In vivo morphometric and mechanical characterization of trabecular bone from high resolution magnetic resonance imaging

La osteoporosis es una enfermedad ósea que se manifiesta con una menor densidad ósea y el deterioro de la arquitectura del hueso esponjoso. Ambos factores aumentan la fragilidad ósea y el riesgo de sufrir fracturas óseas, especialmente en mujeres, donde existe una alta prevalencia. El diagnóstico actual de la osteoporosis se basa en la cuantificación de la densidad mineral ósea (DMO) mediante la técnica de absorciometría dual de rayos X (DXA). Sin embargo, la DMO no puede considerarse de manera aislada para la evaluación del riesgo de fractura o los efectos terapéuticos. Existen otros factores, tales como la disposición microestructural de las trabéculas y sus características que es necesario tener en cuenta para determinar la calidad del hueso y evaluar de manera más directa el riesgo de fractura. Los avances técnicos de las modalidades de imagen médica, como la tomografía computarizada multidetector (MDCT), la tomografía computarizada periférica cuantitativa (HR-pQCT) y la resonancia magnética (RM) han permitido la adquisición in vivo con resoluciones espaciales elevadas. La estructura del hueso trabecular puede observarse con un buen detalle empleando estas técnicas. En particular, el uso de los equipos de RM de 3 Teslas (T) ha permitido la adquisición con resoluciones espaciales muy altas. Además, el buen contraste entre hueso y médula que proporcionan las imágenes de RM, así como la utilización de radiaciones no ionizantes sitúan a la RM como una técnica muy adecuada para la caracterización in vivo de hueso trabecular en la enfermedad de la osteoporosis. En la presente tesis se proponen nuevos desarrollos metodológicos para la caracterización morfométrica y mecánica del hueso trabecular en tres dimensiones (3D) y se aplican a adquisiciones de RM de 3T con alta resolución espacial. El análisis morfométrico está compuesto por diferentes algoritmos diseñados para cuantificar la morfología, la complejidad, la topología y los parámetros de anisotropía del tejido trabecular. En cuanto a la caracterización mecánica, se desarrollaron nuevos métodos que permiten la simulación automatizada de la estructura del hueso trabecular en condiciones de compresión y el cálculo del módulo de elasticidad. La metodología desarrollada se ha aplicado a una población de sujetos sanos con el fin de obtener los valores de normalidad del hueso esponjoso. Los algoritmos se han aplicado también a una población de pacientes con osteoporosis con el fin de cuantificar las variaciones de los parámetros en la enfermedad y evaluar las diferencias con los resultados obtenidos en un grupo de sujetos sanos con edad similar.Los desarrollos metodológicos propuestos y las aplicaciones clínicas proporcionan resultados satisfactorios, presentando los parámetros una alta sensibilidad a variaciones de la estructura trabecular principalmente influenciadas por el sexo y el estado de enfermedad. Por otra parte, los métodos presentan elevada reproducibilidad y precisión en la cuantificación de los valores morfométricos y mecánicos. Estos resultados refuerzan el uso de los parámetros presentados como posibles biomarcadores de imagen en la enfermedad de la osteoporosis.Alberich Bayarri, Á. (2010). In vivo morphometric and mechanical characterization of trabecular bone from high resolution magnetic resonance imaging [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8981Palanci

4D imaging of heart vaso-architecture after myocardial infarction

Cardiovascular diseases remain the number one cause of death globally. There is an ongoing desire to study the distribution and structural changes of the vaso-architecture in the diseased heart in cardiovascular research groups all over the world. The ability to acquire high resolution 3D-images of the heart vasculature enables to study heart diseases more in detail and eventually obtain interesting new findings and new treatments. In this work, we introduce a pipeline for high resolution 3D-imaging of the changes in mouse heart vasculature after a myocardial infarction is produced with Single Plane Illumination Microscopy (SPIM). To achieve high resolution 3D-images, protocols for optical tissue clearing (CUBIC tissue clearing technique) were combined with vasculature labelling methods (IHC and intravenous perfused lectin), enabling the visualization for the very first time of the whole heart vasculature. We here also describe the methods used for image pre-processing of the acquired data, mainly for correction of SPIM-image artifacts and for segmentation of the structures of interest. Finally, the analysis of the changes in vasculature between healthy hearts with the different stages of chronic myocardial infarction (7, 14 and 28 days post-infarction) will provide us a tool to know how this disease affects not only to infarcted region but to the whole heart volume.Ingeniería Biomédic

