 séniors sains issus du programme TAGE (70±4 ans, 81±13kg, 176±6cm) et 2 séniors pathologiques (84±2 ans, 58±11kg, 163±4cm, 1 parkinsonien et 1 ayant subi des fractures aux membres inférieurs suite à une chute). Chaque sujet a réalisé 3 tests de lever de chaise sans aide des bras à vitesse choisie. Une centrale inertielle était fixée sur le buste et a permis de quantifier les paramètres spatiotemporels et énergétiques suivants : énergie cinétique moyenne du buste (Ec_moy), énergie maximum du buste (Ec_max), durée du lever de chaise (T_sts), accélération moyenne de la centrale (Acc_moy), accélération maximale de la

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centrale (Acc_max), amplitude d’inclinaison du tronc (Incl) et vitesse moyenne du CoM du buste (Vit_moy).

3. RESULTATS

Le résultat de comparaison de la méthode d’estimation de la vitesse de translation du centre de masse du buste par rapport à la référence sont présentés dans le tableau 1. L’évolution des différents paramètres étudiés en fonction des différentes populations est proposée sur la figure 1. Les valeurs ont été normalisées par rapport à la valeur maximale mesurée pour l’ensemble des sujets.

4. DISCUSSION

Les résultats permettent de valider la méthode de calcul de l’énergie cinétique du buste lors du test de lever de chaise. L’intégration directe des informations de l’accéléromètre est suffisamment précise pour des acquisitions inférieures à quelques secondes [5]. D’autre part, la quantification du test de lever de chaise montrent que l’âge a un impact limité sur les paramètres mesurés lors du lever de chaise. Cependant, on observe un changement de stratégie suite à l’apparition d’une pathologie affectant la motricité. Les premiers résultats laissent à penser que la stratégie employée dépend de la pathologie. On remarque tout de même que l’énergie cinétique moyenne et la vitesse du buste diminuent significativement pour les deux sujets pathologiques. Pour confirmer cette tendance, il serait intéressant d’augmenter le nombre de sujets pathologiques.

5. CONCLUSIONS

Les résultats sont prometteurs et de futures expérimentations vont être menées afin d’augmenter le nombre de sujets pathologiques. Six sujets féminins séniors pathologiques et deux sains ont participées à l’étude dans ce but.

6. REMERCIEMENTS

Ce travail a reçu le soutien de l’Agence Nationale de la Recherche ANR-11-IDEX-0004-02 sous l’Idex « Sorbonne Universités ». Je remercie également Adrien Letocart de m’avoir autorisé à réaliser des acquisitions avec les sujets du projet TAGE.

7. REFERENCES

[1] American Geriatrics Society, British Geriatrics Society & American Academy of Othopaedic Surgeons Panel on Falls, 2001. Guideline for the prevention of falls in older persons. Journal of the American Geriatrics Society, 49(5), 664-72

[2] Mathias S, Nayak US, Isaacs B. Balance in elderly patients: the get-up and go test. Archives of physical medicine and rehabilitation 1986; 67(6): 387-89

[3] Cameron, D. M., Bohannon, R. W., Garrett, G. E., Owen, S. V., & Cameron, D. A., 2003. Physical impairments related to kinetic energy during sit-to-stand and curb-climbing following stroke. Clinical Biomechanics, 18(4), 332–340

[4] Dumas R, Chèze L, Verriest J. Adjustements to McConville et al. and Young et al. body segment inertial parameters 2007; 40(3): 543-53

[5] Ren, M., Pan, K., Liu, Y., Guo, H., Zhang, X., & Wang, P., 2016. A Novel Pedestrian Navigation Algorithm for a Foot-Mounted Inertial-Sensor-Based System. Sensors, 16(1), 139

[6] Lepetit K, Hansen C, Ben Mansour K, Marin F. 3D location deduced by inertial measurement units: a challenging problem. CMBEE 2003; 18(1): 1984-85

Vitesse nRMSE (%) Corrélation Lente 8,57±4,75 0,98±0,03 Moyenne 5,00±2,09 0,99±0,02 Rapide 3,52±1,12 0,99±0,01

Tableau 1 – nRMSE et corrélations entre la méthode proposée et la méthode de

référence Figure 1 – Evaluation des paramètres en fonction

des différentes populations

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CAPACITES D’ADAPTATIONS TENDINEUSES A L’ENTRAINEMENT – EFFET DE L'AGE

Adrien J. LETOCART1, S. Peter MAGNUSSON2, et Jean-François GROSSET1,3

1: CNRS UMR 7338, Biomécanique et Bioingénierie, Université de Technologie de Compiègne, 60205 Compiègne cedex, France

2: Sports Medicine Research Unit, Bispebjerg Hospital, Bispebjerg bakke 23, 2400 NV, Copenhagen, Denmark

3: Université Paris 13, Sorbonne Paris Cité, UFR Santé Médecine et Biologie Humaine, 93017 Bobigny, France

adrien.letocart@utc.fr;P.Magnusson@mfi.ku.dk; jgrosset@utc.fr

RESUME: L’objectif principal de cette étude est de mieux connaitre/comprendre le processus d’adaptation tendineux et ainsi optimiser l’activité physique proposée à la personne vieillissante pour favoriser son autonomie et de reculer le seuil de dépendance.

1. INTRODUCTION

L’âge est associé à un déclin significatif des fonctions et des performances neuromusculaires, et peut aboutir à une perte de mobilité fonctionnelle et d’indépendance. Afin d’améliorer la qualité de vie (ex : diminuer les risques de chute) chez la personne âgée, ce projet a pour objectif de répondre à de nombreuses questions sur les capacités d’adaptations tendineuses avec l’âge chez l’Homme. Celles-ci étant essentielles à l’optimisation de l’activité physique. Un tendon plus raide permet une transmission de force musculaire et donc un mouvement plus rapide, améliorant ainsi le temps de réaction et diminuant les risques de chute.

