Surface-initiated assembly of protein nanofabrics.

Cells and tissues are self-organized within an extracellular matrix (ECM) composed of multifunctional, nano- to micrometer scale protein fibrils. We have developed a cell-free, surface-initiated assembly technique to rebuild this ECM structure in vitro. The matrix proteins fibronectin, laminin, fibrinogen, collagen type I, and collagen type IV are micropatterned onto thermosensitive surfaces as 1 to 10 nm thick, micrometer to centimeter wide networks, and released as flexible, free-standing nanofabrics. Independent control of microstructure and protein composition enables us to engineer the mechanical and chemical anisotropy. Fibronectin nanofabrics are highly extensible (>4-fold) and serve as scaffolds for engineering synchronously contracting, cardiac muscle; demonstrating biofunctionality comparable to cell-generated ECM.</p

Biohybrid thin films for measuring contractility in engineered cardiovascular muscle.

In vitro cardiovascular disease models need to recapitulate tissue-scale function in order to provide in vivo relevance. We have developed a new method for measuring the contractility of engineered cardiovascular smooth and striated muscle in vitro during electrical and pharmacological stimulation. We present a growth theory-based finite elasticity analysis for calculating the contractile stresses of a 2D anisotropic muscle tissue cultured on a flexible synthetic polymer thin film. Cardiac muscle engineered with neonatal rat ventricular myocytes and paced at 0.5 Hz generated stresses of 9.2 +/- 3.5 kPa at peak systole, similar to measurements of the contractility of papillary muscle from adult rats. Vascular tissue engineered with human umbilical arterial smooth muscle cells maintained a basal contractile tone of 13.1 +/- 2.1 kPa and generated another 5.1 +/- 0.8 kPa when stimulated with endothelin-1. These data suggest that this method may be useful in assessing the efficacy and safety of pharmacological agents on cardiovascular tissue.</p

Nuclear morphology and deformation in engineered cardiac myocytes and tissues.

Cardiac tissue engineering requires finely-tuned manipulation of the extracellular matrix (ECM) microenvironment to optimize internal myocardial organization. The myocyte nucleus is mechanically connected to the cell membrane via cytoskeletal elements, making it a target for the cellular response to perturbation of the ECM. However, the role of ECM spatial configuration and myocyte shape on nuclear location and morphology is unknown. In this study, printed ECM proteins were used to configure the geometry of cultured neonatal rat ventricular myocytes. Engineered one- and two-dimensional tissue constructs and single myocyte islands were assayed using live fluorescence imaging to examine nuclear position, morphology and motion as a function of the imposed ECM geometry during diastolic relaxation and systolic contraction. Image analysis showed that anisotropic tissue constructs cultured on microfabricated ECM lines possessed a high degree of nuclear alignment similar to that found in vivo; nuclei in isotropic tissues were polymorphic in shape with an apparently random orientation. Nuclear eccentricity was also increased for the anisotropic tissues, suggesting that intracellular forces deform the nucleus as the cell is spatially confined. During systole, nuclei experienced increasing spatial confinement in magnitude and direction of displacement as tissue anisotropy increased, yielding anisotropic deformation. Thus, the nature of nuclear displacement and deformation during systole appears to rely on a combination of the passive myofibril spatial organization and the active stress fields induced by contraction. Such findings have implications in understanding the genomic consequences and functional response of cardiac myocytes to their ECM surroundings under conditions of disease.</p

Hierarchical architecture influences calcium dynamics in engineered cardiac muscle.

Changes in myocyte cell shape and tissue structure are concurrent with changes in electromechanical function in both the developing and diseased heart. While the anisotropic architecture of cardiac tissue is known to influence the propagation of the action potential, the influence of tissue architecture and its potential role in regulating excitation-contraction coupling (ECC) are less well defined. We hypothesized that changes in the shape and the orientation of cardiac myocytes induced by spatial arrangement of the extracellular matrix (ECM) affects ECC. To test this hypothesis, we isolated and cultured neonatal rat ventricular cardiac myocytes on various micropatterns of fibronectin where they self-organized into tissues with varying degrees of anisotropy. We then measured the morphological features of these engineered myocardial tissues across several hierarchical dimensions by measuring cellular aspect ratio, myocyte area, nuclear density and the degree of cytoskeletal F-actin alignment. We found that when compared with isotropic tissues, anisotropic tissues have increased cellular aspect ratios, increased nuclear densities, decreased myocyte cell areas and smaller variances in actin alignment. To understand how tissue architecture influences cardiac function, we studied the role of anisotropy on intracellular calcium ([Ca(2+)](i)) dynamics by characterizing the [Ca(2+)](i)-frequency relationship of electrically paced tissues. When compared with isotropic tissues, anisotropic tissues displayed significant differences in [Ca(2+)](i) transients, decreased diastolic baseline [Ca(2+)](i) levels and greater [Ca(2+)](i) influx per cardiac cycle. These results suggest that ECM cues influence tissue structure at cellular and subcellular levels and regulate ECC.</p

Functional differences in engineered myocardium from embryonic stem cell-derived versus neonatal cardiomyocytes.

