X-ray and neutron μCT of biomedical samples: from image acquisition to quantification

Even though the validity of x-ray computed tomography in the analysis of biomedical samples is nowadays undisputed, the more recent imaging techniques and more advanced instruments (such as synchrotrons) are still relatively unknown to many medical doctors that could benefit from them.The doctoral work presented in this thesis joins a collective effort from the imaging community to demonstrate potential applications of advanced x-ray and neutron imaging methods to preclinical medical research, with the hope of contributing to reach a “critical mass” in the medical community and in the public opinion as well.Two main lines of work are detailed, one focused on the ex vivo evaluation of corrosion processes of magnesium-based biodegradable implants for osteosynthesis, the other dedicated to the assessment of neuropathy in human gastroenteric dysmotility. The aimed endpoint was to develop pipelines, from image acquisition all the way to data quantification, that could be used by other research groups with similar questions and may inspire future interdisciplinary collaborations between medicine, natural science and engineering.In the first line of work, we have attempted to employ synchrotron-radiation micro-computed tomography (µCT) coupled with in situ loading tests to assess the mechanical properties of the bone-implant interface (Paper I). We have revealed the crucial importance of the radiation dose deposited on the sample, and that the mechanical loading geometry should be accurately determined in the planning steps of the experiment. Moving away from the mechanical testing, we have also explored a novel three-dimensional analysis of the corrosion by-products of biodegradable implants by combining x-ray µCT, neutron µCT and x-ray fluorescence mapping (Papers IV and V). The second line of work has assessed the potential of x-ray phase-contrast µCT and nano-resolution holotomography as ways to perform virtual histology of unstained peripheral and autonomic neural tissue. In full-thickness biopsies of the myenteric nervous system, qualitative and potentially quantitative differences have been shown between controls and patients affected by gastrointestinal dysmotility (Paper II). In unstained skin biopsies, the methods have failed to visualise peripheral nerves, but we could identify structural changes in the connective tissue of some patients when compared to controls and other patients (Paper III)

Interacción de los tejidos duros y blandos en implantes inmediatos y diferidos conun diseño experimental en un modelo de perro "beagle". Análisis de los cambios de volumen de los tejidos duros y blandos.

Objetivo: Estudiar, por medio de micro--‐CT y análisis de imágenes STL, los cambios en los tejidos duros y blandos utilizando implantes con una forma triangular (test) y una forma cilíndrica (control) en implantes diferidos e inmediatos. Material y Métodos: Se insertaron implantes de titanio test y control en la mandíbula de 8 perros beagle. Cada hemimandíbula recibió dos implantes colocados en crestas cicatrizadas y dos implantes inmediatos. Los implantes test y control se asignaron aleatoriamente a los alveolos post--‐ extracción y crestas cicatrizadas. Se tomaron impresiones de silicona previo a la colocación de los implantes y antes del sacrificio, que tuvo lugar cuatro semanas (T4) o doce semanas (T12) después de la colocación de los implantes. Los modelos dentales de escayola fueron escaneados ópticamente y analizados a través de un software de análisis de imagen para calcular los cambios en los contornos de los tejidos blandos. Las biopsias de tejido se procesaron para el análisis mediante micro‐CT. El contacto hueso--‐implante (BIC) y la relación del volumen del hueso al volumen total de la muestra (BV/TV) se calcularon en un volumen cilíndrico de interés (VOI). A continuación, un VOI especifico en bucal del implante fue seleccionado en todas las muestras para calcular el volumen de hueso , aire e implante y, por último , se seleccionó un tercer dentro del ultimo VOI que solo incluía el hueso bucal y el implante del VOI anterior y se realizó el mismo análisis. Resultados: Al analizar el BIC y BV/TV presentaron valores similares para test y control. Se encontró menor volumen de implante a nivel de los implantes test en todos los sitios sin embargo, 3 no se encontraron diferencias entre los implantes test y control en cuanto al volumen óseo en el VOI bucal. El análisis del VOI bucal óseo dio un porcentaje similar de aire en todas las muestras , lo que indica una composición ósea similar para todos los sitios . El análisis de los contornos de tejido blando no revelo diferencias entre los implantes test y control. Conclusiones: El presente estudio no mostro diferencias entre los implantes de forma triangular y los implantes cónicos con respecto al porcentaje de integración, al volumen del hueso bucal y a los contornos del tejido blando

