Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions ¹. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA ² and PLGA ³ or a number of naturally occurring proteins such as collagen ⁴, hyaluronic acid ⁵ or fibrin ^6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body''s natural wound healing processes ⁸. Fibrin is cell-degradable and potentially autologous ⁹, making it an ideal temporary scaffold for tissue engineering.Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering ⁴. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers ¹⁰. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries ^11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture ¹³.After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions ⁶. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.     

Effect of a mechanical stimulation bioreactor on tissue engineered, scaffold-free cartilage

Achieving sufficient functional properties prior to implantation remains a significant challenge for the development of tissue engineered cartilage. Many studies have shown chondrocytes respond well to various mechanical stimuli, resulting in the development of bioreactors capable of transmitting forces to articular cartilage in vitro. In this study, we describe the production of sizeable, tissue engineered cartilage using a novel scaffold-free approach, and determine the effect of perfusion and mechanical stimulation from a C9-x Cartigen bioreactor on the properties of the tissue engineered cartilage. We created sizable tissue engineered cartilage from porcine chondrocytes using a scaffold-free approach by centrifuging a high-density chondrocyte cell-suspension onto an agarose layer in a 50 mL tube. The gross and histological appearances, biochemical content, and mechanical properties of constructs cultured in the bioreactor for 4 weeks were compared to constructs cultured statically. Mechanical properties were determined from unconfined uniaxial compression tests. Constructs cultured in the bioreactor exhibited an increase in total GAG content, equilibrium compressive modulus, and dynamic modulus versus static constructs. Our study demonstrates the C9-x CartiGen bioreactor is able to enhance the biomechanical and biochemical properties of scaffold-free tissue engineered cartilage; however, no additional enhancement was seen between loaded and perfused groups.     

Biomimetic scaffold combined with electrical stimulation and growth factor promotes tissue engineered cardiac development

Toward developing biologically sound models for the study of heart regeneration and disease, we cultured heart cells on a biodegradable, microfabricated poly(glycerol sebacate) (PGS) scaffold designed with micro-structural features and anisotropic mechanical properties to promote cardiac-like tissue architecture. Using this biomimetic system, we studied individual and combined effects of supplemental insulin-like growth factor-1 (IGF-1) and electrical stimulation (ES). On culture day 8, all tissue constructs could be paced and expressed the cardiac protein troponin-T. IGF-1 reduced apoptosis, promoted cell-to-cell connectivity, and lowered excitation threshold, an index of electrophysiological activity. ES promoted formation of tissue-like bundles oriented in parallel to the electrical field and a more than ten-fold increase in matrix metalloprotease-2 (MMP-2) gene expression. The combination of IGF-1 and ES increased 2D projection length, an index of overall contraction strength, and enhanced expression of the gap junction protein connexin-43 and sarcomere development. This culture environment, designed to combine cardiac-like scaffold architecture and biomechanics with molecular and biophysical signals, enabled functional assembly of engineered heart muscle from dissociated cells and could serve as a template for future studies on the hierarchy of various signaling domains relative to cardiac tissue development.     

Perfusion–compression bioreactor as the optimum choice for growing large-sized engineered bone constructs in vitro

Developing large-sized engineered bone constructs (LEBCs) become nowadays a great challenge of tissue engineering due to the internal cell necrosis in the cultured constructs. Mechanical loadings in bioreactors have improved the development of engineered constructs. We hypothesize that perfusion–compression bioreactor will achieve the growth of LEBCs in vitro. At the early stage of construction, the LEBCs in vitro develop without internal limitations in constructs in the bioreactor. At the after-mineralization stage of construction, the bioreactor provides specific conditions for remaining osteocytes buried within mineralized matrix normal viability by the compression-induced fluid flows in the lacuno-canalicular cavities and mechanical stimuli within constructs. Moreover, the LEBCs in vitro achieve the functional development similar to modeling adaptation of bone in vivo. Therefore, the perfusion–compression bioreactor may be the optimum choice for constructing the LEBCs in vitro.     

