Tesis por compendio / [ES] Actualmente las lesiones del sistema nervioso periférico que conllevan una pérdida de continuidad de los haces axonales suelen implicar secuelas de tipo permanente. Es cierto que en el sistema nervioso periférico existe una cierta regeneración natural de los tractos axonales dañados, pero solamente cuando el espacio entre ambos extremos de la lesión es pequeño, como máximo de 5 mm. Si el espacio es mayor que esta distancia la regeneración no sucede de forma natural y se crea un neuroma traumático. Por tanto, estas lesiones largas requieren de una intervención quirúrgica para puentear la lesión, normalmente con un nervio autógrafo del propio paciente o un nervio alógrafo de un cadáver. No obstante, su uso presenta diversos inconvenientes, como la morbilidad del sitio donante donde puede ocurrir un neuroma, la necesidad de realizar una segunda cirugía, la diferencia de tamaño entre nervio receptor y donante o la necesidad de inmunosupresión en el caso de los nervios alógrafos. Por ello, la ingeniería de tejidos trabaja en el desarrollo de los conductos de guiado nervioso que incorporan estrategias para guiar topográficamente la regeneración, así como células y moléculas bioactivas. La presente tesis doctoral presenta un nuevo conducto de guiado nervioso con una aproximación multimodular para su aplicación en la regeneración de lesiones nerviosas largas (a partir de 15 mm) que hace uso de conductos tubulares huecos modulares de ácido hialurónico (HA) que contienen en su interior una estructura tubular de microfibras de ácido poliláctico (PLA). La estructura fibrilar aporta un guiado topográfico necesario para guiar el crecimiento axonal durante la regeneración a la vez que mantiene unidos los diferentes módulos de HA. Por su parte, los conductos de HA son un hidrogel que evita adherencias con el tejido circundante. A su vez, proporcionan un soporte sobre el que pueden crecer células presembradas. En concreto se ha optado por presembrar células de Schwann, las cuales son unas células gliales de soporte críticas para la regeneración del sistema nervioso periférico. Se ha observado que dichas células son capaces recubrir por completo las paredes internas de los conductos de HA formando una estructura tipo vaina, así como de recubrir las microfibras de PLA creciendo en dirección longitudinal. Los experimentos in vivo en modelo de nervio ciático de conejo han mostrado que la aproximación multimodular mejora significativamente la regeneración nerviosa gracias a proporcionar una mejor neovascularización. A su vez, gracias a las células de Schwann presembradas se ha logrado una mejora adicional de la regeneración nerviosa gracias a su efecto favorecedor del crecimiento axonal. Además, se han estudiado diferentes mejoras aplicables al conducto de guiado nervioso con el objetivo de mejorar los resultados obtenidos in vivo. Gracias a la incorporación de fibroína de seda a los conductos de HA se ha logrado mejorar sus propiedades mecánicas y biológicas. Asimismo, también se ha desarrollado un sustrato electroconductor de microfibras de PLA recubiertas con el polímero electroconductor Polipirrol gracias al cual se ha observado in vitro que es capaz de mejorar el crecimiento axonal al aplicar una estimulación eléctrica. Además, mediante un sistema de modificación génica de las células de Schwann por electrotransfección se ha logrado aumentar su secreción del factor neurotrófico derivado del cerebro (BDNF), gracias a lo cual se ha observado que se incrementa la velocidad de crecimiento axonal in vitro. / [CA] Actualment les lesions del sistema nerviós perifèric que comporten una pèrdua de continuïtat dels feixos axonals solen implicar seqüeles de tipus permanent. És cert que al sistema nerviós perifèric hi ha una certa regeneració natural dels tractes axonals danyats, però només quan l'espai entre ambdós extrems de la lesió és petit, com a màxim de 5 mm. Si l'espai és més gran que aquesta distància la regeneració no succeeix de manera natural i es crea un neuroma traumàtic. Per tant, aquestes lesions llargues requereixen una intervenció quirúrgica per pontejar la lesió, normalment amb un nervi autògraf del pacient o un nervi al·lògraf d'un cadàver. No obstant això, el seu ús presenta diversos inconvenients, com la morbilitat del lloc donant on pot ocórrer un neuroma, la necessitat de fer una segona cirurgia, la diferència de mida entre nervi receptor i donant o la necessitat d'immunosupressió en el cas dels nervis al·lògrafs . Per això, l'enginyeria de teixits treballa en el desenvolupament dels conductes de guiatge nerviós que incorporen estratègies per guiar topogràficament la regeneració, així com cèl·lules i molècules bioactives. Aquesta tesi doctoral presenta un nou conducte de guiatge nerviós amb una aproximació multimodular per a la seva aplicació en la regeneració de lesions nervioses llargues (a partir de 15 mm) que fa ús de conductes tubulars buits modulars d'àcid hialurònic (HA) que contenen al seu interior una estructura tubular de microfibres d'àcid polilàctic (PLA). L'estructura fibril·lar aporta un guiatge topogràfic necessari per guiar el creixement axonal durant la regeneració alhora que manté units els diferents mòduls d'HA. Per part seva, els