Multi-modal matching of 2D images with 3D medical data

Image registration is the process of aligning images of the same object taken at different time points or with different imaging modalities with the aim to compare them in one coordinate system. Image registration is particularly important in biomedical imaging, where a multitude of imaging modalities exist. For example, images can be obtained with X-ray computed tomography (CT) which is based on the object’s X-ray beam attenuation whereas magnetic resonance imaging (MRI) underlines its local proton density. The gold standard in pathology for tissue analysis is histology. Histology, however, provides only 2D information in the selected sections of the 3D tissue. To evaluate the tissue’s 3D structure, volume imaging techniques, such as CT or MRI, are preferable. The combination of functional information from histology with 3D morphological data from CT is essential for tissue analysis. Furthermore, histology can validate anatomical features identified in CT data. Therefore, the registration of these two modalities is indispensable to provide a more complete overview of the tissue. Previously proposed algorithms for the registration of histological slides into 3D volumes usually rely on manual interactions, which is time-consuming and prone to bias. The high complexity of this type of registration originates from the large number of degrees of freedom. The goal of my thesis was to develop an automatic method for histology to 3D volume registration to master these challenges. The first stage of the developed algorithm uses a scale-invariant feature detector to find common matches between the histology slide and each tomography slice in a 3D dataset. A plane of the most likely position is then fitted into the feature point cloud using a robust model fitting algorithm. The second stage builds upon the first one and introduces fine-tuning of the slice position using normalized Mutual Information (NMI). Additionally, using previously developed 2D-2D registration techniques we find the rotation and translation of the histological slide within the plane. Moreover, the framework takes into account any potential nonlinear deformations of the histological slides that might occur during tissue preparation. The application of the algorithm to MRI data is investigated in our third work. The developed extension of the multi-modal feature detector showed promising results, however, the registration of a histological slide to the direct MRI volume remains a challenging task

Mesoscopic three-dimensional hard X-ray imaging of central and peripheral nervous system

Micro computed tomography (μCT), either by means of hard X rays from synchrotronradiation (SR) facilities, or from advanced laboratory sources, has been proven as a powerful method for the nondestructive three-dimensional visualization of biological specimens with isotropic micro- and even nanometer resolution. The established absorption-contrast modality of μCT has been sometimes associated with the need for contrast agents, whereas the more advanced phase-contrast modality has yielded superior results for biological specimens without staining. For around three decades, phase-contrast μCT has been considered between a hundred and a thousand times better than absorption-contrast μCT, based on the ratio of the imaginary and the real part of the complex refractive index that could be determined using the two modalities at desired photon energies. The results of the present study elucidate that for formalin-fixed, paraffin-embedded nervous tissues, conventional μCT delivers a much better contrast than originally expected. Related measurements were performed at a SR facility using monochromatic X rays. The photon energies were not equal for absorption- and phase-contrast measurements, but selected to obtain optimized contrast within a reasonable period of time. The choice of the photon energy, which is much smaller for absorption-contrast μCT, explains that the contrast difference between phase- and absorption-contrast μCT, indicated by the contrast-to-noise ratio of anatomical regions in the respective datasets being about two times better for phase μCT, is much smaller than reported in literature. It should be highlighted that μCT in absorption- and phase contrast are complementary methods and a combination might give additional quantitative insights into the three-dimensional images. For example, one can register the data and build a joint histogram from the common volume to segment anatomical features indistinguishable using just one imaging modality. The main relevance of such results lies in the opportunity to employ laboratory-based μCT, which are much better accessible and cost-effective than μCT at SR facilities. This approach was benchmarked on peripheral nerve reconstruction. The threedimensional visualization of regenerating nerves inside collagen scaffolds was feasible and included the automatic extraction of anatomical features to quantify the regeneration. Indeed, the characteristic parameters, revealed from the conventional μCT data, were significantly different between regenerating and control nerves. The approach including specimen preparation, data acquisition, and analysis has been useful for the investigations of the anatomical alterations in medial temporal epilepsy and the time-critical diagnosis of vasculitis prior to the standard histology

