Cardiac multi-scale investigation of the right and left ventricle ex vivo: a review

The heart is a complex multi-scale system composed of components integrated at the subcellular, cellular, tissue and organ levels. The myocytes, the contractile elements of the heart, form a complex three-dimensional (3D) network which enables propagation of the electrical signal that triggers the contraction to efficiently pump blood towards the whole body. Cardiovascular diseases (CVDs), a major cause of mortality in developed countries, often lead to cardiovascular remodeling affecting cardiac structure and function at all scales, from myocytes and their surrounding collagen matrix to the 3D organization of the whole heart. As yet, there is no consensus as to how the myocytes are arranged and packed within their connective tissue matrix, nor how best to image them at multiple scales. Cardiovascular imaging is routinely used to investigate cardiac structure and function as well as for the evaluation of cardiac remodeling in CVDs. For a complete understanding of the relationship between structural remodeling and cardiac dysfunction in CVDs, multi-scale imaging approaches are necessary to achieve a detailed description of ventricular architecture along with cardiac function. In this context, ventricular architecture has been extensively studied using a wide variety of imaging techniques: ultrasound (US), optical coherence tomography (OCT), microscopy (confocal, episcopic, light sheet, polarized light), magnetic resonance imaging (MRI), micro-computed tomography (micro-CT) and, more recently, synchrotron X-ray phase contrast imaging (SR X-PCI). Each of these techniques have their own set of strengths and weaknesses, relating to sample size, preparation, resolution, 2D/3D capabilities, use of contrast agents and possibility of performing together with in vivo studies. Therefore, the combination of different imaging techniques to investigate the same sample, thus taking advantage of the strengths of each method, could help us to extract the maximum information about ventricular architecture and function. In this review, we provide an overview of available and emerging cardiovascular imaging techniques for assessing myocardial architecture ex vivo and discuss their utility in being able to quantify cardiac remodeling, in CVDs, from myocyte to whole organ

A tomographic microscopy-compatible Langendorff system for the dynamic structural characterization of the cardiac cycle

ntroduction: Cardiac architecture has been extensively investigated ex vivo using a broad spectrum of imaging techniques. Nevertheless, the heart is a dynamic system and the structural mechanisms governing the cardiac cycle can only be unveiled when investigating it as such. Methods: This work presents the customization of an isolated, perfused heart system compatible with synchrotron-based X-ray phase contrast imaging (X-PCI). Results: Thanks to the capabilities of the developed setup, it was possible to visualize a beating isolated, perfused rat heart for the very first time in 4D at an unprecedented 2.75 μm pixel size (10.6 μm spatial resolution), and 1 ms temporal resolution. Discussion: The customized setup allows high-spatial resolution studies of heart architecture along the cardiac cycle and has thus the potential to serve as a tool for the characterization of the structural dynamics of the heart, including the effects of drugs and other substances able to modify the cardiac cycle

Micro-computed tomography for high resolution soft tissue imaging; applications in the normal and failing heart

The normal structure and function of the heart, the common pathological changes that cause abnormal function and the interventions proposed to improve or restore its function are fundamentally based on cardiac anatomy. Therefore in all these areas a detailed and accurate understanding of 3D structure is essential. However there is still disparity over some aspects of the form and function of the healthy heart. Furthermore, in heart failure (HF) the transition from compensated to decompensated HF is poorly understood, and details of ventricular, and particularly atrial, remodelling and their effects on cardiac function are yet to be fully elucidated. In addition little is known on how the 3D morphology of the cardiac conduction system is affected in disease, and further knowledge is required on the structural substrates for arrhythmogenesis associated with HF. Here we have developed contrast enhanced micro-CT for soft tissue imaging, allowing non-invasive high resolution (~5 µm attainable) differentiation of multiple soft tissue types including; muscle, connective tissue and fat. Micro-CT was optimised for imaging of whole intact mammalian hearts and from these data we reveal novel morphological and anatomical detail in healthy hearts and in hearts after experimental HF (volume and pressure overload). Remodelling of the myocardium in HF was dramatic with significant hypertrophy and dilatation observed in both atria and ventricles. The atria showed a 67% increase in myocardial volume, with the left atrium showing a 93% increase. The pectinate muscle: wall thickness ratio was significantly increased in both atria (p

Experimental validation of a novel technique for ultrasound imaging of cardiac fiber orientation

International audienc

DEVELOPMENT OF NANOPARTICLE RATE-MODULATING AND SYNCHROTRON PHASE CONTRAST-BASED ASSESSMENT TECHNIQUES FOR CARDIAC TISSUE ENGINEERING