conductes d'HA són un hidrogel que evita adherències amb el teixit circumdant. Alhora, proporcionen un suport sobre el qual poden créixer cèl·lules presembrades. En concret s'ha optat per presembrar cèl·lules de Schwann, les quals són unes cèl·lules glials de suport crítiques per a la regeneració del sistema nerviós perifèric. S'ha observat que aquestes cèl·lules són capaces de recobrir completament les parets internes dels conductes d'HA formant una estructura tipus beina, així com de recobrir les microfibres de PLA creixent en direcció longitudinal. Els experiments in vivo en model de nervi ciàtic de conill han mostrat que l'aproximació multimodular millora significativament la regeneració nerviosa gràcies a proporcionar una millor neovascularització. Alhora, gràcies a les cèl·lules de Schwann presembrades s'ha aconseguit una millora addicional de la regeneració nerviosa gràcies al seu efecte afavoridor del creixement axonal. A més, s'han estudiat diferents millores aplicables al conducte de guiatge nerviós per tal de millorar els resultats obtinguts in vivo. Gràcies a la incorporació de fibroïna de seda als conductes d'HA s'ha aconseguit millorar les seues propietats mecàniques i biològiques. També s'ha desenvolupat un substrat electroconductor de microfibres de PLA recobertes amb el polímer electroconductor Polipirrol gràcies al qual s'ha observat in vitro que és capaç de millorar el creixement axonal quan s'aplica una estimulació elèctrica. A més, mitjançant un sistema de modificació gènica de les cèl·lules de Schwann per electrotransfecció s'ha aconseguit augmentar la secreció del factor neurotròfic derivat del cervell (BDNF), gràcies a la qual cosa s'ha observat que s'incrementa la velocitat de creixement axonal in vitro. / [EN] Currently, lesions of the peripheral nervous system that lead to a loss of continuity of the axonal bundles usually involve permanent sequelae. It is true that in the peripheral nervous system there is some natural regeneration of damaged axonal tracts, but only when the space between the two ends of the lesion is small, at most 5 mm. If the gap is greater than this distance, regeneration does not occur naturally, and a traumatic neuroma is created. Therefore, these long injuries require surgical intervention to bridge the injury, usually with an autograph nerve from the patient or an allograph nerve from a cadaver. However, its use has various drawbacks, such as the morbidity of the donor site where a neuroma can occur, the need to perform a second surgery, the difference in size between the recipient and donor nerves, or the need for immunosuppression in the case of allograft nerves. For this reason, tissue engineering works on the development of nerve guidance conduits that incorporate strategies to topographically guide the regeneration, as well as cells and bioactive molecules. This doctoral thesis presents a new nerve guidance conduit with a multimodular approach for its application in the regeneration of long nerve lesions (from 15 mm) that makes use of modular hollow tubular conduits of hyaluronic acid (HA) that contain in their inside a tubular structure of microfibers of polylactic acid (PLA). The fibrillar structure provides the necessary topographic guidance to guide axonal growth during regeneration while keeping the different HA modules together. For their part, the HA conduits are a hydrogel that prevents adhesions with the surrounding tissue. In turn, they provide a support on which preseeded cells can grow. Specifically, it has been decided to pre-seed Schwann cells, which are glial support cells that are critical for the regeneration of the peripheral nervous system. It has been observed that these cells are capable of completely covering the inner walls of the HA conduits, forming a sheath-like structure, as well as covering the PLA microfibers by growing in a longitudinal direction. In vivo experiments in a rabbit sciatic nerve model have shown that the multimodular approach significantly improves nerve regeneration by providing better neovascularization. In turn, thanks to the pre-seeded Schwann cells, an additional improvement in nerve regeneration has been achieved thanks to its promoting effect on axonal growth. In addition, different improvements applicable to the nerve guidance conduit have been studied with the aim of improving the results obtained in vivo. Thanks to the incorporation of silk fibroin into HA conduits, their mechanical and biological properties have been improved. Likewise, an electroconductive substrate of PLA microfibers coated with the electroconductive polymer Polypyrrole has also been developed, thanks to which it has been observed in vitro that it is capable of improving axonal growth by applying electrical stimulation. In addition, by means of a gene modification system of Schwann cells by electrotransfection, it has been possible to increase their secretion of brain-derived neurotrophic factor (BDNF), thanks to which it has been observed that the speed of axonal growth is increased in vitro. / Agradezco la ayuda de los diferentes proyectos del Ministerio de Economía y Competitividad del Gobierno de España que han hecho posible la financiación de esta tesis doctoral: MAT2015-66666-C3-1-R, DPI2015-72863-EXP, AEI RTI2018-095872-B-C21-C22/ERDF y FPU16/01833 del Ministerio de Universidades del Gobierno de España, sin la cual no hubiera podido realizar esta tesis doctoral. / Gisbert Roca, F. (2022). Strategies for Guidance and Electrical and Biological Stimulation in a Neural Regeneration Device [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/189937 / Compendio