Verification and Validation of MicroCT-based Finite Element Models of Bone Tissue Biomechanics

Non-destructive 3D micro-computed tomography (microCT) based finite element (microFE) model is popular in estimating bone mechanical properties in recent decades. From a fundamental scientific perspective, as the primary function of the skeleton is mechanical in nature, a lot of related biological and physiological mechanisms are mechano-regulated that becomes evident at the tissue scale. In all these research it is essential to known with the best possible accuracy the displacements, stresses, and strains induced by given loads in the bone tissue. Correspondingly, verification and validation of the microFE model has become crucial in evaluating the quality of its predictions. Because of the complex geometry of cancellous bone tissue, only a few studies have investigated the local convergence behaviour of such models and post-yield behaviour has not been reported. Moreover, the validation of their prediction of local properties remains challenging. Recent technique of digital volume correlation (DVC) combined with microCT images can measure internal displacements and deformation of bone specimen and therefore is able to provide experimental data for validation. However, the strain error of this experimental method tends to be a lot higher (in the order of several thousand microstrains) for spatial resolutions of 10-20 µm, typical element size of microFE models. Strictly speaking no validation of strain is possible. Therefore, the goal of this thesis it to conduct a local convergence study of cancellous bone microFE models generated using three microCT-based tissue modelling methods (homogeneous tetrahedral model, homogeneous hexahedral model and heterogeneous hexahedral model); to validate these models’ prediction in terms of displacement using the novel DVC technique; and finally to compare the strain field predicted by three tissue modelling methods, in order to explore the effect of specific idealisations/simplifications on the prediction of strain