Tissue engineering of functional cardiac muscle: molecular, structural, and electrophysiological studies

The primary aim of this study was to relate molecular and structural properties of in vitro reconstructed cardiac muscle with its electrophysiological function using an in vitro model system based on neonatal rat cardiac myocytes, three-dimensional polymeric scaffolds, and bioreactors. After 1 wk of cultivation, we found that engineered cardiac muscle contained a 120- to 160-microm-thick peripheral region with cardiac myocytes that were electrically connected through gap junctions and sustained macroscopically continuous impulse propagation over a distance of 5 mm. Molecular, structural, and electrophysiological properties were found to be interrelated and depended on specific model system parameters such as the tissue culture substrate, bioreactor, and culture medium. Native tissue and the best experimental group (engineered cardiac muscle cultivated using laminin-coated scaffolds, rotating bioreactors, and low-serum medium) were comparable with respect to the conduction velocity of propagated electrical impulses and spatial distribution of connexin43. Furthermore, the structural and electrophysiological properties of the engineered cardiac muscle, such as cellularity, conduction velocity, maximum signal amplitude, capture rate, and excitation threshold, were significantly improved compared with our previous studies.     

Electromechanical models of the ventricles

Computational modeling has traditionally played an important role in dissecting the mechanisms for cardiac dysfunction. Ventricular electromechanical models, likely the most sophisticated virtual organs to date, integrate detailed information across the spatial scales of cardiac electrophysiology and mechanics and are capable of capturing the emergent behavior and the interaction between electrical activation and mechanical contraction of the heart. The goal of this review is to provide an overview of the latest advancements in multiscale electromechanical modeling of the ventricles. We first detail the general framework of multiscale ventricular electromechanical modeling and describe the state of the art in computational techniques and experimental validation approaches. The powerful utility of ventricular electromechanical models in providing a better understanding of cardiac function is then demonstrated by reviewing the latest insights obtained by these models, focusing primarily on the mechanisms by which mechanoelectric coupling contributes to ventricular arrythmogenesis, the relationship between electrical activation and mechanical contraction in the normal heart, and the mechanisms of mechanical dyssynchrony and resynchronization in the failing heart. Computational modeling of cardiac electromechanics will continue to complement basic science research and clinical cardiology and holds promise to become an important clinical tool aiding the diagnosis and treatment of cardiac disease.     

Effects of C-reactive protein on adipokines genes expression in 3T3-L1 adipocytes

Transplantation of skeletal myoblasts (SMs) has been investigated as a potential cardiac cell therapy approach. SM are available autologously, predetermined for muscular differentiation and resistant to ischemia. Major hurdles for their clinical application are limitations in purity and yield during cell isolation as well as the absence of gap junction expression after differentiation into myotubes. Furthermore, transplanted SMs do not functionally or electrically integrate with the host myocardium. Here, we describe an efficient method for isolating homogeneous SM populations from neonatal mice and demonstrate persistent gap junction expression in an engineered tissue. This method resulted in a yield of 1.4 × 10(8) high-purity SMs (>99% desmin positive) after 10 days in culture from 162.12 ± 11.85 mg muscle tissue. Serum starvation conditions efficiently induced differentiation into spontaneously contracting myotubes that coincided with loss of gap junction expression. For mechanical conditioning, cells were integrated into engineered tissue constructs. SMs within tissue constructs exhibited long term survival, ordered alignment, and a preserved ability to differentiate into contractile myotubes. When the tissue constructs were subjected to passive longitudinal tensile stress, the expression of gap junction and cell adherence proteins was maintained or increased throughout differentiation. Our studies demonstrate that mechanical loading of SMs may provide for improved electromechanical integration within the myocardium, which could lead to more therapeutic opportunities.     

Models of cardiac electromechanics based on individual hearts imaging data Image-based electromechanical models of the heart

Current multi-scale computational models of ventricular electromechanics describe the full process of cardiac contraction on both the micro- and macro- scales including: the depolarization of cardiac cells, the release of calcium from intracellular stores, tension generation by cardiac myofilaments, and mechanical contraction of the whole heart. Such models are used to reveal basic mechanisms of cardiac contraction as well as the mechanisms of cardiac dysfunction in disease conditions. In this paper, we present a methodology to construct finite element electromechanical models of ventricular contraction with anatomically accurate ventricular geometry based on magnetic resonance and diffusion tensor magnetic resonance imaging of the heart. The electromechanical model couples detailed representations of the cardiac cell membrane, cardiac myofilament dynamics, electrical impulse propagation, ventricular contraction, and circulation to simulate the electrical and mechanical activity of the ventricles. The utility of the model is demonstrated in an example simulation of contraction during sinus rhythm using a model of the normal canine ventricles.     

