Traction force microscopy of engineered cardiac tissues.
ABSTRACT: Cardiac tissue development and pathology have been shown to depend sensitively on microenvironmental mechanical factors, such as extracellular matrix stiffness, in both in vivo and in vitro systems. We present a novel quantitative approach to assess cardiac structure and function by extending the classical traction force microscopy technique to tissue-level preparations. Using this system, we investigated the relationship between contractile proficiency and metabolism in neonate rat ventricular myocytes (NRVM) cultured on gels with stiffness mimicking soft immature (1 kPa), normal healthy (13 kPa), and stiff diseased (90 kPa) cardiac microenvironments. We found that tissues engineered on the softest gels generated the least amount of stress and had the smallest work output. Conversely, cardiomyocytes in tissues engineered on healthy- and disease-mimicking gels generated significantly higher stresses, with the maximal contractile work measured in NRVM engineered on gels of normal stiffness. Interestingly, although tissues on soft gels exhibited poor stress generation and work production, their basal metabolic respiration rate was significantly more elevated than in other groups, suggesting a highly ineffective coupling between energy production and contractile work output. Our novel platform can thus be utilized to quantitatively assess the mechanotransduction pathways that initiate tissue-level structural and functional remodeling in response to substrate stiffness.
Project description:Engineering 3D human cardiac tissues is of great importance for therapeutic and pharmaceutical applications. As cardiac tissue substitutes, extracellular matrix-derived hydrogels have been widely explored. However, they exhibit premature degradation and their stiffness is often orders of magnitude lower than that of native cardiac tissue. There are no reports on establishing interconnected cardiomyocytes in 3D hydrogels at physiologically-relevant cell density and matrix stiffness. Here we bioengineer human cardiac microtissues by encapsulating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in chemically-crosslinked gelatin hydrogels (1.25 × 108/mL) with tunable stiffness and degradation. In comparison to the cells in high stiffness (16 kPa)/slow degrading hydrogels, hiPSC-CMs in low stiffness (2 kPa)/fast degrading and intermediate stiffness (9 kPa)/intermediate degrading hydrogels exhibit increased intercellular network formation, ?-actinin and connexin-43 expression, and contraction velocity. Only the 9 kPa microtissues exhibit organized sarcomeric structure and significantly increased contractile stress. This demonstrates that muscle-mimicking stiffness together with robust cellular interconnection contributes to enhancement in sarcomeric organization and contractile function of the engineered cardiac tissue. This study highlights the importance of intercellular connectivity, physiologically-relevant cell density, and matrix stiffness to best support 3D cardiac tissue engineering.
Project description:Hydrogels are a class of biomaterials used for a wide range of biomedical applications, including as a three-dimensional (3D) scaffold for cell culture that mimics the extracellular matrix (ECM) of native tissues. To understand the role of the ECM in the modulation of cardiac cell function, alginate was used to fabricate crosslinked gels with stiffness values that resembled embryonic (2.66?±?0.84 kPa), physiologic (8.98?±?1.29 kPa) and fibrotic (18.27?±?3.17 kPa) cardiac tissues. The average pore diameter and hydrogel swelling were seen to decrease with increasing substrate stiffness. Cardiomyocytes cultured within soft embryonic gels demonstrated enhanced cell spreading, elongation, and network formation, while a progressive increase in gel stiffness diminished these behaviors. Cell viability decreased with increasing hydrogel stiffness. Furthermore, cells in fibrotic gels showed enhanced protein expression of the characteristic cardiac stress biomarker, Troponin-I, while reduced protein expression of the cardiac gap junction protein, Connexin-43, in comparison to cells within embryonic gels. The results from this study demonstrate the role that 3D substrate stiffness has on cardiac tissue formation and its implications in the development of complex matrix remodeling-based conditions, such as myocardial fibrosis.
Project description:Cardiac cells mature in the first postnatal week, concurrent with altered extracellular mechanical properties. To investigate the effects of extracellular stiffness on cardiomyocyte maturation, we plated neonatal rat ventricular myocytes for 7 days on collagen-coated polyacrylamide gels with varying elastic moduli. Cells on 10 kPa substrates developed aligned sarcomeres, whereas cells on stiffer substrates had unaligned sarcomeres and stress fibers, which are not observed in vivo. We found that cells generated greater mechanical force on gels with stiffness similar to the native myocardium, 10 kPa, than on stiffer or softer substrates. Cardiomyocytes on 10 kPa gels also had the largest calcium transients, sarcoplasmic calcium stores, and sarcoplasmic/endoplasmic reticular calcium ATPase2a expression, but no difference in contractile protein. We hypothesized that inhibition of stress fiber formation might allow myocyte maturation on stiffer substrates. Treatment of maturing cardiomyocytes with hydroxyfasudil, an inhibitor of RhoA kinase and stress fiber-formation, resulted in enhanced force generation on the stiffest gels. We conclude that extracellular stiffness near that of native myocardium significantly enhances neonatal rat ventricular myocytes maturation. Deviations from ideal stiffness result in lower expression of sarcoplasmic/endoplasmic reticular calcium ATPase, less stored calcium, smaller calcium transients, and lower force. On very stiff substrates, this adaptation seems to involve RhoA kinase.