La surveillance des rejets gazeux des installations nucléaires dans l'environnement et de contrôle des dispositifs d'épuration reposent sur des mesures régulières de concentrations des contaminants en sortie de cheminées et dans les réseaux de ventilation. La répartition de la concentration peut être hétérogène au niveau du point de mesure si la distance d'établissement du mélange est insuffisante. La question se pose sur l'évaluation du positionnement des points de piquage et sur l'erreur commise par rapport à la concentration homogène en cas de non-respect de cette distance. Cette étude définit cette longueur dite de « bon mélange » à partir d'expériences menées en laboratoire. Le banc dimensionné pour ces essais a permis de reproduire des écoulements dans des conduits longs circulaire et rectangulaire, comprenant chacun un coude. Une technique de mesure optique a été développée, calibrée puis utilisée pour mesurer la distribution de la concentration d'un traceur injecté dans l'écoulement. Les résultats expérimentaux en conduit cylindrique ont validé un modèle analytique basé sur l'équation de convection-diffusion d'un traceur, et ont permis de proposer des modèles de longueur de bon mélange et de représentativité de points de prélèvement. Dans le conduit à section rectangulaire, les mesures acquises constituent une première base de données sur l'évolution de l'homogénéisation d'un traceur, dans la perspective de simulations numériques explorant des conditions plus réalistes des mesures in situ. / Monitoring of gaseous releases from nuclear installations in the environment and air cleaning efficiency measurement are based on regular measurements of concentrations of contaminants in outlet chimneys and ventilation systems. The concentration distribution may be heterogeneous at the measuring point if the distance setting of the mixing is not sufficient. The question is about the set up of the measuring point in duct and the error compared to the homogeneous concentration in case of non-compliance with this distance. This study defines the so-called "well mixing length" from laboratory experiments. The bench designed for these tests allowed to reproduce flows in long circular and rectangular ducts, each including a bend. An optical measurement technique has been developed, calibrated and used to measure the concentration distribution of a tracer injected in the flow. The experimental results in cylindrical duct have validated an analytical model based on the convection-diffusion equation of a tracer, and allowed to propose models of good mixing length and representativeness of sampling points. In rectangular duct, the acquired measures constitute a first database on the evolution of the homogenization of a tracer, in the perspective of numerical simulations exploring more realistic conditions for measurements in situ.

In comparison to the flow in a rigid channel, there is a multi-fold reduction in the transition Reynolds number for the flow in a micro channel when one of the walls is made sufficiently soft, due to a dynamical instability induced by the fluid-wall coupling. The flow after transition is characterized using Particle Image Velocimetry (PIV) in the x − y plane where x is the stream-wise direction and y is the cross-stream co-ordinate along the small dimension of the channel of height 0.2 − 0.3mm. For the two different soft walls of shear modulus 18 kPa and 2.19 kPaused here, the transition Reynolds number is about 250 and 330 respectively. The deformation of the microchannel due to the applied pressure gradient is measured in the experiments, and is used to predict the laminar mean velocity profiles for comparison with the experimental results. The mean velocity profiles in the microchannel are in quantitative agreement with those predicted for the laminar flow before transition, but are flatter near the centerline and have higher gradients at the wall after transition. The flow after transition is characterized by a mean velocity profile that is flatter at the center and steeper at the walls in comparison to that for a laminar flow. The root mean square of the stream-wise fluctuating velocity shows the characteristic sharp increase from the wall and a maximum close to the wall, as observed in turbulent flows in rigid-walled channels. However, the profile is asymmetric with a significantly higher maximum close to the soft wall in comparison to that close to the hard wall, and the Reynolds stress is found to be non-zero at the soft wall, indicating that there is a stress exerted by fluid velocity fluctuations on the wall. The turbulent energy production profile has a maximum at the soft wall, in contrast to the flow at a rigid surface where the turbulent energy production is zero at the wall (due to the zero Reynolds stress). The maximum of the root mean square of the velocity fluctuations and the