2. METHODES

Ce projet repose sur une investigation multi-échelles des capacités d’adaptations tendineuses chez l’Homme afin de mettre en évidence les effets de l’entrainement en force et de l’âge. Pour ce faire, l’architecture et les propriétés mécaniques des tendons d’Achille et Patellaire sont évalués chez l’Homme pour différentes catégories d’âge (Groupe âgé : plus de 65 ans ; groupe jeune : de 20 à 30 ans). Ces groupes ont été répartis dans des programmes d’entraînements différents. L’effet de l’âge et des différents programmes d’entrainement sur les adaptations des tendons d’Achille et Patellaire ont été évalués avant/pendant/après une période d’entrainements de 12 semaines. Les paramètres suivis sont : l’architecture musculaire et tendineuse (imagerie IRM et échographique), les propriétés mécaniques des tendons d’Achille et Patellaire (sur ergomètre avec imagerie échographique), un certain nombre de paramètres sanguins (prise de sang), et l’analyse du mouvement (plateforme du CI). Chaque sujet fut évalué 4 semaines avant le début du programme d’entrainement (T-4), au début (T0) et toutes les 4 semaines durant les 12 semaines de la période d’entrainement (T4, T8 et T12) (Figure 1). L’évaluation réalisée 4 semaines avant le début du programme d’entrainement (T-4) permet d’utiliser chaque sujet comme contrôle et ainsi a permis de ne pas recruter3 groupes de sujets supplémentaires. Chaque sujet fut ensuite affecté à un programme d’entrainement de 12 semaines (à 80% ou 55% de 1 Répétition Maximale) à raison de 3 séances par semaine.

3. RESULTATS

Durant cette deuxième année de thèse, la phase expérimentale du projet TAGE pour la partie UTC a été réalisée. Durant les mois de septembre et octobre 2016 des réunions d’information ont eu lieu et ont permis d’organiser la visite médicale d’inclusion. 11 jeunes et 35 séniors ont finalement répondu à nos critères et ont ainsi pu être inclus dans le projet. A partir de mi-novembre 2016 et jusqu’à début juillet 2017 les sujets ont été répartis en 2 vagues d’entrainements (programme d’entrainement en force du membre inférieur sur des machines de musculation afin de solliciter les tendons d’Achille et Patellaire, 3 fois par semaine durant 3 mois). Ils ont réalisé tous les mois un grand nombre de tests en suivant le protocole expérimental illustré Figure 1. Ce qui a permis d’acquérir toutes les données nécessaires à l’objectif. Au final, 27 volontaires âgés et 10 jeunes sont allés au bout de l’étude. La dernière année sera donc focalisée sur le traitement de ses données et sa valorisation.

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4. DISCUSSION

Nous pouvons émettre l’hypothèse qu’après un entrainement de longue durée à haute intensité, des adaptations tendineuses seront observées [1],[2]. Il n’est toutefois pas à exclure que pour un même volume d’entrainement, un entrainement à plus faible intensité soit suffisant pour induire des adaptations significatives. Il a été montré que le taux de synthèse protéique musculaire atteint un maximum à 60% de la contraction maximale chez la personne âgée contrairement aux sujets plus jeunes qui voient leur taux croître jusqu’à 90% [3]. Nous pouvons donc nous attendre à un comportement similaire pour le tissu tendineux.

5. CONCLUSION

Une meilleure connaissance de l’ensemble des paramètres analysés dans cette étude, grâce aux différents protocoles d’entrainement, apportera une contribution majeure dans la réduction possible des frais de santé associés à l’autonomie et au bien-être des personnes âgées. Cette étude renforcera la région Picardie (nouvellement Hauts de France) comme un acteur majeur dans la recherche sur le vieillissement en bonne santé. Ce projet permettra également à l’UTC de devenir un acteur national majeur pour l’investigation des adaptations du tissu tendineux in vivo.

6. REMERCIEMENTS

Je remercie la région Nord–Pas de Calais–Picardie qui est financeur de ce projet, le Ministère de l’Enseignemant Supérieur et de la Recherche pour l’allocation doctoral, et l’UTC les conditions d’exercice et l’implication de ses différents acteurs dans ce projet. Je remercie Jean-François Grosset pour son soutien dans cette thèse. Je remercie tout particulièrement tous les volontaires sans qui, cette étude n’aurait pas eu lieu.

Figure 1 – Déroulement et contenu des différents protocoles expérimentaux de l’étude.

7. REFERENCES

[1] : Kubo K, Kanehisa H, Miyatani M, et al (2003) Effect of low-load resistance training on the tendon properties in middle-aged and elderly women. Acta Physiol Scand 178:25–32. doi: 10.1046/j.1365-201X.2003.01097.x

[2] : Grosset J-F, Breen L, Stewart CE, et al (2014) Influence of exercise intensity on training-induced tendon mechanical properties changes in older individuals. Age (Omaha) 36:9657. doi: 10.1007/s11357-014-9657-9

[3] : Kumar V, Selby A, Rankin D, et al (2009) Age-related differences in the dose-response relationship of muscle protein synthesis to resistance exercise in young and old men. J Physiol 587:211–217. doi: 10.1113/jphysiol.2008.164483

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PRECLINICAL STUDIES ON AN EXTRACORPOREAL BIOARTIFICIAL LIVER

Mattia PASQUA, Ulysse PEREIRA, Marie José FLEURY, Quentin DERMIGNY, Cécile LEGALLAIS

UTC UMR CNRS 7338, France

mattia.pasqua@utc.fr, ulysse.pereira@utc.fr, cecile.legallais@utc.fr

ABSTRACT: Acute liver failure (ALF) is a life-threatening critical illness with an incidence of fewer than 10 cases per million persons per year in the developed world. The growing gap between the number of patients on waiting lists, and the number of donor organs available, has highlighted the need for alternative therapies as a bridge to transplantation or liver regeneration. Aim of this work is thus the development of an extracorporeal liver support systems as an alternative to liver transplantation, in order to support patients with ALF

1. INTRODUCTION

In the context of extracorporeal liver support systems, two types of devices are nowadays available: artificial and bioartificial liver (BAL). If artificial liver supports are designed to replace the detoxification liver functions, BALs based on tissue engineering, are expected to fulfil the majority of liver functions (Carpentier et al., 2009). The key component of a bioartificial liver is the bioreactor, the cell-housing component. Its role is to keep hepatic cells working physiologically for prolonged period of time. In this scenario, hepatic cell microencapsulation in alginate beads has been recognized as an interesting alternative to classical cell immobilization in hollow fiber membranes. The mechanical properties still need to be tuned to offer the best microenvironment to cells. The biomass is the main pitfall of this promising treatment. Although primary human hepatocytes are still considered the gold standard, their limited availability, as well as logistical issues, hampers their use in BAL. In this context, hepatocyte-like cells (HepaRG, iPS) need to be considered as an alternative. However, the challenge is to induce/maintain the hepatic functions over time. Recently, some authors successfully extended hepatic functions cultivating cells as spheroids before encapsulation. This strategy appears a better way than the use of isolate cells. However, the majority of these studies are focused on toxicology approaches and they do not consider logistic obstacles or costs due to additional manipulation. Therefore, in this study, we propose to compare the biological response of either isolated HepaRG or spheroids encapsulated in alginate beads, to choose the best process for further use in fluidized bed BAL.