Investigating the role of Rac1 in cardiovascular development

PhD ThesisRac1 is a Rho GTPase which is involved in a variety of fundamental cellular processes, such as cell proliferation, adhesion and migration. In the adult heart, Rac1 is upregulated in cardiovascular disease and is required for the development of hypertrophy through production of oxidative stress. However, its expression and function during cardiovascular development remains unclear. Rac1 gene expression was characterised during mouse cardiac development, and using Cre-loxP technology it was identified that Rac1 is essential in the cardiomycoytes within the myocardium, but not in the epicardium, for the normal development of the mouse embryonic heart. Deletion of Rac1 specifically in the cells of the myocardium, the cardiomyocytes, using the TnT-Cre transgenic mouse line, resulted in severe defects of the ventricular myocardial wall as well as disrupted cardiac outflow tract alignment. Rac1TnTCre mutants displayed disrupted formation of ventricular trabeculae early in development. This resulted in abnormalities in the mature structures that form from the trabeculae, including the compact myocardial wall and interventricular septum. As a consequence of these myocardial defects, Rac1TnTCre mutant hearts become dilated and ballooned in shape leading to malalignment of the cardiac outflow tract, but with no overall loss of cardiomyocytes. This thesis shows that Rac1 and interacting partners, Mena and Vav2, are required for the organisation and movement of cardiomyocytes in the ventricular myocardial wall during early trabeculae formation. Congenital heart defects, including those affecting the myocardium and outflow tract, are the most common form of birth abnormalities. Additionally, defects occurring in utero can predispose to heart disease in later life. Therefore, understanding the role of genes that regulate myocardial wall and outflow tract formation could facilitate the development of preventative measures and treatments for congenital heart defects and cardiovascular disease

Quantifying heart development

This thesis presents a series of papers on quantified heart development. It contains an atlas of human embryonic heart development, covering the first 8 weeks after conception. This atlas gives graphs of growth in size and volume of the various cardiac compartments. Such measures are still scarce in literature as illustrated in a review about ventricular wall development. The atlas also shows that by quantification of growth, new insights in developmental processes, such as sinus venosus incorporation can be gained. It, together with a series of ventricular wall growth curves covering foetal development, illustrates that a hypertrabeculated ventricle is the result of differential growth rather than a failure of compaction as has been presumed to underlie left ventricular non-compaction cardiomyopathy. Additionally, this thesis shows that trabecular myocardium is not necessarily weaker or ill-adapted to force generation compared to the compact wall as is assumed to be the case in aforementioned cardiomyopathy. Furthermore, quantification of atrioventricular canal growth on foetal ultrasounds lend support to the theory that aberrant atrioventricular canal development can lead to tricuspid valve agenesis. Finally, this thesis shows that there is a role for comparative anatomy, in a broader sense than just mouse and chicken, in understanding mammalian and human heart development by comparing a series of bird hearts from different species

Multiscale Quantitative Imaging of Human Femoral Heads Using X-ray Microtomography

PhDClinical diagnostic tools provide limited information on the underlying structural and mechanical properties of bone-tissue affected by degenerative and bone metabolic diseases. In-vivo bone failure studies provide limited information due to constraints such as X-ray dosage, cost and various other practicalities. In-vitro studies are thus required to enhance understanding of this phenomenon. The aims of this study were to use quantitative high-definition X-ray Micro-Tomography (XMT) to assess factors contributing to pathological and non-pathological bone failure and repair in relation to the mechanics of whole human femoral heads. XMT images of one normal and six pathological femoral heads were collected at 26 – 8.8 μm voxel resolution and evaluated to determine structural features; bone mineral concentration (BMC); and using image analysis, identify microcallus formations. In addition, in-vitro compression tests were carried out on specimens taken from regions with different anatomical loading. Bone quality was then related to the anatomical loading and BMC. Results from non-pathological tissue where used to establish a baseline for measurements of structural features. Microcallus formations where identified and used as markers to map the occurrence of bone damage. In osteoarthritic (OA) heads, the damage was found to be concentrated in localised clusters. Conversely, in the osteoporotic head damage was distributed homogeneously throughout the entire specimen. No significant difference in the BMC was observed, however there was a iii significant difference in the bone quality values between the non-pathological and pathological heads, and also between the pathologies. In-vitro mechanical testing revealed a difference in the mechanical properties of OA trabecular bone in relation to bone quality measurements but the samples exhibited no significant correlation to anatomical loading. X-ray Ultra Microscopy (XuM) at 200nm and 775nm voxel resolution was used to investigate the nano-morphology of individual trabeculae. The XuM images showed differences in bone structure and fewer osteocyte lacunae present close to fracture site. XuM also identified micro-cracks within trabeculae that were encased by microcallus formations. The application of novel quantitative high definition X-ray imaging to clinically relevant tissue at multiple length scales has provided new metrological data on the distribution of damage within pathological tissue. Insight into the vulnerability of diseased tissue to damage could ultimately lead to improved diagnosis from clinical radiographs