Reynolds stress (divided by the fluid density) in the soft-walled microchannel for Reynolds numbers in the range 250-400, when scaled by suitable powers of the maximum velocity, are comparable to those in a rigid channel at Reynolds numbers in the range 5000-20000. The near-wall velocity profile shows no evidence of a viscous sub-layer for (yv∗/ν) as low as 2, but there is a logarithmic layer for (yv∗/ν) up to about 30, where the von Karman constants are very deferent from those for a rigid-walled channel. Here, v∗ is the friction velocity, ν is the kinematic viscosity and y is the distance from the soft surface. . The surface of the soft wall in contact with the fluid is marked with dye spots to monitor the deformation and motion along the fluid-wall interface. The measured displacement of the surface in the stream-wise direction, which is of the order of 5 − 12µm, is consistent with that calculated on the basis of linear elasticity. Low-frequency oscillations in the displacement of the surface are observed after transition in both the stream-wise and span-wise directions, indicating that the turbulent velocity fluctuations are dynamically coupled to motion in the solid. Modification of soft-wall turbulence in a micro channel due to the addition of small amounts of polymer The modification of soft-wall turbulence in a microchannel due to the addition of small amounts of polymer is experimentally studied using Particle Image Velocimetry (PIV) to measure the mean and the fluctuating velocities. The micro channels are of rectangular cross-section with height about 160 µm, width about 1.5 mm and length about 3 cm, with three walls made of hard Poly-dimethylsiloxane (PDMS) gel, and one wall made of soft PDMS gel with an elasticity modulus of about 18 kPa. A dynamical instabilty of the laminar flow due to the fluid-wall coupling, and a transition to turbulence, is observed at a Reynolds number of about 290 for the flow of pure water in the soft-walled microchannel (Verma and Kumaran, J. Fluid Mech., 727, 407-455, 2013). Solutions of polyacrylamide of molecular weight 5 × 106 and mass fraction up to 50 ppm, and of molecular weight 4 × 104 and mass fraction up to 1500 ppm, are used in the experiments. In all cases, the solutions are in the dilute limit be-low the critical concentration where the interactions between polymer molecules become important. The modification of the fluid viscosity due to addition of polymer molecules is small; the viscosity of the solutions with the highest polymer concentration exceed those for pure water by about 10% for the polymer with molecular weight 5 × 106, and by about 5% for the polymer with molecular weight 4 × 104. Two distinct types of flow modifications below and above a threshold mass fraction for the polymer, cTHRESHOLD , which is about 1 ppm for the polyacrylamide with molecular weight 5 × 106, and about 500 ppm for the polyacrylamide with molecular weight 4 × 104. As the polymer mass fraction increases up to the threshold value, there is no change in the transition Reynolds number, but there is significant turbulence attenuation the root mean square velocities in the stream wise and cross-stream directions decrease by a factor of 2, and the Reynolds stress decreases by a factor of 4 in comparison to that for pure water. When the polymer concentration increases beyond the threshold value, there is a decrease in the decrease in the transition Reynolds number by nearly one order of magnitude, and a further decrease in the intensity of the turbulent fluctuations. The lowest transition Reynolds number of about 35 for the solution of polyacrylamide with molecular weight 5 × 106 and mass fraction 50 ppm. For the polymer solutions with the highest concentrations, the fluctuating velocities in the stream wise and cross-stream direction are lower by a factor of 5, and the Reynolds stress is lower by a factor of 10, in comparison to pure water. Despite the significant turbulence attenuation, a sharp increase in the intensity of the fluctuating velocities is evident at transition for all polymer concentrations. Transitions to deferent kinds of turbulence in a channel with soft walls The flow in a rectangular channel with walls made of soft polyacrylamide gel is studied to examine the effect of soft walls on transition and turbulence. The width of the channel is much larger than the height, so that the flow can be considered approximately two-dimensional, the wall thickness is much larger than the channel height (smallest dimension), the bottom wall is fixed to a substrate and the top wall is unrestrained. The fluid velocity is measured using Particle Image Velocimetry, while the wall motion is studied by embedding beads in the soft wall, and measuring the time-variation of the displacement both parallel and perpendicular to the surface. As the Reynolds number increases, two different flow regimes are observed in sequence. The first is the ‘soft-wall turbulence’ resulting from a dynamical instability of the base flow due to the fluid-wall coupling. The flow in this case exhibits many of the features of the turbulent flow in a rigid channel, including the departure of the velocity profile from the parabolic profile, and the near-wall maxima in the