<p>Stem cell-derived cardiomyocytes represent unique tools for cell- and tissue-based regenerative therapies, drug discovery and safety, and studies of fundamental heart-failure mechanisms. However, the degree to which stem cell-derived cardiomyocytes compare to mature cardiomyocytes is often debated. We reasoned that physiological metrics of engineered cardiac tissues offer a means of comparison. We built laminar myocardium engineered from cardiomyocytes that were differentiated from mouse embryonic stem cell-derived cardiac progenitors or harvested directly from neonatal mouse ventricles, and compared their anatomy and physiology in vitro. Tissues assembled from progenitor-derived myocytes and neonate myocytes demonstrated similar cytoskeletal architectures but different gap junction organization and electromechanical properties. Progenitor-derived myocardium had significantly less contractile stress and slower longitudinal conduction velocity than neonate-derived myocardium, indicating that the developmental state of the cardiomyocytes affects the electromechanical function of the resultant engineered tissue. These data suggest a need to establish performance metrics for future stem cell applications.</p

Controlling the contractile strength of engineered cardiac muscle by hierarchal tissue architecture.

<p>The heart is a muscular organ with a wrapping, laminar structure embedded with neural and vascular networks, collagen fibrils, fibroblasts, and cardiac myocytes that facilitate contraction. We hypothesized that these non-muscle components may have functional benefit, serving as important structural alignment cues in inter- and intra-cellular organization of cardiac myocytes. Previous studies have demonstrated that alignment of engineered myocardium enhances calcium handling, but how this impacts actual force generation remains unclear. Quantitative assays are needed to determine the effect of alignment on contractile function and muscle physiology. To test this, micropatterned surfaces were used to build 2-dimensional myocardium from neonatal rat ventricular myocytes with distinct architectures: confluent isotropic (serving as the unaligned control), confluent anisotropic, and 20 μm spaced, parallel arrays of multicellular myocardial fibers. We combined image analysis of sarcomere orientation with muscular thin film contractile force assays in order to calculate the peak sarcomere-generated stress as a function of tissue architecture. Here we report that increasing peak systolic stress in engineered cardiac tissues corresponds with increasing sarcomere alignment. This change is larger than would be anticipated from enhanced calcium handling and increased uniaxial alignment alone. These results suggest that boundary conditions (heterogeneities) encoded in the extracellular space can regulate muscle tissue function, and that structural organization and cytoskeletal alignment are critically important for maximizing peak force generation.</p

A tissue-engineered jellyfish with biomimetic propulsion.

<p>Reverse engineering of biological form and function requires hierarchical design over several orders of space and time. Recent advances in the mechanistic understanding of biosynthetic compound materials, computer-aided design approaches in molecular synthetic biology 4,5 and traditional soft robotics, and increasing aptitude in generating structural and chemical micro environments that promote cellular self-organization have enhanced the ability to recapitulate such hierarchical architecture in engineered biological systems. Here we combined these capabilities in a systematic design strategy to reverse engineer a muscular pump. We report the construction of a freely swimming jellyfish from chemically dissociated rat tissue and silicone polymer as a proof of concept. The constructs, termed 'medusoids', were designed with computer simulations and experiments to match key determinants of jellyfish propulsion and feeding performance by quantitatively mimicking structural design, stroke kinematics and animal-fluid interactions. The combination of the engineering design algorithm with quantitative benchmarks of physiological performance suggests that our strategy is broadly applicable to reverse engineering of muscular organs or simple life forms that pump to survive.</p

Generation of functional ventricular heart muscle from mouse ventricular progenitor cells.

The mammalian heart is formed from distinct sets of first and second heart field (FHF and SHF, respectively) progenitors. Although multipotent progenitors have previously been shown to give rise to cardiomyocytes, smooth muscle, and endothelial cells, the mechanism governing the generation of large numbers of differentiated progeny remains poorly understood. We have employed a two-colored fluorescent reporter system to isolate FHF and SHF progenitors from developing mouse embryos and embryonic stem cells. Genome-wide profiling of coding and noncoding transcripts revealed distinct molecular signatures of these progenitor populations. We further identify a committed ventricular progenitor cell in the Islet 1 lineage that is capable of limited in vitro expansion, differentiation, and assembly into functional ventricular muscle tissue, representing a combination of tissue engineering and stem cell biology.</p