2. METHODS

Alginate microbeads formation Alginate Manucol LKX (FMC Biopolymer, Brussels, Belgium) was solubilized in extrusion media (NaCl 154 mM and HEPES 10 mM, pH 7.4) for 24 hours at concentrations of 1 %(w/v) and 1.3% (w/v). Microbeads preparation was achieved using the extrusion method adapted in our laboratory (Gautier et al., 2011). Briefly, the solutions were extruded through a 24 G nozzle with a coaxial air flow. The droplets fell into their respective gelation solution bath (NaCl 154 mM, HEPES 10 mM and CaCl2 115 mM, pH 7.4) and were allowed to gelify for 15 min at room temperature. Then the beads were washed twice with 710 HepaRG media. Compression study The beads were subjected to a classical compression assay following the method previously described by David et al. The beads are compressed at constant speed using a computer controlled device fitted with a 2 N force transducer (machine BOSE Electroforce 3230). Cell culture (amplification) HepaRG from Biopredict (Rennes, France) cells were routinely propagated in static conditions following the indications reported by the supplier. Cells were passaged every 2 weeks until passage 19, with medium (Biopredic 710 proliferation media) replenishment twice per week. After that, cells were detached and used in different conditions. 2D static culture HepaRG cells were maintained in conventional 2D culture for 14 days with 710 proliferation media. Cells encapsulated HepaRG cells were directly encapsulated as aforementioned at density of 5x106 cells by mL alginate, and maintained until 14 days in proliferation media. Spheroid encapsulated Cells were suspended in 710 Biopredic proliferation media and inoculated at a density of 5 x 106 cells/dish into glass petri dishes (ø x h = 60 x 12 mm) coated with Sigmacote® (Sigma-Aldrich). Cells were subject to continuous orbital agitation at 100 rpm with oscillation amplitude of 16 mm (SSL1 orbital shaker, Stuart) in a humidified environment at 37°C and 5% CO2. After 3 days of aggregation, spheroids were encapsulated with the same process and density, as previously descried. After encapsulation, beads are maintained until 14 days in 710 media.

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Viability and metabolic assay Viability was assessed by propidium iodide and Hoechst staining. The secretion of albumin was measured by an enzyme-linked immunosorbent assay (ELISA) (human/ rat antibody, Bethyl laboratories, Euromedex). The urea synthesis was quantified by colorimetric urea kit (QuantiChrom Urea Assay Kit, BioAssay Systems). The results were expressed as µg/days/106 cells. Xenobiotics function were also measured by EROD assay (phase I) UGT assay (Phase II) and indocyanine green assay (Phase 0 and III).

3. RESULTS

About the alginate microbeads, after different trials, we were able to set the device in order to obtain beads with a diameter of 900 µm. In order to measure the microbeads mechanical properties, we performed compression tests on alginate microbeads and for 1% (w/v) their Young’s modulus was closer to the physiological one than 1.5% (w/v) (concentration currently used in our laboratory). For the spheroids production and encapsulation, three days after continuous stirring in petri dishes, spheroids with a mean diameter of 60-70 µm were obtained. The technique, which was first applied to a hepatoma human cell line (C3A) (Figaro et al., 2015) was then successfully applied to HepaRG.

4. DISCUSSION

HepaRG cells have the ability to develop, during cell culture, from epithelial phenotype to a dual phenotype containing both hepatocyte- and biliary-like cells at confluence (figure 1A). Hepatocyte-like cells seeded at low density reverts to a more undifferentiated phenotype and biliary cells, after removal of hepatocytes, also give rise to both cell populations. The HepaRG cells are thus considered to be progenitor cells. About the alginate microbeads, the aim was to obtain beads with a mean diameter of 900 µm and a standard deviation within the 5% of the mean value, by means of the encapsulation device developed in our laboratory. About the spheroids production, three days after continuous stirring in petri dishes, spheroids compact enough to be encapsulated were obtained (figure 1B).

5. CONCLUSIONS

Some preliminary tests have been carried out but still a lot of work is in progress, regarding compilation data of the viability and metabolic assay. The aim of this work is the microencapsulation of HepaRG cells into alginate microbeads either as single cells or spheroids, in order to understand which is the best condition, in terms of cell viability and functionality, over time. The evaluation of the impact of alginate matrix stiffness on encapsulated cells and spheroids and also the influence of cells or spheroids on the mechanical properties of the alginate beads will be deeply studied. After this investigation, we will be able to understand which is the best solution for bioartificial liver purposes and in vitro characterization of encapsulate cells or spheroids, in a prototype of bioreactor, will be explored and the behaviour of cells evaluated. Finally, the goal will be the development of a bioartificial liver for small size animal: ALF will be induced in the animal model and the efficacy of the BAL will be evaluated in terms of animal survival over time. 6. ACKNOWLEDGMENTS

The project is funded by PIA-RHU ILite (ANR16-RHUS-0005).

Fig. 1: HepaRG at day 14 (A, 14 days after seeding) and spheroids at 4 days of cell culture (B)

7. REFERENCES

x Carpentier B., Gautier A., Legallais C. Artificial and bioartificial liver devices: present and future. Gut. 58,1690-1702 (2009). x Gautier A., Carpentier B., Dufresne M., et al.. Impact of alginate type and bead diameter on mass transfers and the metabolic

activities of encapsulated C3A cells in bioartificial liver applications. Eur. Cells & Materials 21, 94-106 (2011). x Figaro S., Pereira U., Rada H., et al. Development and validation of a bioartificial liver device with fluidized bed bioreactors

hosting alginate-encapsulated hepatocyte spheroids. Conf. Proc. IEEE Eng. Med. Biol. Soc.,1335-1338 (2015). x David B., Barbe I., Barthès-Biesel D., Legallais C., Mechanical properties of alginate beads hosting hepatocytes in a fluidized

bed bioreactor. Int. J. Artif. Organs. 29(8):756-63 (2006).

