Towards an in vitro cardiac model: 3D environment, co-culture, alignment, and mechanical stimulation impact on cell behavior

Our aim is to develop a 3D model unit of cardiac muscle: an in-vitro analog of the trabeculae carneae found in vivo. As a base hydrogel matrix for cardiomyocyte culture in 3D, we develop a blend of decellularized extracellular matrix (dECM) and fibrin. This blend contains essential components of cardiac ECM, provides rapid, cell-friendly coagulation, and closely matches the mechanical properties of native myocardium. Co-culture of the H9c2 model cell line with fibroblast cells in this hydrogel showed enhancement in attachment, spreading, and cardiogenic differentiation of H9c2 cells. This is ascribed primarily to the collagen content of the dECM. Calcium imaging and analysis of beating motion of primary rat neonatal cardiomyocytes cultured in the 3D hydrogel showed specific improvement in recovery, frequency, synchronicity, and beating rates compared to a series of common hydrogel controls, including collagen-fibrin composites. This establishes the dECM-fibrin hydrogel as an optimal base matrix for cardiac tissue culture and engineering. The trabeculae carneae are cardiac muscle fibers at the inner surface of the ventricles. They are an accessible representation of the tiniest building blocks of the cardiac tissue because of their dimensions and cellular orientation. Molding cell-seeded dECM-fibrin hydrogel in microfabricated grooves, we fabricated in vitro analogs of the trabeculae carneae. In these 3D structures, propagation of cell alignment due to the corner contact guidance successfully addresses the challenge of 3D cell orientation. The effect provides alignment 250-300 µm from the corners, enabling full 3D orientation in 350 µm by 350 µm square section microgrooves. The cell-laden hydrogel can be detached from the PDMS surface while maintaining cell alignment. The alignment enhanced the functionality of rat neonatal cardiomyocytes beating by maintaining the contractility of the cells for longer time compared to the random distribution of the cells in the hydrogel. Mechanical forces play key roles in the development and cardiac tissue morphogenesis. Relatively well-known in 2D cultures, knowledge about mechanical effects in 3D is scarcer. We investigate the combined effect of topography and mechanical stimulation on 3D cardiac cell culture. For application of cyclic stretch, we designed and fabricated a user-friendly mechanical stimulator. In 2D cultures, the cells orient perpendicularly to the direction of applied cyclic stretch in agreement with known strain-avoidance mechanisms. In 3D, the cells react to combined topography and mechanical stimulation by adopting an orientation around 45°. This reflects the integration of the conflicting stimuli of alignment along the grooves but perpendicular to the stretch direction. Off-axis alignment may be a novel mechanism for maintenance of helicoidal fiber alignment in the heart. As anticipated, mechanical stimulation also improved the maturation and functionality of the neonatal cardiac cells. Overall, we provide a novel biomaterial for 3D cardiac cell culture and find an effective, yet simple approach to encourage 3D cell alignment. Adding mechanical stretching enhances the maturation and functionality of the bioengineered tissue in vitro, and provides the possibility of off-axis alignment reminiscent of the helicoidal fiber arrangement in the heart. Our trabeculae carneae unit model therefore provides an enhanced 3D environment for investigating cell fate and tissue functionality

Toward a Physiologically Relevant 3D Helicoidal-Oriented Cardiac Model: Simultaneous Application of Mechanical Stimulation and Surface Topography