Project description:Activation of cardiac fibroblasts into myofibroblasts is considered to play an essential role in cardiac remodeling and fibrosis. A limiting factor in studying this process is the spontaneous activation of cardiac fibroblasts when cultured on two-dimensional (2D) culture plates. In this study, a simplified three-dimensional (3D) hydrogel platform of contractile cardiac tissue, stimulated by transforming growth factor-?1 (TGF-?1), is presented to recapitulate a fibrogenic microenvironment. It is hypothesized that the quiescent state of cardiac fibroblasts can be maintained by mimicking the mechanical stiffness of native heart tissue. To test this hypothesis, a 3D cell culture model consisting of cardiomyocytes and cardiac fibroblasts encapsulated within a mechanically engineered gelatin methacryloyl hydrogel, is developed. The study shows that cardiac fibroblasts maintain their quiescent phenotype in mechanically tuned hydrogels. Additionally, treatment with a beta-adrenergic agonist increases beating frequency, demonstrating physiologic-like behavior of the heart constructs. Subsequently, quiescent cardiac fibroblasts within the constructs are activated by the exogenous addition of TGF-?1. The expression of fibrotic protein markers (and the functional changes in mechanical stiffness) in the fibrotic-like tissues are analyzed to validate the model. Overall, this 3D engineered culture model of contractile cardiac tissue enables controlled activation of cardiac fibroblasts, demonstrating the usability of this platform to study fibrotic remodeling.
Project description:To investigate the transcriptomic profiling of human trabecular meshwork cells by increasing substrate stiffness and construct a cell model in vitro for the molecular mechanism exploration of glaucoma, normal HTM cells were cultured on collagen-coated polyacrylamide gels of tunable stiffness with Young’s moduli ranging from 1.1 kPa to 50 kPa. Then next-generation RNA sequencing (RNA-seq) was performed to obtain the transcriptomic profile of HTMCs. The expression of selected differentially expressed genes was validated by quantitative polymerase chain reaction (qPCR). Human normal and glaucomatous TM tissues were also obtained to perform RNA-seq analysis. After substrate stiffness treatment, genes related to glaucoma, including DCN, SPARC and CTGF, were significantly increased. RNA-seq analysis also confirmed the global alternation of gene transcriptional alternation. Cells under different densities exhibited similar profile change. We found ECM related genes were greatly activated by substrate stiffness, which was consistent with the molecular alternation of glaucoma. RNA-seq data from clinical samples also support the profiling alternation of cell model.The in vitro cell model could efficiently mimic the pathogenetic process of TM cells for glaucoma patients. Overall design: Normal HTM cells with two different densities were cultured on collagen-coated polyacrylamide gels of tunable stiffness with Young’s moduli ranging from 1.1 kPa to 50 kPa. TM tissues of glaucoma patients were also obtained. RNA-seq experiments were performed for all the samples.
Project description:The mechanical properties of the cellular microenvironment play a crucial role in modulating cell function, and many pathophysiological processes are accompanied by variations in extracellular matrix (ECM) stiffness. Lysyl oxidase (LOx) is one of the enzymes involved in several ECM-stiffening processes. Here, we engineered poly(ethylene glycol) (PEG)-based hydrogels with controlled mechanical properties in the range typical of soft tissues. These hydrogels were functionalized featuring free primary amines, which allows an additional chemical LOx-responsive behavior with increase in crosslinks and hydrogel elastic modulus, mimicking biological ECM-stiffening mechanisms. Hydrogels with elastic moduli in the range of 0.5-4 kPa were obtained after a first photopolymerization step. The increase in elastic modulus of the functionalized and enzyme-responsive hydrogels was also characterized after the second-step enzymatic reaction, recording an increase in hydrogel stiffness up to 0.5 kPa after incubation with LOx. Finally, hydrogel precursors containing HepG2 (bioinks) were used to form three-dimensional (3D) in vitro models to mimic hepatic tissue and test PEG-based hydrogel biocompatibility. Hepatic functional markers were measured up to 7 days of culture, suggesting further use of such 3D models to study cell mechanobiology and response to dynamic variation of hydrogels stiffness. The results show that the functionalized hydrogels presented in this work match the mechanical properties of soft tissues, allow dynamic variations of hydrogel stiffness, and can be used to mimic changes in the microenvironment properties of soft tissues typical of inflammation and pathological changes at early stages (e.g., fibrosis, cancer).