A Multiscale In Silico Study to Characterize the Atrial Electrical Activity of Patients With Atrial Fibrillation : A Translational Study to Guide Ablation Therapy

The atrial substrate undergoes electrical and structural remodeling during atrial fibrillation. Detailed multiscale models were used to study the effect of structural remodeling induced at the cellular and tissue levels. Simulated electrograms were used to train a machine-learning algorithm to characterize the substrate. Also, wave propagation direction was tracked from unannotated electrograms. In conclusion, in silico experiments provide insight into electrograms\u27 information of the substrate

A Multiscale in Silico Study to Characterize the Atrial Electrical Activity of Patients With Atrial Fibrillation. A Translational Study to Guide Ablation Therapy

[ES] La fibrilación auricular es la arritmia cardíaca más común. Durante la fibrilación auricular, el sustrato auricular sufre una serie de cambios o remodelados a nivel eléctrico y estructural. La remodelación eléctrica se caracteriza por la alteración de una serie de canales iónicos, lo que cambia la morfología del potential de transmembrana conocido como potencial de acción. La remodelación estructural es un proceso complejo que involucra la interacción de varios procesos de señalización, interacción celular y cambios en la matriz extracelular. Durante la remodelación estructural, los fibroblastos que abundan en el tejido cardíaco, comienzan a diferenciarse en miofibroblastos que son los encargados de mantener la estructura de la matriz extracelular depositando colágeno. Además, la señalización paracrina de los miofibroblastos afecta a los canales iónicos de los miocitos circundantes. Se utilizaron modelos computacionales muy detallados a diferentes escalas para estudiar la remodelación estructural inducida a nivel celular y tisular. Se realizó una adaptación de un modelo de fibroblastos humanos a nivel celular para reproducir la electrofisiología de los miofibroblastos durante la fibrilación auricular. Además, se evaluó la exploración de la interacción del calcio en la electrofisiología de los miofibroblastos ajustando el canal de calcio a los datos experimentales. A nivel tisular, se estudió la infiltración de miofibroblastos para cuantificar el aumento de vulnerabilidad a una arritmia cardíaca. Los miofibroblastos cambian la dinámica de la reentrada. Una baja densidad de miofibroblastos permite la propagación a través del área fibrótica y crea puntos de salida de actividad focal y roturas de ondas dentro de esta área. Además, las composiciones de fibrosis juegan un papel clave en la alteración del patrón de propagación. La alteración del patrón de propagación afecta a los electrogramas recogidos en la superficie del tejido. La morfología del electrograma se alteró dependiendo de la disposición y composición del tejido fibrótico. Se combinaron modelos detallados de tejido cardíaco con modelos realistas de los catéteres de mapeo disponibles comercialmente para comprender las señales registradas clínicamente. Se generó un modelo de ruido a partir de señales clínicas para reproducir los artefactos de señal en el modelo. Se utilizaron electrogramas de modelos de dos dominios altamente detallados para entrenar un algoritmo de aprendizaje automático para caracterizar el sustrato fibrótico auricular. Las características que cuantifican la complejidad de las señales fueron extraídas para identificar la densidad fibrótica y la transmuralidad fibrótica. Posteriormente, se generaron mapas de fibrosis utilizando el registro del paciente como prueba de concepto. El mapa de fibrosis proporciona información sobre el sustrato fibrótico sin utilizar un valor único de corte de 0,5 milivoltios. Además, utilizando la medición del flujo de información como la entropía de transferencia combinada con gráficos dirigidos, en este estudio, se siguió la dirección de propagación del frente de onda. La transferencia de entropía con gráficos dirigidos proporciona información crucial durante la electrofisiología para comprender la dinámica de propagación de ondas durante la fibrilación auricular. En conclusión, esta tesis presenta un estudio in silico multiescala que proporciona información sobre los mediadores celulares responsables de la remodelación de la matriz extracelular y su electrofisiología. Además, proporciona una configuración realista para crear datos in silico que pueden ser usados para aplicaciones clínicas y servir de soporte al tratamiento de ablación.[CA] La fibril·lació auricular és l'arrítmia cardíaca més freqüent, en la qual el substrat auricular patix una sèrie de remodelacions elèctriques i estructurals. La remodelació de tipus elèctric es caracteritza per l'alteració d'un conjunt de canals iònics que modifica la morfologia del voltatge transmembrana, conegut com a potencial d'acció. La remodelació estructural és un fenomen complex que implica la relació entre diversos processos de senyalització, interaccions cel·lulars i canvis en la matriu extracel·lular. Durant la remodelació estructural, els abundants fibroblasts presents en el teixit cardíac comencen a diferenciar-se en miofibroblasts, els quals s'encarreguen de mantenir l'estructura de la matriu extracel·lular dipositant-hi col·lagen. A més, la senyalització paracrina dels miofibroblasts amb els miòcits circumdants també afectarà els canals iònics. Es van utilitzar models computacionals molt detallats a diferents escales per estudiar la remodelació estructural induïda a nivell tissular i cel·lular. Es va fer una adaptació a nivell cel·lular d'un model de fibroblasts humans per reproduir-hi l'electrofisiologia dels miofibroblasts durant la fibril·lació auricular. A més, l'exploració de la interacció del calci amb l'electrofisiologia dels miofibroblasts va ser avaluada mitjançant l'adequació del canal de calci a