Mechanical and morphometric characterization of cancellous bone

[EN] Bone fracture is a social health problem of increasing magnitude because of its prevalence in aged population due to osteoporosis. Bone quality is often characterized by bone mineral density (BMD) measured at cancellous bone regions using dual-energy X-ray absorptiometry (DXA). However, BMD alone cannot predict several cases because not only density is important, but also microstructure plays an important role in cancellous bone strength. The mechanical properties can be used as indicators of bone integrity as a function of age, disease or treatment. Therefore, cancellous bone fracture characterization and its relationship to microstructure has not been completely solved in the literature and is relevant to improve fracture prediction. In this thesis, we aim at characterizing cancellous bone morphometry and mechanical behavior. Morphometry is estimated through the analysis of micro-computed tomography (micro-CT) images of vertebral cancellous bone specimens. With regards to the mechanical behavior, we calculate elastic, yield and failure properties at the apparent and tissue levels. To determine them, we followed different approaches: compression tests, finite element models and micro-CT phantoms. We have developed finite element models that reproduce the elastic and failure response of cancellous bone under compression conditions. We modeled failure as a combination of continuum damage mechanics and the element deletion technique. The numerical models permitted to estimate elastic and failure properties. Failure properties were consistent with results reported in the literature. Specifically, our results revealed that yield strain is relatively constant (0.7 %) over a range of apparent densities, while failure strain presents a wider range of variation. A single strain parameter (equivalent strain) was found as an accurate descriptor of cancellous bone compression failure. Image-based numerical models usually need for the action of a technician to segment the images. Therefore, we studied the sensitivity to variations of the segmentation threshold on the morphometry and the elastic properties of vertebral cancellous bone specimens of different bone volume fractions. The apparent modulus is highly sensitive to the segmentation threshold. We report variations between 45 and 120 % for a ± 15 % threshold variation. Other parameters, such as BS/BV, BS/TV, Tb.Sp, Tb.N, Conn.D and fractal dimension were influenced significantly. Digital image correlation (DIC) was applied to images taken during compression testing to analyze displacement fields at failure and characterize them. Some variables were explored to describe failure and a study is done about how DIC parameters influence the strain field obtained. Facet and step sizes have a relevant effect on the failure strain estimation, and an increment of both parameters reduces the strain estimation up to 40 %. Besides, several parameters combination led to correct failure pattern detection, so values reported in the literature should be referred to the parameters used. Furthermore, we explored if cancellous bone microstructure acts (non-speckle/texture approach) as a proper pattern to calculate displacements using DIC technique. As regards relationships between microstructure and mechanics, single and multiple parameter analysis were performed to assess the morphometric variables that control the explanation of mechanical properties variation. Bone volume fraction (BV/TV), bone surface to volume ratio (BS/BV), mean trabecular thickness (Tb.Th) and fractal dimension (D) presented the best linear correlations to the elastic properties, while both the yield and failure strains did not show correlation to any morphometric parameter. The regressions obtained permit to estimate those mechanical properties that describe the state of a specimen.[ES] Las fracturas óseas constituyen un problema social de salud con magnitud creciente por su prevalencia en la población de edad avanzada debido a la osteoporosis. La calidad del hueso suele caracterizarse mediante la estimación de la densidad mineral ósea (DMO) en regiones de hueso trabecular, utilizando absorciometría de rayos X de energía dual (DXA). No obstante, la DMO por si sola no es capaz de predecir numerosos casos de fractura porque no solo importa la pérdida de densidad, sino que la microestructura también tiene un papel principal en la resistencia del hueso. Las propiedades mecánicas del hueso pueden usarse como indicadores de su integridad en función de la edad, enfermedad o tratamiento. Por lo tanto, la caracterización de la fractura de hueso trabecular y su relación con la microestructura no se ha resuelto de forma completa en la literatura y es relevante para mejorar las predicciones de fractura. En esta tesis, nuestro principal objetivo es caracterizar la morfometría y el comportamiento mecánico del hueso trabecular. Estimamos la morfometría a través del análisis de imágenes obtenidas por micro tomografía computerizada (micro-CT) de muestras de hueso trabecular vertebral de cerdo. Respecto al comportamiento mecánico, calculamos propiedades elásticas, de plasticidad y fractura a escala aparente y de tejido. Para determinar esas propiedades, hemos seguido diferentes procedimientos: ensayos a compresión, modelos de elementos finitos y fantomas de calibración micro-CT. Los modelos de elementos finitos desarrollados reproducen la respuesta elástica y de fallo bajo condiciones de compresión en hueso trabecular, modelando el fallo como combinación de mecánica del daño contínuo y la técnica de eliminación de elementos. Los modelos numéricos desarrollados han permitido estimar propiedades elásticas y de fallo. En concreto, las deformaciones de inicio de fallo estimadas son relativamente constantes para las muestras analizadas (0.7 %), mientras que las deformaciones últimas de fallo presentan