Project description:Cells that line major tissues in the body such as blood vessels, lungs and gastrointestinal tract experience deformation from mechanical strain with our heartbeat, breathing, and other daily activities. Tissues also remodel in both development and disease, changing their mechanical properties. Taken together, cells can experience vastly different mechanical cues resulting from the combination of these interdependent stimuli. To date, most studies of cellular mechanotransduction have been limited to assays in which variations in substrate stiffness and strain were not combined. Here, we address this technological gap by implementing a method that can simultaneously tune both substrate stiffness and mechanical strain. Substrate stiffness is controlled with different monomer and crosslinker ratios during polyacrylamide gel polymerization, and strain is transferred from the underlying silicone platform when stretched. We demonstrate this platform with polyacrylamide gels with elastic moduli at 6 kPa and 20 kPa in combination with two different silicone formulations. The gels remain attached with up to 50% applied strains. To validate strain transfer through the gels into cells, we employ particle-tracking methods and observe strain transmission via cell morphological changes.
Project description:We undertook mRNA microarray and gene ontology analyses to screen out substrate stiffness-dependent genes. Total mRNA were extracted from E17 cortical neurons grown on soft or stiff substrates at 5 or 16 hr time points. We identified 114 differentially-expressed mRNA transcripts in cells grown on 0.1 kPa and 20 kPa gels at the 5 hr time-point. Among them, 66 were upregulated in 0.1 kPa gel cultures and the remainder were downregulated (compared to cells grown on stiffer substrates). The expressions of three endocytic genes (Cltc, Dab2, and Myo6) and four adhesion genes(Vcl, Robo2, Nrcam, and Cad11) were confirmed by QGP and smRNA FISH. Overall design: We collected total RNA extracted from E17 cortical neurons on different substrates with various stiffness ( 0.1,1,20 kPa gels and glass). Three independent experiments were performed at two time points (5 hr and 16 hr).
Project description:Macrophages participate in immunity, tissue repair and tissue homeostasis. Activation of Toll-like receptors (TLRs) by conserved exogenous or endogenous structures initiates signaling cascades that result in the release of cytokines such as tumor necrosis factor ? (TNF?). Extracellular substrate stiffness is known to regulate functions of non-immune cells through a process called mechanotransduction, yet less is known about how physical cues affect macrophage function or TLR signaling. To investigate this question, we cultured murine primary bone marrow-derived macrophages (BMMs) and RAW264.7 cells on fibronectin-coated polyacrylamide (PA) gels of defined stiffnesses (1, 20 and 150 kPa) that approximate the physical properties of physiologic tissues. BMMs on all gels were smaller and more circular than those on rigid glass. Macrophages on intermediate stiffness 20 kPa PA gels were slightly larger and less circular than those on either 1 or 150 kPa. Secretion of the pro-inflammatory cytokine, TNF?, in response to stimulation of TLR4 and TLR9 was increased in macrophages grown on soft gels versus more rigid gels, particularly for BMMs. Inhibition of the rho-associated coiled-coil kinase 1/2 (ROCK1/2), key mediators in cell contractility and mechanotransduction, enhanced release of TNF? in response to stimulation of TLR4. ROCK1/2 inhibition enhanced phosphorylation of the TLR downstream signaling molecules, p38, ERK1/2 and NF?B. Our data indicate that physical cues from the extracellular environment regulate macrophage morphology and TLR signaling. These findings have important implications in the regulation of macrophage function in diseased tissues and offer a novel pharmacological target for the manipulation of macrophage function in vivo.
Project description:RATIONALE:Diabetes causes cardiac dysfunction, and understanding of its mechanism is still incomplete. One reason could be limitations in modeling disease conditions by current in vitro cardiomyocyte culture. Emerging evidence suggests that the mechanical properties of the microenvironment affect cardiomyocyte function. Nevertheless, the impact of high glucose on cardiomyocytes cultured on substrates whose stiffness matches that of the heart (approximately 15 kPa) is untested. OBJECTIVE:To test the hypothesis that cardiomyocytes cultured in microenvironments that mimic the mechanical properties of those for cardiomyocytes in vivo may reproduce the pathophysiology characteristics of diabetic cardiomyocytes ex vivo, such as the morphological appearance, ROS accumulation, mitochondrial dysfunction, apoptosis and insulin-stimulated glucose uptake. METHODS AND RESULTS:Isolated neonatal rat cardiomyocytes were seeded on 15 kPa polyacrylamide (PAA) gels, whose stiffness mimics that of heart tissues, or on glass coverslips, which represent conventional culture devices but are unphysiologically stiff. Cells were then cultured at 5 mM glucose, corresponding to the normal blood glucose level, or at high glucose levels (10 to 25 mM). Cytoskeletal disorganization, ROS accumulation, attenuated mitochondrial membrane potential and attenuated ATP level caused by high glucose and their reversal by a ROS scavenger were prominent in cells on gels, but not in cells on coverslips. The lack of response to ROS scavenging could be attributable to enhanced apoptosis in cells on glass, shown by enhanced DNA fragmentation and higher caspase 3/7 activity in cells on glass coverslips. High-glucose treatment also downregulated GLUT4 expression and attenuated insulin-stimulated glucose uptake only in cells on 15 kPa gels. CONCLUSION:Our data suggest that a mechanically compliant microenvironment increases the susceptibility of primary cardiomyocytes to elevated glucose levels, which enables these cells to serve as an innovative model for diabetic heart research.