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INTERACTIVE AND CONNECTED REHABILITATION SYSTEMS FOR E-HEALTH

Halim TANNOUS*, Tien Tuan DAO*, Dan ISTRATE*, Aziz BENLARBI-DELAI** et Julien SARRAZIN**

*: Université de technologie de Compiègne, CNRS, UMR 7338 Biomechanics and Bioengineering (BMBI) **: Université Pierre et Marie Curie, L2E

halim.tannous@utc.fr, tien-tuan.dao@utc.fr, mircea-dan.istrate@utc.fr, aziz.benlarbi_delai@upmc.fr,

julien.sarrazin@upmc.fr.

RESUME: Serious games have been recently investigated as a complementary tool for clinical rehabilitation. In our current study, we introduce the concept of a multisensory serious game, with a system of systems approach. We will describe an energy consumption study that we are currently conducting in order to assess the ability of this system to be implemented at home. Finally, we will mention our newly developed games, and the multiple campaign trials held at the Centre “Hospitalier de Limoges” (CHU-Limoges) on pathological patients, to validate this new tool.

1. INTRODUCTION

Physical rehabilitation is a long process that requires the intervention of a specific team of experts. Usually, a patient undergoing this process will execute some sessions at the clinic, with expert supervision, and will be required later to perform some exercises at home to remain active after clinical sessions. However, there are no current solution for experts to monitor patient movements while performing exercises at home. In addition, patients drop home sessions due to the lack of motivation, and the high repetitiveness in these assigned exercises. Recently, these challenges have been the center of interest for many engineers and scientists, who used serious games as a complimentary tool for rehabilitation. In this work, we highlight the implementation of our previously developed fusion algorithm, between Inertial ShimmerTM sensors and the KinectTM camera [1], in our developed serious games [2]. The implementation was done using a system of systems approach, where the system works with the Kinect alone if no inertial sensors are available and uses the fusion algorithm when inertial sensors are available. We also studied the energy consumption of the inertial sensors, in different conditions, to try to optimize the use of our system. Finally, we describe the 2 campaign trials that were conducted at the CHU Limoges, where we tested the games on pathological patients, and the expert interface on experts.

2. METHODS

A system of systems approach presents a promising solution for applying sensor fusion to a serious game, or to any system, that requires communication with sensors. In our case, IMU sensors and Kinect sensors (which contains several sensors) are used. When these two systems are available, data fusion is performed on the data sent separately from each sensor. If the Kinect is available alone, the data sent from it will be directly used by the application. Our idea is to offer the opportunity to select joints that require more precision, in a configuration panel. This will help reduce the number of sensors on the body. For instance, if an expert requires additional precision on knee angles, the user configures the application to add only 4 sensors on each thigh and shank. This represents an intermediate solution between the high precision that we gain from using 10 to 12 sensors on the whole body, which reduces portability and comfort, and the low precision that we get from the Kinect alone. Next, we studied the battery life and current consumption of the ShimmerTM sensors. The battery life study includes 3 tests: 1) Effect of communication distance and sampling rate on battery life; 2) Effect of motion on battery life; and 3) Effect of multisensory streaming on battery life. The current consumption study includes 5 tests: 1) Current consumption until battery depletion at 51.2 Hz; 2) Effect of communication distance and sampling rate on current consumption; 3) Effect of motion on current consumption; 4) Effect of multisensory streaming on current consumption; and 5) Effect of placing sensor behind human body on current consumption. Finally, to evaluate our system we held 2 evaluation campaigns with pathological patients. The first campaign was held in June 2016 with 20 pathological patients (13 males and 7 females with a mean age of 49.75 [SD 18.68]), where the patients tried our two serious games (football and object manipulation). The second campaign was held in June 2017 with 8 Hemiplegic patients (6 males and 2 females with a mean age of 66.37 [SD 7.52]), where the patients tried the two games with and without the fusion algorithm. Four experts (all female with a mean age of 42 [SD 13.58]) tested the interface that we developed, in June 2017.

3. RESULTS

The interface to configure the game is presented in Figure 1 (Right). This interface allows adding inertial sensors to the game, using our previously described system of systems approach. The numerical results of our current consumption study will not be detailed in this abstract; however, we have not found that any external environmental factor influenced the battery life and current consumption of Shimmer sensors. Finally, our trial campaigns (Figure 1) were validated using several questionnaires and interviews with patients and experts. Some of the patient comments were the following: 1) “Interesting game and this game

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needs to be developed in bigger scales”. 2) “The games are amusing, motivational and not bad at all. It made me really move my legs.” 3) “The football scene is excellent. I am a football fan and I watch all the games”. Some of the expert comments were the following: “I do not know if we need all this information, but it is a future system that can be used for the elderly of the new generation”. 4. DISCUSSION In this work, we implemented inertial and visual sensor data fusion in a serious game. This type of implementation has never been done previously, to our knowledge, in a scientific study. The battery life study showed that the sensors are not really affected by environmental changes, and that the only constraints to the implementation of our system is a maximum number of 7 sensors, and a 5m radius between the person and the gaming system. Finally, our two evaluation campaigns were promising and showed that the patients really appreciate a serious game concept for physical rehabilitation, as they found the visual feedback and the virtual environment motivational. The experts also agreed that a similar system can be very beneficial if deployed at home, for certain types of pathological patients in certain states of rehabilitation, which could help keep an eye on their movements and progress.

5. CONCLUSIONS

In the next steps of our study, we will finalize our energy consumption study and study the possibility to predict and correct the movement of a patient, in real time, using machine-learning algorithms.

6. ACKNOWLEDGMENTS

This work was funded within the framework of E-BIOMED Chair – IUIS (Institut Universitaire d’Ingénierie en Santé). This work was carried out in the framework of the Labex MS2T, which is funded by the French Government. We would like to thank the CHU of Limoges for setting up the tests with patients and Experts.