Rotating versus perfusion bioreactor for the culture of engineered vascular constructs based on hyaluronic acid

It is generally accepted that dynamic culture conditions are required for vascular tissue engineering. We compared the effects of two dynamic culture systems, a perfusion and a rotating bioreactor, using tubular constructs based on hyaluronic acid seeded with porcine aortic smooth muscle cells (SMC), that we recently showed to be adequate for the generation of vascular tissue. In perfused constructs mechanical stimulation importantly affected cell morphology, increased the incidence of cell proliferation and reduced apoptosis. However, extracellular matrix deposition, cytoskeletal organization and mechanical properties were poor. In rotated constructs cell proliferation was also higher and apoptosis lower than in static controls. Rotated constructs showed the highest ultimate stress and the lowest elastic modulus. Our data indicate that the rotating bioreactor is more efficient than the perfusion bioreactor and we then suggest that this method can be considered a valid alternative to complex bioreactor systems described in the literature.     

Electromechanical model of excitable tissue to study reentrant cardiac arrhythmias

We introduce the concept of a contracting excitable medium that is capable of conducting non-linear waves of excitation that in turn initiate contraction. Furthermore, these kinematic deformations have a feedback effect on the excitation properties of the medium. Electrical characteristics resemble basic models of cardiac excitation that have been used to successfully study mechanisms of reentrant cardiac arrhythmias in electrophysiology. We present a computational framework that employs electromechanical and mechanoelectric feedback to couple a three-variable FitzHugh–Nagumo-type excitation-tension model to the non-linear stress equilibrium equations, which govern large deformation hyperelasticity. Numerically, the coupled electromechanical model combines a finite difference method approach to integrate the excitation equations, with a Galerkin finite element method to solve the equations governing tissue mechanics. We present example computations demonstrating various effects of contraction on stationary rotating spiral waves and spiral wave break. We show that tissue mechanics significantly contributes to the dynamics of electrical propagation, and that a coupled electromechanical approach should be pursued in future electrophysiological modelling studies.     

Engineered heart tissue enables study of residual undifferentiated embryonic stem cell activity in a cardiac environment

Embryonic stem cell (ESC) derivatives are a promising cell source for cardiac cell therapy. Mechanistic studies upon cell injection in conventional animal models are limited by inefficient delivery and poor cell survival. As an alternative, we have used an engineered heart tissue (EHT) based on neonatal rat cardiomyocytes (CMs) cultivated with electrical field stimulation as an in vitro model to study cell injection. We injected (0.001, 0.01, and 0.1 million) and tracked (by qPCR and histology) undifferentiated yellow‐fluorescent protein transgenic mouse ESCs and Flk1 + /PDGFRα+ cardiac progenitor (CPs) cells, to investigate the effect of the cardiac environment on cell differentiation, as well as to test whether our in vitro model system could recapitulate the formation of teratoma‐like structures commonly observed upon in vivo ESC injection. By 8 days post‐injection, ESCs were spatially segregated from the cardiac cell population; however, ESC injection increased survival of CMs. The presence of ESCs blocked electrical conduction through the tissue, resulting in a 46% increase in the excitation threshold. Expression of mouse cardiac troponin I, was markedly increased in CP injected constructs compared to ESC injected constructs at all time points and cell doses tested. As early as 2 weeks, epithelial and ganglion‐like structures were observed in ESC injected constructs. By 4 weeks of ESC injection, teratoma‐like structures containing neural, epithelial, and connective tissue were observed in the constructs. Non‐cardiac structures were observed in the CP injected constructs only after extended culture (4 weeks) and only at high cell doses, suggesting that these cells require further enrichment or differentiation prior to transplantation. Our data indicate that the cardiac environment of host tissue and electrical field stimulation did not preferentially guide the differentiation of ESCs towards the cardiac lineage. In the same environment, injection of CP resulted in a more robust cardiac differentiation than injection of ESC. Our data demonstrate that the model‐system developed herein can be used to study the functional effects of candidate stem cells on the host myocardium, as well as to measure the residual activity of undifferentiated cells present in the mixture. Biotechnol. Bioeng. 2011; 108:704–719. © 2010 Wiley Periodicals, Inc.     