un rango de variación mayor. Por otro lado, encontramos que la deformación equivalente es el descriptor más preciso del fallo a compresión del hueso trabecular. Normalmente, los modelos numéricos basados en imágenes suelen necesitar la acción de un técnico para segmentar las imágenes. En este sentido, estudiamos la sensibilidad de la morfometría y la estimación de propiedades elásticas ante variaciones en el umbral de segmentación en muestras con distinta fracción en volumen. Hemos obtenido que la rigidez aparente es muy sensible a cambios en el umbral de segmentación, con variaciones entre 45 y 120 % para una variación de ± 15 % del umbral de segmentación. Otros parámetros, como BS/BV, BS/TV, Tb.Sp, Tb.N, Conn.D y la dimensión fractal se ven afectados significativamente. Por otro lado, hemos aplicado la técnica correlación digital por imagen (DIC) para caracterizar campos de desplazamientos en el fallo a compresión del hueso trabecular, a partir del análisis de imágenes tomadas durante el ensayo de las muestras. Además, estudiamos la influencia de algunos parámetros de la técnica DIC en el campo de deformaciones obtenido. También, hemos explorado la aplicación DIC sin el uso de moteado, utilizando como patrón de reconocimiento la propia microestructura trabecular. En relación al estudio de la influencia de la microestructura en la respuesta mecánica, hemos calculado correlaciones de uno y varios parámetros para analizar qué variables morfométricas explican la variación de las propiedades mecánicas. La fracción en volumen de hueso (BV/TV), la relación entre el área y el volumen de hueso (BS/BV), el espesor trabecular medio (Tb.Th) y la dimensión fractal (D) presentan las mejores correlaciones lineales respecto a las propiedades elásticas, mientras que las deformaciones de inicio de plasticidad y fractura no mostraron correlación con ningún parámetro morfométrico.[CA] Les fractures òssies constitueixen un problema social de salut amb magnitud creixent per la seua prevalença en la població d'edat avançada a causa de l'osteoporosi. La qualitat de l'os sol caracteritzar-se mitjançant l'estimació de la densitat mineral òssia (DMO) en regions d'os trabecular, utilitzant absorciometria de raigs X d'energia dual (DXA). No obstant això, la DMO per si sola no és capaç de predir nombrosos casos de fractura perquè no sols importa la pèrdua de densitat, sinó que la microestructura també té un paper principal en la resistència de l'os. Les propietats mecàniques de l'os poden usar-se com a indicadors de la seua integritat en funció de l'edat, malaltia o tractament. Per tant, la caracterització de la fractura d'os trabecular i la seua relació amb la microestructura no s'ha resolt de manera completa en la literatura i és rellevant per a millorar les prediccions de fractura. En aquesta tesi, el nostre principal objectiu és caracteritzar la morfometria i el comportament mecànic de l'os trabecular. Estimem la morfometria a través de l'anàlisi d'imatges obtingudes per micro tomografia automatitzada (micro-CT) de mostres d'os trabecular vertebral de porc. Respecte al comportament mecànic, calculem propietats elàstiques, de plasticitat i fractura a escala aparent i de teixit. Per a determinar aqueixes propietats, hem seguit diferents procediments: assajos a compressió, models d'elements finits i fantomas de calibratge micro-CT. Hem desenvolupat models d'elements finits que reprodueixen la resposta elàstica i de fallada sota condicions de compressió en os trabecular, modelant la fallada com a combinació de mecànica del dany continu i la tècnica d'eliminació d'elements. Els models numèrics desenvolupats han permés estimar propietats elàstiques i de fallada. Les nostres estimacions respecte a propietats de fallada són consistents amb valors reportats en la literatura. En concret, les deformacions d'inici de fallada estimades són relativament constants per a les mostres analitzades (0.7 %), mentre que les deformacions últimes de fallada presenten un rang de variació major. D'altra banda, trobem que la deformació equivalent és el descriptor més precís de la fallada a compressió de l'os trabecular. Els models numèrics basats en imatges solen necessitar l'acció d'un tècnic per a segmentar les imatges. En aquest sentit, estudiem la sensibilitat de la morfometria i l'estimació de propietats elàstiques davant variacions en el llindar de segmentació en mostres amb diferent fracció en volum. Hem obtingut que la rigidesa aparent és molt sensible a canvis en el llindar de segmentació, amb variacions entre 45 i 120 % per a una variació de ± 15 % del llindar de segmentació. Altres paràmetres, com BS/BV, BS/TV, Tb.Sp, Tb.N, Conn.D i la dimensió fractal es veuen afectats significativament. D'altra banda, hem aplicat la tècnica correlació digital per imatge (DIC) per a caracteritzar camps de desplaçaments en la fallada a compressió de l'os trabecular, a partir de l'anàlisi d'imatges preses durant l'assaig de les mostres. A més, estudiem la influència d'alguns paràmetres de la tècnica DIC en el camp de deformacions obtingut. També, hem explorat l'aplicació DIC sense l'ús de clapejat, utilitzant com a patró de reconeixement la pròpia microestructura trabecular. En relació a l'estudi de la influència de la microestructura en la resposta mecànica, hem calculat correlacions d'un i diversos paràmetres per a analitzar quines variables morfomètriques expliquen la variació de les propietats mecàniques. La fracció en volum d'os (BV/TV), la relació entre l'àrea i el volum d'os (BS/BV), la espessor trabecular mitjà (Tb.th) i la dimensió fractal (D) presenten les millors correlacions lineals respecte a les propietats elàstiques, mentre que les deformacions d'inici de plasticitat i fractura no van mostrar correlació amb cap paràmetre morfomètric.Belda González, R. (2020). Mechanical and morphometric characterization of cancellous bone [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/149376TESI