Figure 1 – Patient trying the Object manipulation game, using inertial sensors on his left upper body (Left), the new interface for fusion between IMU and Kinect for game configuration (Right)

7. REFERENCES

[1] H. Tannous, D. Istrate, A. Benlarbi-Delai, J. Sarrazin, D. Gamet, M. C. Ho Ba Tho, and T. T. Dao, A new multi-sensor fusion scheme to improve the accuracy of knee flexion kinematics for functional rehabilitation movements, Sensors (Switzerland), 2016; 11. [2] Idriss, M., Tannous, H., Istrate, D., Perrochon, A., Salle J. Y., Ho Ba Tho, M. C. & Dao, T. T. Rehabilitation-Oriented Serious Game Development and Evaluation Guidelines for Musculoskeletal Disorders, JMIR Serious Games, 2017 (in Press). 8. PUBLICATIONS

x Journal Papers: 2 (Sensors, JMIR Serious Games) and 2 in Preparation (IRBM, CMPB). x National Conferences: 3 (JETSAN 2015, 2017, SFT 2015) x International Conferences: 3 (IEEE ICABME 2015, IEEE EMBC 2016, ICCB 2017)

1

DEFORMABILITY-BASED MICROFLUIDIC CELL SORTING: APPLICATION TO IN VITRO PLATELET PRODUCTION

Doriane VESPERINI*, Anne LE GOFF*

*: UTC Sorbonne Universités, BMBI, IFSB

doriane.vesperini@utc.fr

anne.le-goff@utc.fr

Abstract: I am working on a microfluidic method to sort micro-objects such as capsules and cells based on their mechanical properties. The sorting principle consists in confining objects in a gap between an obstacle and the wall. Downstream of the obstacle, micro-objects flow in a diffuser and are collected in 5 different outlets. Trajectories in the diffuser depend on several parameters such as micro-object size and deformability. Soft micro-objects are less deflected than softer ones. I have already proven the efficiency of my device to sort large capsules (50-80 µm in diameter). I have now downscaled the microfluidic system to adapt its geometry to cell size and I am improving the method to sort megakaryocyte (Mk) cells, progenitors of platelets.

1. INTRODUCTION

Cell mechanical properties depend on their differentiation stage or pathologies such as cancer or infections. Sorting cells according to their mechanical properties is thus particularly interesting in diagnostic applications or tissue engineering. The context of the present study is in vitro platelet production. Although researchers have demonstrated the feasibility of producing platelets in microfluidic devices, the yield is still low [1]. In the device used by our team it is partially due to the heterogeneity of the Mk suspension [2]. Sorting Mk according to their maturity stage would present a significant advantage to improve platelets production. Since we expect Mk to have different mechanical properties depending on their maturity, we develop a deformability-based sorting method. In the lab, we have designed a microfluidic device inspired by the work of Zhu et al. [3]. It consists of an obstacle at the end of a straight channel. The gap between the wall and the obstacle confines micro-objects. Last year, I studied experimentally the ability of our microfluidic system to sort microcapsules [4]. I proved the sensitivity of the device and I have now adaped it to cell-size.

2. MATERIALS AND METHODS

Non-adhesive Mk DAMI cell line is cultured in αMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells are maintained at 37°C and 5% CO2. To modify the cytoskeleton organization in DAMI cells, they are treated with 40 µM blebbistatin (BB) (Sigma Aldrich) during 1h. Untreated cells are marked during 2 hours with neutral red (1:40 v/v, prepared one day before markage). We prepare the solutions as explained below: we centrifuge cells during 5 minutes at 100 rcf and resuspend each population in 2 ml MEM medium supplemented with only BSA 1%. Then we prepare a cell suspension of 4 ml with 200 000 cell/ml, mixing in a 1:2 ratio of treated and untreated cells. During perfusion cells are agitated at 200 rpm. All experiments with cells have been done following strict safety rules and cleaning specifications. We use a solution of Dextran 10% as a viscous fluid in cell experiments. Capsules suspension preparation is detailed elsewhere [4]. All experiments are done at 20°C. The microfluidic device is produced in Polydimethylsiloxane (PDMS) using standard soft lithography techniques [5]. The device consists of an asymmetrical version of a cylindrical obstacle in a straigth channel (Figure 1B). Downtream of the obstacle, a diffuser leads to 5 outlets to collect cells. Perfusion of cells (or capsules) is performed monitoring pressure flow with a pressure controller (MFCS, Fluigent, France). In the case of cell perfusion, the outlet 1 is overpressured. The solution reservoirs are connected to the microfluidic chip using PTFE tubes of internal diameter 0.3 mm. The chip is placed on a stage of an inverted microscope (DMIL LED, Leica Microsystems GmbH, Germany). A high-speed camera (Fastcam SA3, Photron, USA) records videos.

3. RESULTS AND DISCUSSION

In previous work, we have demonstrated the efficiency of our system to sort capsules according to their size at low flow strengths and according to their stiffness at high flow strengths, using capsule populations that are heterogeneous in size and mechanical properties. The device consists of a diffuser located downstream an obstacle and several outlets to collect micro-objects. The principle is based on capsules ability to deforme around an obstacle and the position of their center of mass in the constriction. We first adapted the device geometry to the size of DAMI cells. The length of the straight channel has been reduced to avoid diffusion between the two fluids. One limit in symmetrical devices is the flow perturbations, either because not perfectly centred obstacle or clogging in the constriction between the wall and the obstacle. An asymmetrical version (Figure 1B) has been designed in order to avoid clogging. Also, it limits centering uncertainty and facilitates the fabrication. We evaluate the sorting efficiency by counting the cells passing through outlets 1 or 2 (Figure 1C).

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DAMI cell line is similar to immature Mk and is homogeneous in size (14 ± 1.5 µm). Half of the population is treated with blebbistatin (40µM), which disorganizes the cytoskeleton [6] but does not modify cell size. The other half is stained with neutral red. Tracking the two populations is not trivial; cells are flowing very fast in the microfluidic device. The high-speed camera is monochrome and not compatible with fluorescent observations. The staining I had to find should be: efficient, integrated by living cells and detectable in shade of gray. We selected neutral red, which marks lysosomes in living cells (Figure 1A). The perfusion of the cell suspension, containing treated and untreated cells, is performed with constant cell pressure Pin and dextran pressure Pext, and variable pressure in outlet 1 (PO1). I count cells reaching outlets 1 or 2. Without any overpressure (PO1 = 0), cells are massively concentrating in outlet 1. An increase in pressure PO1 deviates them towards outlet 2. Treated and untreated cells behave the same way (Figure 1D). That is similar to what we observed in capsule sorter at low flow strengths [4]. I expect that increasing the flow strengths will separate treated and untreated cells. To do so I will increase dextran viscosity, Pin and Pext.