Myocardial infarction (MI) is the most common cause of heart failure. Despite advancements in cardiovascular treatments and interventions, current therapies can only slow down the progression of heart failure, but not tackle the progressive loss of cardiomyocytes after MI. One aim of cardiac tissue engineering is to develop implantable constructs (e.g. cardiac patches) that provide physical and biochemical cues for myocardium regeneration. To this end, vascularization in these constructs is of great importance and one key issue involved is the spatiotemporal control of growth-factor (GF)-release profiles. The other key issue is to non-invasively quantitatively monitor the success of these constructs in-situ, which will be essential for longitudinal assessments as studies are advanced from ex-vivo to animal models and human patients. To address these issues, the present research aims to develop nanoparticles to modulate the temporal control of GF release in cardiac patches, and to develop synchrotron X-ray phase contrast tomography for visualization and quantitative assessment of 3D-printed cardiac patch implanted in a rat MI model, with four specific objectives presented below. The first research objective is to optimize nanoparticle-fabrication process in terms of particle size, polydispersity, loading capacity, zeta potential and morphology. To achieve this objective, a comprehensive experimental study was performed to examine various process parameters used in the fabrication of poly(lactide-co-glycolide) (PLGA) nanoparticles, along with the development of a novel computational approach for the nanoparticle-fabrication optimization. Results show that among various process parameters examined, the polymer and the external aqueous phase concentrations are the most significant ones to affect the nanoparticle physical and release characteristics. Also, the limitations of PLGA nanoparticles such as initial burst effect and the lack of time-delayed release patterns are identified. The second research objective is to develop bi-layer nanoparticles to achieve the controllable release of GFs, meanwhile overcoming the above identified limitations of PLGA nanoparticles. The bi-layer nanoparticle is composed of protein-encapsulating PLGA core and poly(L-lactide) (PLLA)-rate regulating shell, thus allowing for low burst effect, protein structural integrity and time-delayed release patterns. The bi-layer nanoparticles, along with PLGA ones, were successfully fabricated and then used to regulate simultaneous and/or sequential release of multiple angiogenic factors with the results demonstrating that they are effective to promote angiogenesis in fibrin matrix. The third objective is to develop novel mathematical models to represent the controlled-release of bioactive agents from nanoparticles. For this, two models, namely the mechanistic model and geno-mechanistic model, were developed based on the local and global volume averaging approaches, respectively, and then validated with experiments on both single- and bi-layer nanoparticles, by which the ovalbumin was used as a protein model for the release examination. The results illustrates the developed models are able to provide insight on the release mechanism and to predict nanoparticle transport and degradation properties of nanoparticles, thus providing a means to regulate and control the release of bioactive agents from the nanoparticles for tissue engineering applications. The fourth objective of this research is to develop a synchrotron-based phase contrast non-invasive imaging technique for visualization and quantitative assessment of cardiac patch implanted in a rat MI model. To this end, the patches were created from alginate strands using the three-dimensional (3D) printing technique and then surgically implanted on rat hearts for the assessment based on phase contrast tomography. The imaging of samples was performed at various sample-to-detector distances, CT-scan time, and areas of the region of interest (ROI) to examine their effects on imaging quality. Phase-retrieved images depict visible and quantifiable structural details of the patch at low radiation dose, which, however, are not seen from the images by means of dual absorption-phase and a 3T clinical magnetic resonance imaging. Taken together, this research represents a significant advance in cardiac tissue engineering by developing novel nano-guided approaches for vascularization in myocardium regeneration as well as non-invasive and quantitative monitoring techniques for longitudinal studies on the cardiac patch implanted in animal model and eventually in human patients

An Analytical Fiber ODF Reconstruction in 3D Polarized Light Imaging

International audienceThree dimensional polarized light imaging (3D-PLI) utilizes the birefringence in postmortem tissue to map its spatial fiber structure at a submillimeter resolution. We propose an analytical method to compute the fiber orientation distribution function (ODF) from high-resolution vector data provided by 3D-PLI. This strategy enables the bridging of high resolution 3D-PLI to diffusion magnetic resonance imaging with relatively low spatial resolution. First, the fiber ODF is modeled as a sum of K orientations on the unit sphere and expanded with a high order spherical harmonics series. Then, the coefficients of the spherical harmonics are derived directly with the spherical Fourier transform. We quantitatively validate the accuracy of the reconstruction against synthetic data and show that we can recover complex fiber configurations in the human heart at different scales

Modular Instrumentation for Controlling and Monitoring In-Vitro Cultivation Environment and Image-based Functionality Measurements of Human Stem Cells