les dades experimentals. A nivell tissular es va estudiar la infiltració de miofibroblasts per tal de quantificar l'augment de vulnerabilitat que això conferia per patir una arrítmia cardíaca. Els miofibroblasts canvien la dinàmica de la reentrada, i presentar-ne una baixa densitat permet la propagació a través de la zona fibròtica, tot creant punts de sortida d'activitat focal i trencaments d'ones dins d'aquesta àrea. A més, les composicions de fibrosi tenen un paper clau en l'alteració del patró de propagació, afectant els electrogrames recollits en la superfície del teixit. La morfologia dels electrogrames es va veure alterada en funció de la disposició i la composició del teixit fibròtic. Per comprendre els senyals clínicament registrats es van combinar models detallats de teixits cardíacs amb models realistes dels catèters de cartografia disponibles comercialment. Es va generar un model de soroll a partir de senyals clínics per reproduir-hi els artefactes de senyal. Es van utilitzar electrogrames de models de bidominis molt detallats per entrenar un algoritme d'aprenentatge automàtic destinat a caracteritzar el substrat fibròtic auricular. Les característiques que quantifiquen la complexitat dels senyals van ser extretes per identificar la densitat i transmuralitat fibròtica. Posteriorment, es van generar mapes de fibrosi mitjançant la gravació del pacient com a prova de concepte. El mapa de fibrosi proporciona informació sobre el substrat fibròtic sense utilitzar un sol valor de tensió de tall de 0,5 mV. A més, utilitzant la mesura del flux d'informació com l'entropia de transferència combinada amb gràfics dirigits, en aquest estudi es va fer un seguiment de la direcció de propagació de l'ona. L'entropia de transferència amb gràfics dirigits proporciona informació crucial durant l'electrofisiologia per entendre la dinàmica de propagació d'ones durant la fibril·lació auricular. En conclusió, aquesta tesi presenta un estudi multi-escala in silico que proporciona informació sobre els mediadors cel·lulars responsables de la remodelació de la matriu extracel·lular i la seva electrofisiologia. A més, proporciona una configuració realista per crear dades in silico que es poden traduir a aplicacions clíniques que puguen donar suport al tractament de l'ablació.[EN] Atrial fibrillation is the most common cardiac arrhythmia. During atrial fibrillation, the atrial substrate undergoes a series of electrical and structural remodeling. The electrical remodeling is characterized by the alteration of specific ionic channels, which changes the morphology of the transmembrane voltage known as action potential. Structural remodeling is a complex process involving the interaction of several signalling pathways, cellular interaction, and changes in the extracellular matrix. During structural remodeling, fibroblasts, abundant in the cardiac tissue, start to differentiate into myofibroblasts, which are responsible for maintaining the extracellular matrix structure by depositing collagen. Additionally, myofibroblasts paracrine signalling with surrounding myocytes will also affect ionic channels. Highly detailed computational models at different scales were used to study the effect of structural remodeling induced at the cellular and tissue levels.At the cellular level, a human fibroblast model was adapted to reproduce the myofibroblast electrophsyiology during atrial fibrillation. Additionally, the calcium handling in myofibroblast electrophysiology was assessed by fitting calcium ion channel to experimental data. At the tissue level, myofibroblasts infiltration was studied to quantify the increase of vulnerability to cardiac arrhythmia. Myofibroblasts alter the dynamics of reentry. A low density of myofibroblasts allows the propagation through the fibrotic area and creates focal activity exit points and wave breaks inside this area. Moreover, fibrosis composition plays a key role in the alteration of the propagation pattern. The alteration of the propagation pattern affects the electrograms computed at the surface of the tissue. Electrogram morphology was altered depending on the arrangement and composition of the fibrotic tissue. Detailed cardiac tissue models were combined with realistic models of the commercially available mapping catheters to understand the clinically recorded signals. A noise model from clinical signals was generated to reproduce the signal artifacts in the model. Electrograms from highly detailed bidomain models were used to train a machine learning algorithm to characterize the atrial fibrotic substrate. Features that quantify the complexity of the signals were extracted to identify fibrotic density and fibrotic transmurality. Subsequently, fibrosis maps were generated using patient recordings as a proof of concept. Fibrosis map provides information about the fibrotic substrate without using a single cut-off voltage value of 0.5 mV. Furthermore, in this study, using information theory measurements such as transfer entropy combined with directed graphs, the wave propagation direction was tracked. Transfer entropy with directed graphs provides crucial information during electrophysiology to understand wave propagation dynamics during atrial fibrillation. In conclusion, this thesis presents a multiscale in silico study atrial fibrillation mechanisms providing insight into the cellular mediators responsible for the extracellular matrix remodeling and its electrophysiology. Additionally, it provides a realistic setup to create in silico data that can be translated to clinical applications that could support ablation treatment.Sánchez Arciniegas, JP. (2021). A Multiscale in Silico Study to Characterize the Atrial Electrical Activity of Patients With Atrial Fibrillation. A Translational Study to Guide Ablation Therapy [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171456TESI