4. CONCLUSION

Our microfluidic sorter has been downscaled and modified to be adapted to DAMI cell size. We have shown that we are now able to collect and identify 2 populations of cells at the end of the microfluidic device. We are not yet ready to sort them according to their deformability, but the system is very promising. I have 3 main tasks to performe to improve sorting. First I have to find better flow conditions to separate the two populations of cells. Second, I will check how blebbistatin treatment modifies mechanical properties. Third I would like to quantify the efficiency of the sorting method.

5. ACKNOWLEDGEMENT

I would like to thank I. BIHI who often takes care of my DAMI cells. Thanks to M. Dufresne who kindly answers my questions about cell culture. Thanks to A. LE GOFF who is always ready to give me great advices and with whom it is great to work with. And I thank L. Dubois, C. Védrine and Y. Bounab from Bioaster, with whom I have collaborated to improve cell sorting. I thank A.-V. Salsac supervising IFSB group. Finally thanks to all my colleagues in BMBI for nice scientific (or personnal) discussions.

Figure 1 – A- Picture of the two populations of cells, before perfusion. Untreated cells are coloroured with neutral red and the others are treated with 40µM blebbistatin. B- Sketch of the sorting device and a zoom in the constriction, channel depth is 27 µm. C- Zoom on the 2 first outlets were the capsules are flowing in. D- Graph representating the percentage of treated cells (circle) (respectively untreated cells (square)) collected in outlet 1 (green) or outlet 2 (red).

6. REFERENCES

[1] C.A. Di Buduo, D.L. Kaplan, A. Balduini, Transfusion Clinique et Biologique (2017) [2] A. Blin, A. Le Goff, A. Magniez, et al., Scientific Reports (2016) [3] L. Zhu, C. Rorai, D. Mitra, et al., Soft Matter (2014). [4] D. Vesperini, P. Maire, N. Munier, et al., Medical Engineering and Physics (in press) [5] G. M. Whitesides, E. Ostuni, S. Takayama, et al., Annual Review Biomedical Engineering (2001) [6] J-W. Shin, J. Swift, K. R. Spinler, et al., PNAS (2011)

1

2D TIME-RESOLVED PHASE-CONTRAST MRI MEASUREMENTS IN INTRACRANIAL ANEURYSM PHANTOM

Quan YUAN, François LANGEVIN, Bruno RAMOND and Jean-Yves GAUVRIT

quan.yuan@utc.fr, francois.langevin@utc.fr, bruno.ramond@utc.fr, jean-yves.gauvrit@chu-rennes.fr

RESUME: Aneurysm is widely believed as a fatal pathology. As one type of aneurysms, an intracranial aneurysm is notably dangerous

due to its thin vessel wall and high rate of rupture. The objective of this study in vitro is to analyze the hemodynamics in an intracranial aneurysm using a silicone phantom. In order to visualize and quantify the flow in the phantom, time-resolved 2-dimensional phase-contrast MRI sequence is applied. With post-processing of images, velocity profile and 3-dimensional path lines are obtained and analyzed.

1. INTRODUCTION

Intracranial aneurysms are frequently the cause of cerebrovascular disease. It is also a fatal malformation which is already demonstrated as a result of the reaction between vessel wall and blood flow [1], for example, subarachnoid hemorrhage (SAH), which often arises from a rupture of intracranial aneurysms, has a mortality of 50% [2]. It has a global prevalence of 2.3% according to a research based on 23 reports from year 1955 to 1996, and there is a trend that this ratio increases with age [3]. It is hence extremely important to estimate the risk of an unruptured intracranial aneurysm and study the hemodynamic states which have evidently an impact to a rupture. There are studies found in publications which are carried out by studying the hemodynamics in intracranial aneurysms computationally and experimentally. For computational works, the method of Computed Fluid Dynamics (CFD) has been widely applied to simulate and analyze the flow within an aneurysm and obtain the velocity field and wall shear stress (WSS) [4, 5]. The computational method is advantageous owing to its operability and more economic, nevertheless, calculating errors exist always during a simulation which leads to inaccuracy of the results [6]. On the other hand, for experimental works, many tests in vitro are carried out by the application of vascular phantoms. In this study, we have fabricated an elastic phantom in silicone of intracranial aneurysm. Hemodynamic tests are carried out by a 1.5-T MR scanner (Optima MR450w, GE Healthcare, Waukesha, WI, USA), in order to analyze the flow quantitively, 2D time-resolved phase-contrast technique is applied.

2. METHODES

2.1 Fabrication of phantom The aneurysm phantom consists of 2 principle parts: 1) a trunk with a diameter of 6 mm which is equal to that of internal

carotid artery [7]; 2) an aneurysm in the form of sphere with a diameter of 7.6 mm which present the mean size of ruptured aneurysm [8]. The wall thickness of trunk is 1 mm while that of the aneurysmal part is 0.7 mm according to the literature [9]. The fabrication methodology is based lost-wax casting and moulding methods. The whole fabrication process consists of 3 steps: fabrication of the internal mould, fabrication of the external mould and injection of silicone.

2.2 Imaging and analysis method The phantom is connected to the testing platform driven by an artificial ventricle which is described in our publication [10].

Hemodynamic measurements are performed using ECG-gated 2D cine phase contrast MR imaging with velocity encoding in 3 directions. The axial and sagittal images along the trunk and images of the central sections of the aneurysm are acquired. The imaging parameters were as follows: repetition time (TR)/ echo time (TE)/ flip angle: 7.0/4.4/15; field of view (FOV): 160×160 mm; acquisition matrix: 192×192; slice thickness: 4 mm; cardiac rate: 60 bpm; number of phases during one cardiac cycle: 30. Concerning the encoding velocity (VENC), it is set as 150 cm/s for the tests in trunk and 30 cm/s for those in aneurysm.