Artificial animal cell culture was successfully developed by Ross Harrison in 1907. But it was not until the 1940’s and 1950’s that several developments occurred, which expedited the cell culturing in-vitro (C-Vitro) to be a consistent and reproducible technique to study isolated living-cells in a controlled environment. Currently, CVitro is one of the major tools in cellular and molecular biology both in the academia and industry. They are extensively utilised to study the cellular physiology/biochemistry, to screen drugs/therapeutic compounds, to understand the effects of drugs/toxic compounds and also to identify the pathways of carcinogenesis/mutagenesis. It is also used in large scale manufacturing of vaccines and therapeutic proteins. In any experimental setup, it is important that the C-Vitro model should represent the physiological phenomena of interest with reasonable accuracy so that all experimental results are statistically consistent and reproducible. In this direction, sensors and measurement systems play important roles in in-situ detection and/or control/manipulation of cells/tissues/environment. This thesis aimed to develop new technology for tailored cell culturing and integrated measurements. Firstly, design and assembly of a portable Invert-upright microscope interchangeable modular cell culturing platform (iuCMP) was envisioned. In contrast to conventional methods, micro-scaled systems mimic the cells' natural microenvironment more precisely, facilitating accurate and tractable models. The iuCMP integrates modular measurement schemes with a mini culture chamber using biocompatible cell-friendly materials, automated environment-control (temperature and gas concentrations), oxygen sensing and simultaneous functional measurements (electrophysiological and image-based). Time lapse microscopy is very useful in cell biology, but integration of advanced >i>in-vitro/device based biological systems (e.g. lab/organ/body-on-chips, or mini-bioreactors/microfluidic systems) into conventional microscopes can be challenging in several circumstances due to multiple reasons. But in iuCMP the main advantage is, the microscope can be switched either as an inverted or as an upright system and therefore can accommodate virtually any in-vitro device. It can capture images from regions that are otherwise inaccessible by conventional microscopes, for example, cells cultured on physical or biochemical sensor systems. The modular design also allows accommodating more sensor or measurement systems quite freely. We have demonstrated the system for video-based beating analysis of cardiomyocytes, cell orientation analysis on nanocellulose, and simultaneous long-term in-situ microscopy with oxygen and temperature sensing in hypoxia. In an example application, the system was utilised for long-term temperature stressing and simultaneous mechanobiological analysis of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). For this the iuCMP together with a temperature sensor plate (TSP) and a novel non-invasive beating analysis software (CMaN—cardiomyocyte function analysis tool, scripted as a subpart of this thesis), was applied for automated temperature response studies in hiPSC-CM cultures. In-situ temperature sensing is usually challenging with bulky external sensors, but TSPs solved this issue. In the temperature response study, we showed that the relationship between hiPSC-CM beating frequency and temperature is non-linear and measured the Q10 temperature coefficients. Moreover, we observed the hiPSC-CM contractile networking, including propagation of the action potential signal between dissociated clusters and their non-invasive measurements. It was the first case where these events were reported in hiPSC-CM clusters and their noninvasive measurements by image processing. The software CMaN comes with a user-friendly interface and, is equipped with features for batch processing, movement centre detection and cluster finding. It can extract six different signals of the contractile motion of cardiomyocytes (clusters or single cells) per processing. This ensures a minimum of one useful beating signal even in the cases of complex beating videos. On the processing end, compared to similar tools, CMaN is faster, more sensitive, and computationally less expensive and allows ROI based processing. In the case of healthy cells, the waveform of the signal from the CMaN resembles an ECG signal with positive and negative segments, allowing the computation of contraction and relaxation features separately. In addition to iuCMP, a Modular optical pH measurement system (MO-pH) for 24/7 non-contact cell culture measurements was also developed. The MO-pH incorporates modular sterilisable optical parts and is used in phenol-red medium cell cultures. The modular assembly of MO-pH cassettes is unique and reusable. Measurements are carried out in a closed flow system without wasting any culture medium and requires no special manual attention or recalibrations during culture. Furthermore, a new absorption correction model was put forward that minimised errors caused e.g. by biolayers in spectrometric pH measurement, which improved the pH measurement accuracy. MO-pH has been applied in long-term human adipose stem cells (hASC) expansion cultures in CO2 dependent and independent media. Additionally, the MO-pH was also utilised to comprehend the behaviour of pH, temperature and humidity in water jacked incubators as well as to record the pH response as a function of temperature in the presence and absence of CO2 in the context of stem cell cultures. The resulting plots clearly showed the interplay between measured parameters indicating a few stress sources present all through the culture. Additionally, it provided an overall picture of behaviour of critical control parameters in an incubator and pointed out the need for bioprocess systems with automatic process monitoring and smart control for maximum yield, optimal growth and maintenance of the cells. Besides, we also integrated MO-pH into flasks with reclosable lids (RL-F) and tested its applicability in stem cell cultures. A standalone system around an RL-F flask was built by combining the cell culture, medium perfusion and optical measurements. The developed RL-F system has been successfully tested in ASC-differentiation cultures. Finally, a few trial experiments for image-based pH estimation aimed for iuCMP have also been carried out. This includes tests with LCD illumination, optical projection tomography, and webcam systems. In reality, the pH is not distributed uniformly in tissues, and has shown a gradient of up to 1.0 pH unit within 1 cm distance. Therefore, producing reliable pH maps also in in-vitro can be important in understanding various common pathologies and location of lesions. A reliable and adequately developed long-term pH mapping method will be an important addition into the iuCMP

Modified mass-spring system for physically based deformation modeling

Mass-spring systems are considered the simplest and most intuitive of all deformable models. They are computationally efficient, and can handle large deformations with ease. But they suffer several intrinsic limitations. In this book a modified mass-spring system for physically based deformation modeling that addresses the limitations and solves them elegantly is presented. Several implementations in modeling breast mechanics, heart mechanics and for elastic images registration are presented