Myocardium consists of cardiac cells that interact with their environment through physical, biochemical, and electrical stimulations. The physiology, function, and metabolism of cardiac tissue are affected by this dynamic structure. Within the myocardium, cardiomyocytes’ orientations are parallel, creating a dominant orientation. Additionally, local alignments of fibers, along with a helical organization, become evident at the macroscopic level. For the successful development of a reliable in vitro cardiac model, evaluation of cardiac cells’ behavior in a dynamic microenvironment, as well as their spatial architecture, is mandatory. In this study, we hypothesize that complex interactions between long-term contraction boundary conditions and cyclic mechanical stimulation may provide a physiological mechanism to generate off-axis alignments in the preferred mechanical stretch direction. This off-axis alignment can be engineered in vitro and, most importantly, mirrors the helical arrangements observed in vivo. For this purpose, uniaxial mechanical stretching of dECM-fibrin hydrogels was performed on pre-aligned 3D cultures of cardiac cells. In view of the potential development of helical structures similar to those in native hearts, the possibility of generating oblique alignments ranging between 0° and 90° was explored. Indeed, our investigations of cell alignment in 3D, employing both mechanical stimulation and groove constraint, provide a reliable mechanism for the generation of helicoidal structures in the myocardium. By combining cyclic stretch and geometric alignment in grooves, an intermediate angle toward favored direction can be achieved experimentally: while cyclic stretch produces a perpendicular orientation, geometric alignment is associated with a parallel one. In our 2D and 3D culture conditions, nonlinear cellular addition of the strains and strain avoidance concept reliably predicted the preferred cellular alignment. The 3D dECM-fibrin model system in this study shows that cyclical stretching supports cell survival and development. Using mechanical stimulation of pre-aligned heart cells, maturation markers are augmented in neonatal cardiomyocytes, while the beating culture period is prolonged, indicating an improved model function. We propose a simplified theoretical model based on numerical simulation and nonlinear strain avoidance by cells to explain oblique alignment angles. Thus, this work lays a possible rational basis for understanding and engineering oblique cellular alignments, such as the helicoidal layout of the heart, using approaches that simultaneously enhance maturation and function

Highly Efficient Cardiac Differentiation and Maintenance by Thrombin-Coagulated Fibrin Hydrogels Enriched with Decellularized Porcine Heart Extracellular Matrix

Biochemical and biophysical properties instruct cardiac tissue morphogenesis. Here, we are reporting on a blend of cardiac decellularized extracellular matrix (dECM) from porcine ventricular tissue and fibrinogen that is suitable for investigations employing an in vitro 3D cardiac cell culture model. Rapid and specific coagulation with thrombin facilitates the gentle inclusion of cells while avoiding sedimentation during formation of the dECM-fibrin composite. Our investigations revealed enhanced cardiogenic differentiation in the H9c2 myoblast cells when using the system in a co-culture with Nor-10 fibroblasts. Further enhancement of differentiation efficiency was achieved by 3D embedding of rat neonatal cardiomyocytes in the 3D system. Calcium imaging and analysis of beating motion both indicate that the dECM-fibrin composite significantly enhances recovery, frequency, synchrony, and the maintenance of spontaneous beating, as compared to various controls including Matrigel, pure fibrin and collagen I as well as a fibrin-collagen I blend

Highly Efficient Cardiac Differentiation and Maintenance by Thrombin-Coagulated Fibrin Hydrogels Enriched with Decellularized Porcine Heart Extracellular Matrix

Biochemical and biophysical properties instruct cardiac tissue morphogenesis. Here, we are reporting on a blend of cardiac decellularized extracellular matrix (dECM) from porcine ventricular tissue and fibrinogen that is suitable for investigations employing an in vitro 3D cardiac cell culture model. Rapid and specific coagulation with thrombin facilitates the gentle inclusion of cells while avoiding sedimentation during formation of the dECM-fibrin composite. Our investigations revealed enhanced cardiogenic differentiation in the H9c2 myoblast cells when using the system in a co-culture with Nor-10 fibroblasts. Further enhancement of differentiation efficiency was achieved by 3D embedding of rat neonatal cardiomyocytes in the 3D system. Calcium imaging and analysis of beating motion both indicate that the dECM-fibrin composite significantly enhances recovery, frequency, synchrony, and the maintenance of spontaneous beating, as compared to various controls including Matrigel, pure fibrin and collagen I as well as a fibrin-collagen I blend