Electrosensitive polyacrylic acid/fibrin hydrogel facilitates cell seeding and alignment

Three-dimensional cell culture and conditioning is an effective means to guide cell distribution and patterning for tissue engineered constructs such as vascular grafts. Polyacrylic acid is known as an electroresponsive polymer, capable of transforming environmental stimuli like electrical energy to mechanical forces. In this study, we developed an electrosensitive and biocompatible hydrogel-based smart device composed of acrylic acid and fibrin as a tissue engineered construct to mechanically stimulate cells. Structural properties of the hydrogel were assessed by FTIR-ATR, scanning electron microscopy, prosimetry, and swelling measurement. Distribution and alignment of porcine smooth muscle cells (pSMCs) seeded on the surface of lyophilized hydrogels were evaluated and quantified by two-photon laser scanning microscopy. Smooth muscle cell tissue constructs exposed to 2 h of pulsatile electrical stimulation showed significantly enhanced cell penetration and alignment due to dynamic changes produced by alternative swelling and deswelling, in comparison with static samples. On the basis of the results, this hydrogel under electrical stimulation works as a mechanical pump, which can direct SMC alignment and facilitate infiltration and distribution of cells throughout the structure.     

Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts

The concept of regenerating diseased myocardium by implantation of tissue-engineered heart muscle is intriguing, but convincing evidence is lacking that heart tissues can be generated at a size and with contractile properties that would lend considerable support to failing hearts. Here we created large (thickness/diameter, 1-4 mm/15 mm), force-generating engineered heart tissue from neonatal rat heart cells. Engineered heart tissue formed thick cardiac muscle layers when implanted on myocardial infarcts in immune-suppressed rats. When evaluated 28 d later, engineered heart tissue showed undelayed electrical coupling to the native myocardium without evidence of arrhythmia induction. Moreover, engineered heart tissue prevented further dilation, induced systolic wall thickening of infarcted myocardial segments and improved fractional area shortening of infarcted hearts compared to controls (sham operation and noncontractile constructs). Thus, our study provides evidence that large contractile cardiac tissue grafts can be constructed in vitro, can survive after implantation and can support contractile function of infarcted hearts.     

Mathematical models for the study of electromechanical and mechanoelectrical coupling in the myocardium

Mathematical models have been developed to describe interactions of electrical, mechanical and chemical processes in cardiomyocytes. The models simulate wide range of experimental data on excitation-contraction coupling and, more importantly, on mechanoelectric feedback in heart muscle. The model results clearly show that mechano-dependence of intracellular calcium handling due to cooperative effects of contractile proteins activation plays a key role in cardiac mechanoelectric coupling. At the same time, mechanosensitive currents can also contribute to action potential responses to mechanical perturbations. Using this model to study the heterogeneous myocardium we have shown that temporal and functional electromechanical heterogeneity of coupled cardiomyocytes can essentially determine the myocardium contractility. Optimization of the electromechanical function of contractile system emerges from the fine coordination between the activation sequence of cardiomyocytes, their local electromechanical properties and the mechanical interaction during contraction.     

Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation

In vitro studies have used protein markers to distinguish between myogenic cells isolated from fast and slow skeletal muscles. The protein markers provide some support for the hypothesis that satellite cells from fast and slow muscles are different, but the data are equivocal. To test this hypothesis directly, three-dimensional skeletal muscle constructs were engineered from myogenic cells isolated from fast tibialis anterior (TA) and slow soleus (SOL) muscles of rats and functionality was tested. Time to peak twitch tension (TPT) and half relaxation time (RT_1/2) were 30% slower in constructs from the SOL. The slower contraction and relaxation times for the SOL constructs resulted in left shift of the force-frequency curve compared with those from the TA. Western blot analysis showed a 60% greater quantity of fast myosin heavy chain in the TA constructs. 14 days of chronic low-frequency electrical stimulation resulted in a 15% slower TPT and a 14% slower RT_1/2, but no change in absolute force production in the TA constructs. In SOL constructs, slow electrical stimulation resulted in an 80% increase in absolute force production with no change in TPT or RT_1/2. The addition of cyclosporine A did not prevent the increase in force in SOL constructs after chronic low-frequency electrical stimulation, suggesting that calcineurin is not responsible for the increase in force. We conclude that myogenic cells associated with a slow muscle are imprinted to produce muscle that contracts and relaxes slowly and that calcineurin activity cannot explain the response to a slow pattern of electrical stimulation. tissue engineering; calcineurin; electrical stimulation; engineered muscle; bioreactors     

Successful development of small diameter tissue-engineering vascular vessels by our novel integrally designed pulsatile perfusion-based bioreactor