A Novel In Vivo Synchrotron Radiation Micro-CT Imaging Platform For The Direct Tracking Of Remodeling Events In Cortical Bone

Throughout life, bone tissue continuously alters its microarchitecture in response to microdamage and other stimuli through remodeling. Specialized cellular groupings, Basic Multicellular Units (BMUs), conduct remodeling through ‘coupling’ bone resorption to formation. Osteoclasts within a BMU’s cutting cone create a localized cylindrical space which osteoblasts concentrically refill, creating a secondary osteon (a.k.a. Haversian system). Continual production of secondary osteons by multitudes of BMUs creates a vast interconnected vascular network that permeates the cortex of bone, and therefore, BMUs are essential components in the overall maintenance of bone health. However, with increasing age or diseased states, such as osteoporosis (OP), remodeling can destabilize where resorbed bone is not entirely replaced (unbalanced) and/or where BMUs become ‘uncoupled’ preventing initiation of the bone formation following resorption. This increases porosity and thins cortices, leading to fragile, brittle bones much more susceptible to fracture. BMU behavior has never been replicated in vitro nor directly observed in vivo. The resorptive characteristics of BMUs, such as Longitudinal Erosion Rate (LER) – the rate of the advance of the cutting cone over time – are particularly poorly understood as our current understanding is inferred from indirect histological assessment of bone formation. Critically, BMUs have never been imaged in 4D (3D over time) due to limitations imposed by the radiation dose associated with conventional absorption-based imaging. This thesis explores in-line phase contrast synchrotron radiation micro- CT (SR micro-CT) as means of overcoming the limitations of conventional imaging. The goal was to develop a novel pre-clinical (animal) platform capable of directly tracking individual BMUs. The specific objectives of my thesis research were: 1) develop an in vivo imaging protocol to target individual BMU remodeling events within rabbit tibiae cortical bone to permit longitudinal imaging, using in-line phase contrast SR micro-CT; 2) Within rabbits, implement OP models of ovariectomy, glucocorticoids, a combination thereof and parathyroid hormone (PTH) to elevate cortical bone remodeling rates and, thus, the ability to observe BMU behavior on a large scale; and 3) directly measure BMU LER in 4D for the first time. A novel SR micro-CT protocol capable of detecting cortical porosity without any apparent radiation impacts was successfully developed on the BioMedical Imaging and Therapy Beamline of the Canadian Light Source. Compared to sham controls, elevated remodeling was found for all the OP models. PTH induced the highest rate of remodeling and it was selected as the model for direct assessment of LER. Through a novel co-registration technique, where in vivo SR micro-CT and follow-up ex vivo micro-CT scans acquired two weeks later were combined, LER (23.79 µm/day) was directly assessed for the first time. This novel platform establishes a means of investigating BMU spatio- temporal behavior and thus has great potential to advance our understanding of the role of remodeling in bone aging, adaptation, and disease