Effects of chronic hypoxia on the expression of seladin-1/Tuj1 and the number of dark neurons of hippocampus

Background: There are evidences showing the relation between chronic hypoxia and Alzheimer&#39;s disease (AD) as a metabolic neurodegenerative disease. This study was designed to evaluate the effects of chronic hypoxia on factors which characterized in AD to introduce a new model of AD-dementia. Methods and materials: Twenty-four male rats were randomly divided in three groups: Control group (Co), Sham group (Sh), Hypoxia induction group (Hx, exposed to hypoxic chamber [oxygen 8% and nitrogen 92%] for 30 days, 4 h/day). Spatial learning and memory were analyzed using the Morris water maze task. At day 30 after hypoxia period, animals were sacrificed and serum was gathered for pro-inflammatory cytokines (interleukin-1 beta and tumor necrosis factor) measurements and brains were used for molecular and histopathological investigations. Results: According to behavioral studies, a significant impairment was seen in Hx group (P &lt; 0.05). TNF-alpha and IL-1 beta showed a significant enhanced in Hx group comparing with Co group and Sh group (P &lt; 0.05). As well, the gene expression of seladin-1, Tuj1 and the number of seladin-1 +, Tuj1 +neurons significantly decreased and also the mean number of dark neurons significantly increased in CA1 and CA3 regions of hippocampus. Conclusions: In this study, a new model of AD was developed which showed the underlying mechanisms of AD and its relations with chronic hypoxia. Hypoxia for 30 days decreased seladin-1, Tuj1 expression, increased the number of dark neurons, and also induced memory impairment. These results indicated that chronic hypoxia mediated the dementia underlying AD and AD-related pathogenesis in rat.</p

Electrochemical performance of polymer-derived SiOC and SiTiOC ceramic electrodes for artificial cardiac pacemaker applications

In an implantable electrode, such as a pacemaker electrode, fibrotic tissue formation due to a foreign body reaction is an important challenge affecting the efficiency to transmit the electrical signal of the device. The chemical inertness, biocompatibility, and electrical conductivity of polymer-derived ceramics (PDCs) are promising features in terms of overcoming this challenge. Here, the electrochemical behavior of polymer-derived silicon oxycarbide (SiOC) and titanium-doped SiOC (SiTiOC) ceramic electrodes for use as pacemaker electrodes is investigated by measuring impedance spectroscopy and cyclic voltammetry. In addition, typical stimulation electrodes such as iridium oxide, titanium nitride, platinum, and glassy carbon were prepared and loaded simultaneously into a custom-made electrochemical testing platform for comparison with SiOC and SiTiOC electrodes under identical conditions. The SiOC and SiTiOC electrodes shows a wide electrochemical stability window in the range of −0.9 to 1.2 V with a double layer capacitance as the charge injection mechanism at the electrode/phosphate-buffered saline interface. Also, analyzing the voltage transient shows that the maximum charge injection of the SiTiOC electrode was about 28 μC/cm2. The results of the electrochemical evaluation and comparison of SiOC and SiTiOC stimulating electrodes will be helpful to understand fundamental characteristics for the potential of this material as candidate for next-generation pacemaker electrodes

A Three-Dimensional Engineered Cardiac In Vitro Model: Controlled Alignment of Cardiomyocytes in 3D Microphysiological Systems

Cardiomyocyte alignment in myocardium tissue plays a significant role in the physiological, electrical, and mechanical functions of the myocardium. It remains, however, difficult to align cardiac cells in a 3D in vitro heart model. This paper proposes a simple method to align cells using microfabricated Polydimethylsiloxane (PDMS) grooves with large dimensions (of up to 350 µm in width), similar to the dimensions of trabeculae carneae, the smallest functional unit of the myocardium. Two cell groups were used in this work; first, H9c2 cells in combination with Nor10 cells for proof of concept, and second, neonatal cardiac cells to investigate the functionality of the 3D model. This model compared the patterned and nonpatterned 3D constructs, as well as the 2D cell cultures, with and without patterns. In addition to alignment, we assessed the functionality of our proposed 3D model by comparing beating rates between aligned and non-aligned structures. In order to assess the practicality of the model, the 3D aligned structures should be demonstrated to be detachable and alignable. This evaluation is crucial to the use of this 3D functional model in future studies related to drug screening, building blocks for tissue engineering, and as a heart-on-chip by integrating microfluidics