Novel cardiovascular magnetic resonance phenotyping of the myocardium

INTRODUCTION Left ventricular (LV) microstructure is unique, composed of a winding helical pattern of myocytes and rotating aggregations of myocytes called sheetlets. Hypertrophic cardiomyopathy (HCM) is a cardiovascular disease characterised by left ventricular hypertrophy (LVH), however the link between LVH and underlying microstructural aberration is poorly understood. In vivo cardiovascular diffusion tensor imaging (cDTI) is a novel cardiovascular MRI (CMR) technique, capable of characterising LV microstructural dynamics non-invasively. In vivo cDTI may therefore improve our understanding microstructural-functional relationships in health and disease. METHODS AND RESULTS The monopolar diffusion weighted stimulated echo acquisition mode (DW-STEAM) sequence was evaluated for in vivo cDTI acquisitions at 3Tesla, in healthy volunteers (HV), patients with hypertensive LVH, and HCM patients. Results were contextualised in relation to extensively explored technical limitations. cDTI parameters demonstrated good intra-centre reproducibility in HCM, and good inter-centre reproducibility in HV. In all subjects, cDTI was able to depict the winding helical pattern of myocyte orientation known from histology, and the transmural rate of change in myocyte orientation was dependent on LV size and thickness. In HV, comparison of cDTI parameters between systole and diastole revealed an increase in transmural gradient, combined with a significant re-orientation of sheetlet angle. In contrast, in HCM, myocyte gradient increased between phases, however sheetlet angulation retained a systolic-like orientation in both phases. Combined analysis with hypertensive patients revealed a proportional decrease in sheetlet mobility with increasing LVH. CONCLUSION In vivo DW-STEAM cDTI can characterise LV microstructural dynamics non-invasively. The transmural rate of change in myocyte angulation is dependent on LV size and wall thickness, however inter phase changes in myocyte orientation are unaffected by LVH. In contrast, sheetlet dynamics demonstrate increasing dysfunction, in proportion to the degree of LVH. Resolving technical limitations is key to advancing this technique, and improving the understanding of the role of microstructural abnormalities in cardiovascular disease expression.Open Acces