Small-diameter (<4 mm) vascular constructs are urgently needed for patients requiring replacement of their peripheral vessels. However, successful development of constructs remains a significant challenge. In this study, we successfully developed small-diameter vascular constructs with high patency using our integrally designed computer-controlled bioreactor system. This computer-controlled bioreactor system can confer physiological mechanical stimuli and fluid flow similar to physiological stimuli to the cultured grafts. The medium circulating system optimizes the culture conditions by maintaining fixed concentration of O(2) and CO(2) in the medium flow and constant delivery of nutrients and waste metabolites, as well as eliminates the complicated replacement of culture medium in traditional vascular tissue engineering. Biochemical and mechanical assay of newly developed grafts confirm the feasibility of the bioreactor system for small-diameter vascular engineering. Furthermore, the computer-controlled bioreactor is superior for cultured cell proliferation compared with the traditional non-computer-controlled bioreactor. Specifically, our novel bioreactor system may be a potential alternative for tissue engineering of large-scale small-diameter vascular vessels for clinical use.     

Development of large engineered cartilage constructs from a small population of cells

Confronted with articular cartilage's limited capacity for self‐repair, joint resurfacing techniques offer an attractive treatment for damaged or diseased tissue. Although tissue engineered cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for the repair of large defects. As routine cell expansion methods tend to elicit negative effects on chondrocyte function, we have developed an approach to generate phenotypically stable, large‐sized engineered constructs (≥3 cm²) directly from a small amount of donor tissue or cells (as little as 20,000 cells to generate a 3 cm² tissue construct). Using rabbit donor tissue, the bioreactor‐cultivated constructs were hyaline‐like in appearance and possessed a biochemical composition similar to native articular cartilage. Longer bioreactor cultivation times resulted in increased matrix deposition and improved mechanical properties determined over a 4 week period. Additionally, as the anatomy of the joint will need to be taken in account to effectively resurface large affected areas, we have also explored the possibility of generating constructs matched to the shape and surface geometry of a defect site through the use of rapid‐prototyped defect tissue culture molds. Similar hyaline‐like tissue constructs were developed that also possessed a high degree of shape correlation to the original defect mold. Future studies will be aimed at determining the effectiveness of this approach to the repair of cartilage defects in an animal model and the creation of large‐sized osteochondral constructs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Electrical stimulation of cultured lepidopteran dorsal vessel tissue: an experiment for development of bioactuators

An insect dorsal vessel (DV) is well suited for a bioactuator since it is capable of contracting autonomously, and its tissue and cells are more environmentally robust under culturing conditions compared with mammalian tissue. In this study, electrical pulse stimulation was examined so as to regulate a bioactuator using the DV tissue. The DV tissue of a larva of Ctenoplusia agnate was assembled on a micropillar array, which was stimulated after culturing for about 3 wk. The contraction of the DV tissue was evaluated by image analysis to measure lateral displacements at the micropillar top. As a result, suitable stimulation conditions in a 35-mm petri dish were determined as: applied voltage of 10 V with 20-ms duration. Next, the time lag between the onset of electrical stimulus and the onset of mechanical contraction (electromechanical delay (EMD)) was estimated. A light-emitting diode (LED) was connected serially with the petri dish, and the LED flashed when electrical pulses were given. Movie images were analyzed in which electrical pulses made the DV tissue contract and the LED flashed virtually simultaneously; from these, the EMD was estimated as approximately 50 ms. These results suggest that the electrical pulse stimulation is capable of regulating the DV tissue, and the micropillar array is a useful biological tool to investigate physiological properties of muscle tissue.     

Induction of atrial natriuretic factor and myosin light chain-2 gene expression in cultured ventricular myocytes by electrical stimulation of contraction.

While hormonal stimuli and mechanical stretch can induced cardiac-specific gene expression and in some cases cellular hypertrophy, the relationship between myocyte contraction frequency, gene expression, and myocyte growth has not been well characterized. In this study a new model system was developed in which cultures of neonatal rat ventricular myocytes were subjected to long term pacing of contractions with pulsatile electrical stimulation. Myocytes submitted to electrical stimulation for 3 days displayed dramatic increases in cellular size and myofibrillar organization, and a 5-10-fold increase in the expression of the cardiac genes atrial natriuretic factor and myosin light chain-2. Atrial natriuretic factor expression induced by electrical stimulation of contractions was inhibited by nifedipine or W7, indicating a dependence on calcium influx and calmodulin activity. Phosphoinositide hydrolysis and cAMP formation were not affected by electrical stimulation suggesting that gene induction occurred independently of the activation of protein kinases C or A above basal levels. These findings show that the cellular events associated with contraction, such as changes in cytoplasmic free calcium levels and/or cellular stretch, may serve as important determinants of myocyte growth and cardiac gene expression.