Toward a Physiologically Relevant 3D Helicoidal-Oriented Cardiac Model: Simultaneous Application of Mechanical Stimulation and Surface Topography

Myocardium consists of cardiac cells that interact with their environment through physical, biochemical, and electrical stimulations. The physiology, function, and metabolism of cardiac tissue are affected by this dynamic structure. Within the myocardium, cardiomyocytes’ orientations are parallel, creating a dominant orientation. Additionally, local alignments of fibers, along with a helical organization, become evident at the macroscopic level. For the successful development of a reliable in vitro cardiac model, evaluation of cardiac cells’ behavior in a dynamic microenvironment, as well as their spatial architecture, is mandatory. In this study, we hypothesize that complex interactions between long-term contraction boundary conditions and cyclic mechanical stimulation may provide a physiological mechanism to generate off-axis alignments in the preferred mechanical stretch direction. This off-axis alignment can be engineered in vitro and, most importantly, mirrors the helical arrangements observed in vivo. For this purpose, uniaxial mechanical stretching of dECM-fibrin hydrogels was performed on pre-aligned 3D cultures of cardiac cells. In view of the potential development of helical structures similar to those in native hearts, the possibility of generating oblique alignments ranging between 0° and 90° was explored. Indeed, our investigations of cell alignment in 3D, employing both mechanical stimulation and groove constraint, provide a reliable mechanism for the generation of helicoidal structures in the myocardium. By combining cyclic stretch and geometric alignment in grooves, an intermediate angle toward favored direction can be achieved experimentally: while cyclic stretch produces a perpendicular orientation, geometric alignment is associated with a parallel one. In our 2D and 3D culture conditions, nonlinear cellular addition of the strains and strain avoidance concept reliably predicted the preferred cellular alignment. The 3D dECM-fibrin model system in this study shows that cyclical stretching supports cell survival and development. Using mechanical stimulation of pre-aligned heart cells, maturation markers are augmented in neonatal cardiomyocytes, while the beating culture period is prolonged, indicating an improved model function. We propose a simplified theoretical model based on numerical simulation and nonlinear strain avoidance by cells to explain oblique alignment angles. Thus, this work lays a possible rational basis for understanding and engineering oblique cellular alignments, such as the helicoidal layout of the heart, using approaches that simultaneously enhance maturation and function

Oxygen-rich Environment Ameliorates Cell Therapy Outcomes of Cardiac Progenitor Cells for Myocardial Infarction

To some extent, cell therapy for myocardial infarction (MI) has supported the idea of cardiac repair; however, further optimizations are inevitable. Combined approaches that comprise suitable cell sources and supporting molecules considerably improved its effect. Here, we devised a strategy of simultaneous transplantation of human cardiac progenitor cells (CPCs) and an optimized oxygen generating microparticles (MPs) embedded in fibrin hydrogel, which was injected into a left anterior descending artery (LAD) ligating-based rat model of acute myocardial infarction (AMI). Functional parameters of the heart, particularly left ventricular systolic function, markedly improved and reached pre-AMI levels. This functional restoration was well correlated with substantially lower fibrotic tissue formation and greater vascular density in the infarct area. Our novel approach promoted CPCs retention and differentiation into cardiovascular lineages. We propose this novel co-transplantation strategy for more efficient cell therapy of AMI which may function by providing an oxygen-rich microenvironment, and thus regulate cell survival and differentiation