Post-processing of the phase-contrast images in this study is carried out by the aid of GT Flow software (Gyrotools LLC, Zurich, Switzerland), it is used to analyze the velocity profiles and visualize the flow in the phantom.

3. RESULTATS

3.1 Analysis of flow in the trunk Images of the whole trunk and of each axial section with an interval of 7 mm are analyzed. The velocity of all sections varies

along with the cardiac cycle, it begins to increase rapidly when systole starts (around 9th phase out of 30) and decrease after the systolic peak (around 18th phase). The maximum velocity varies from 108.47 to 116.86 cm/s.

3.2 Analysis of flow in the aneurysm The GT Flow software has been used to visualize the flow from the phase-contrast images. The 3D path-lines are generated

based on the images of aneurysms as shown in Fig. 1(a), the direction of the flow in trunk are marked as a white arrow. We can see from the figure that the flow goes in at the downstream side of the ostium of the aneurysm, it then reverses along the inner surface and goes out from the upstream side. Path lines in the form of helix are therefore presented. Remarkably, as a result of gravity, flow pattern in the aneurysm shows a small stream in the center of the aneurysm which goes down vertically, but the velocity is extremely weak (less than 10 cm/s) who shows as blue in the velocity mapping.

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The quantitative analysis of the flow in the aneurysm is also carried out, the result shows in Fig. 1(b). Compared with the velocity in the trunk, that in the aneurysm is much lower. The velocity peak comes around 25th phase, there is a delay of 7 phases with that in the trunk.

4. CONCLUSIONS

In this study, we have performed 2D phase-contrast MRI using a silicone intracranial aneurysm phantom. With acquired images, hemodynamic information of an intracranial aneurysm and parent artery (the trunk) in a silicone aneurysm model, such as 2D velocity field and 3D path-lines can be obtained by post-processing software. In conclusion, feasibility of the methodology is confirmed.

5. REMERCIEMENTS

The authors wish to thank Yvan Duhamel and Nicolas Piton of the innovation center of University of Technology of Compiegne for their helpful assistance that greatly contributed to improving the results of this study.

Figure 1 – The analysis of the flow in the aneurysm: (a) Reconstructed 3D path-lines; (b) Mean flow velocity in the aneurysm during a cardiac cycle.

6. REFERENCES

[1] Rudin, S., Wang, Z., Kyprianou, I., Hoffmann, K. R., Wu, Y., Meng, H., Guterman, L. R., Nemes, B., Bednarek, D. R., Dmochowski, J. and Hopkins, L. N. Measurement of flow modification in phantom aneurysm model: comparison of coils and a longitudinally and axially asymmetric stent--initial findings. Radiology, 231, 1 (Apr 2004), 272-276. [2] Hop, J. W., Rinkel, G. J. E., Algra, A. and van Gijn, J. Case-Fatality Rates and Functional Outcome After Subarachnoid Hemorrhage: A Systematic Review. Stroke, 28, 3 (March 1, 1997 1997), 660-664. [3] Rinkel, G. J., Djibuti, M., Algra, A. and van Gijn, J. Prevalence and risk of rupture of intracranial aneurysms: a systematic review. Stroke, 29, 1 (Jan 1998), 251-256. [4] Shojima, M., Oshima, M., Takagi, K., Torii, R., Hayakawa, M., Katada, K., Morita, A. and Kirino, T. Magnitude and role of wall shear stress on cerebral aneurysm: computational fluid dynamic study of 20 middle cerebral artery aneurysms. Stroke, 35, 11 (Nov 2004), 2500-2505. [5] Shojima, M., Oshima, M., Takagi, K., Torii, R., Nagata, K., Shirouzu, I., Morita, A. and Kirino, T. Role of the bloodstream impacting force and the local pressure elevation in the rupture of cerebral aneurysms. Stroke, 36, 9 (Sep 2005), 1933-1938. [6] Lee, B.-K. Computational fluid dynamics in cardiovascular disease. Korean circulation journal, 41, 8 2011), 423-430. [7] Williams, M. A. and Nicolaides, A. N. Predicting the normal dimensions of the internal and external carotid arteries from the diameter of the common carotid. European Journal of Vascular Surgery, 1, 2 (1987/04/01 1987), 91-96. [8] Ohashi, Y., Horikoshi, T., Sugita, M., Yagishita, T. and Nukui, H. Size of cerebral aneurysms and related factors in patients with subarachnoid hemorrhage. Surgical neurology, 61, 3 (3// 2004), 239-245. [9] Thomas, J. B., Rutt, B. K., Ladak, H. M. and Steinman, D. A. Effect of black blood MR image quality on vessel wall segmentation. Magnetic resonance in medicine, 46, 2 (Aug 2001), 299-304. [10] Yuan, Q., Zalc, V., Ramond, B. and Langevin, F. A newly designed MR-compatible guidewire for intracranial aneurysmal interventional therapy: Initial researchs and results. City, 2015.

(a) (b)

Predictive models for induced pluripotent stem cells susceptibility to xenobiotics.

Elias ZGHEIB* and Frédéric BOIS**

*: Université de Technologie de Compiègne (UTC), Compiègne

**: Institut National de l’Environnement Industriel et des Risques (INERIS), Verneuil-en-Halatte

elias.zgheib@utc.fr, frederic.bois@ineris.fr

ABSTRACT: Within the works for the European program “StemBANCC”, this thesis focuses on the development of mathematical models for the main toxicity pathways (especially the Nrf2 pathway for control of oxidative stress) using induced pluripotent stem cells. The main achievement of this thesis was to create a new “systems biology” model of the transcriptional regulations of the glutathione pathway by the Nrf2–Keap1 signaling cascade. This model was built of 2 separate models of Nrf2 and Glutathione that were improved in our lab and then calibrated to fit data on exposure ofrenal TPTEC/TERT1 cells to Cyclosporine A (CsA).