stream-wise root mean square fluctuating velocity. However, there are also significant differences. The turbulence intensities, when scaled by suitable powers of the mean velocity, are much larger than those after the hard-wall laminar-turbulent transition at a Reynolds number of about 1000. The Reynolds stress profiles do not decrease to zero at the walls, indicating that the wall motion plays a role in the generation of turbulent fluctuations. There is no evidence of a viscous sub-layer close to the wall to within the experimental resolution. The mean velocity profile does satisfy a logarithmic law close to the surface within a region between 2-30 wall units from the surface, but the von Karman constants are very different from those for the hard-wall turbulence. The wall displacement measurements indicate that there is no observable motion perpendicular to the surface, but displacement fluctuations parallel to the surface are observed after transition, coinciding with the onset of velocity fluctuations in the fluid. The fluid velocity fluctuations are symmetric about the center line of the channel, and they show relatively little downstream variation after a flow development length of about 5 cm. As the Reynolds number is further increased, there is a second ‘wall flutter’ transition, which involves visible downstream traveling waves in the top (unrestrained) wall alone. Wall displacement fluctuations of low frequency (less than about 500 rad/s) are observed both parallel and perpendicular to the wall. The mean velocity profiles and turbulence intensities are asymmetric, with much larger turbulence intensities near the top wall. There is no evident logarithmic profile close to either the top or bottom wall. Fluctuations are initiated at the entrance of the test section, and the fluctuation intensities decrease with downstream distance, the fluctuation intensities first rapidly increase and then decrease as the Reynolds number is increased. For a channel with relatively small height (0.6 mm), the transition Reynolds number for the soft-wall instability is lower the hard-wall transition Reynolds number of about 1000, and the laminar flow becomes unstable to the soft-wall instability leading to soft-wall turbulence and then to wall flutter as the Reynolds number is increased. For a channel with relatively large height (1.8 mm), the transition Reynolds number for the soft-wall instability is higher than 1000, the flow first undergoes the hard-wall laminar-turbulent transition at a Reynolds number of about 1000, the turbulent flow undergoes the soft-wall transition leading to soft-wall turbulence, and then to wall flutter.

Il est communément admis que les écoulements dans les aquifères karstiques sont particulièrement concentrés dans les conduits et les fractures principaux. Dans ce travail, on cherche à décrire le comportement du flux à différentes échelles. A l'échelle locale, on considère une modélisation des écoulements en milieu continu. A cela s'ajoute à l'échelle régionale l'inclusion de drains comme éléments discrets (approche hybride). Nous avons recours pour ceci à l'utilisation d'un modèle d'écoulement défini par des équations aux dérivées partielles qui décrivent l'écoulement dans un milieu à double-porosité. La solution analytique de ce modèle nous permet d'étudier les phases de transition des flux selon une condition à la limite de type charge imposée. Ceci correspond à une perturbation hydraulique tel qu'un puits maintenu à charge constante. Afin d'inclure des conditions aux limites spécifiques (par exemple une limite imperméable) et une géométrie de domaine irrégulière, nous développons et employons un modèle numérique basée sur la méthode des éléments intégrales de frontière. Ceci permet de mettre en oeuvre l'approche hybride (milieu continu et discret) dans laquelle nous considérons les intersections entre conduits verticaux et horizontaux. Ce type de modélisation permet ainsi de considérer une échelle plus importante qu'un modèle continu seul. Par ailleurs, un modèle expérimental de laboratoire est conçu et réalisé afin d'étudier expérimentalement les phases de transition du flux issu de milieux hétérogènes. Cette approche est complémentaire de celle développée à l'aide de la solution analytique et du modèle numérique. Enfin, nous proposons le développement et les essais préliminaires d'un nouvel instrument de mesure. Il s'agit d'une sonde autonome qui a pour objectif de cartographier les conduits noyés dans les aquifères karstiques. Ainsi la géométrie des drains discrets pourra être représentée de façon plus réaliste dans le modèle numérique hybride. Les conclusions principales de ce travail sont les suivantes : à l'échelle locale et régionale, les phases de transition du flux dépendent des propriétés hydrauliques du milieu. La solution numérique permet d'étudier l'influence des géométries de domaine et de conditions aux limites complexes. Le modèle expérimental permet de reproduire qualitativement les phases de transition du flux analysées théoriquement. Enfin, la sonde permet de cartographier son parcours dans des conditions expérimentales contrôlées. Ces nouvelles méthodes contribuent à la caractérisation de l'hydrodynamique dans les aquifères karstiques.