INTRODUCTION

Oxidative stress, linked to the production of reactive oxygen species (ROS) for example, is one of the main aspects of toxicity studied by our team [1]. Oxidative stress is a major factor in the development of chemical induced injury and some chronic diseases (e.g., cancer and Parkinson's disease) [2]. Glutathione is an antioxidant protecting cells against these ROS. Such detoxification processes extensively consumes glutathione. Sufficient glutathione concentrations are maintained by a few enzymes whose expressions are partly regulated by the Nrf2–Keap1 signaling system through specific transcriptional mechanisms. Glutathione depletion makes cells more susceptible to oxidative stress that may damage DNA or impair cell viability [3]. Nrf2 exists in an inactive, cytoplasm-localized state, as a consequence of binding to the cytoskeleton-associated Kelch-like-ECH-associated protein 1 (Keap1) which facilitates Nrf2 degradation. Upon oxidative stress, the cytoplasmic retention mechanism is inactivated, and Nrf2 can migrate to the nucleus [4], where it activates a set of target genes implicated in xenobiotics' metabolism and transport, and ROS scavenging by glutathione [5].

This thesis is a part of the European Innovative Medicine Initiative research program “StemBANCC” (www.stembancc.org) that aims at developing a biobank of induced pluripotent stem (iPS) cells, and at using them for therapeutic or drug safety research. In more specific way, our work aims to develop a mathematical model that characterizes the Nrf2 pathway behavior in pluripotent stem cells facing exposures at different concentrations, frequencies and degrees of mixture complexity.

METHODS

In 2012, Geenen et al [6] proposed a systems biology model of glutathione synthesis inspired by the work of Reed et al [7]. Geenen has simplified folate cycles, added 2 biomarkers (5-oxoproline and ophthalmic acid) and adapted the model to describe paracetamol metabolism. The only changes we made to Geenen’s model were the definitive suppression of the folate cycle and of paracetamol metabolism (Figure 1).

To better understand the behavior of glutathione under the control of the Nrf2 signaling pathway, we have merged Geenen’s model with a Nrf2 pathway model developed by Zhang et al [8], improved by Hamon et al [9]. The Hamon's model equations describing cyclosporine A were not taken into consideration. To simplify that part of the model, we modeled transcription control cascades according to Hill equations, fitted by Markov-Chain-Monte-Carlo simulations in the GNU MCSim version 5.6.5. Finally, we added 2 extra genes (HMOX1 and SRXN1) which are often used as activation markers for Nrf2 pathway.

On the other hand we calibrated our model on data from the Medical University of Innsbruck of the measured oxidative stress production in RPTEC/TERT1 cells after exposure Cyclosporine A. A sensitivity analysis according to Morris’s method in order to do the data fitting.

RESULTS

The model we obtained provides a wider understanding of the glutathione pathway by covering both its transcriptional and biochemical aspects. Figure 1 shows a representation of our model: the big blue compartment represents the cell cytosol and the smaller red one its nucleus. Blue arrows show reactant(s): product(s) exchange in biochemical or transport reactions, and red arrows indicate enzymatic catalysis (diamond heads) or genes’ transcription (round heads). In the nucleus, black boxes represent genes, orange arrows indicate activation by nrf2 and red arrows activation by XAhR. Also, this model was completed by Pharmacokinetic in vitro model to understand the entry of the CsA molecules to the cytosol, their interactions and metabolisms.

After the sensitivity analysis, 37 out of the 229 parameters of our model were selected to participate to the “Markov-Chain Monte Carlo” simulations for the calibration process that is still in process. DISCUSSION

Next, we plan to improve the calibration of our model with the current data and with further data provided by our StemBANCC partners. The parameters will be optimized so that our model provides predictive time-dose response curves for oxidative stress. After that, we should be able to finely model oxidative stress in renal tubular epithelial cells following exposures to single molecules or mixtures of compounds. CONCLUSION

The ultimate goal of this model is to acquire the capacity to predict chemical stress in vivo, on the basis of in vitro data collected for example with the StemBANCC cell lines.

Figure 1 – Control of Glutathione synthesis by the Nrf2 pathway

REFERENCES

[1] Leclerc E, Hamon J, Legendre A, and Bois F. Integration of Pharmacokinetic and NRF2 System Biology Models to Describe Reactive Oxygen Species Production and Subsequent Glutathione Depletion in Liver Microfluidic Biochips after Flutamide Exposure. Toxicology in Vitro 2014; 28 (7): 1230–41. [2] Kong, Yahui, Trabucco S, and Zhang H. Oxidative Stress, Mitochondrial Dysfunction and the Mitochondria Theory of Aging. Interdisciplinary Topics in Gerontology 2014; 39:86–107. [3] He CH, Gong P, Hu B, Stewart D, Choi ME, Choi AM, and Alam J. Identification of Activating Transcription Factor 4 (ATF4) as a Nrf2-Interacting Protein. Implication for Heme Oxygenase-1 Gene Regulation. The Journal of Biological Chemistry 2001; 276 (24): 20858–65. [4] Huang HC, Nguyen T, and Pickett CB. Regulation of the Antioxidant Response Element by Protein Kinase C-Mediated Phosphorylation of NF-E2-Related Factor 2. Proceedings of the National Academy of Sciences of the United States of America 97 2000; (23): 12475–80. [5] Andrews NC, Erdjument-Bromage H, Davidson MB, Tempst P, and Orkin SH. Erythroid Transcription Factor NF-E2 Is a Haematopoietic-Specific Basic-Leucine Zipper Protein. Nature 1993; 362 (6422): 722–28. [6] Geenen, S., du Preez, F. B., Reed, M., Frederik Nijhout, H., Gerry Kenna, J., Wilson, I. D., Westerhoff, H. V., and Snoep, J. L. (2012a). A mathematical modelling approach to assessing the reliability of biomarkers of glutathione metabolism. European journal of pharmaceutical sciences, 46(4):233–243. [7] Reed et al. Theor Biol Med Model. 2008;5:8. [7] Reed, M. C., Thomas, R., Pavisic, J., James, S., Ulrich, C. M., and Nijhout, H. F. (2008). A mathematical model of glutathione metabolism. Theoretical Biology and Medical modelling, 5(8):1–16. [8] Zhang Q, Pi J, Woods CG, Andersen ME. Phase I to II cross-induction of xenobiotic metabolizing enzymes: a feedforward control mechanism for potential hormetic responses. Toxicol Appl Pharmacol. 2009;237:345–356.. [9] Hamon J, Jennings P, and Bois F. Systems Biology Modeling of Omics Data: Effect of Cyclosporine a on the Nrf2 Pathway in Human Renal Cells. BMC Systems Biology 2014; 8 (1): 76.