Substrate mechanics unveil early structural and functional pathology in iPSC micro-tissue models of hypertrophic cardiomyopathy.

Hypertension is a major cause of morbidity and mortality in patients with hypertrophic cardiomyopathy (HCM), suggesting a potential role for mechanics in HCM pathogenesis. Here, we developed an in vitro physiological model to investigate how mechanics acts together with HCM-linked myosin binding protein C (MYBPC3) mutations to trigger disease. Micro-heart muscles (µHM) were engineered from induced pluripotent stem cell (iPSC)-derived cardiomyocytes bearing MYBPC3+/- mutations and challenged to contract against substrates of different elasticity. µHMs that worked against substrates with stiffness at or exceeding the stiffness of healthy adult heart muscle exhibited several hallmarks of HCM, including cellular hypertrophy, impaired contractile energetics, and maladaptive calcium handling. Remarkably, we discovered changes in troponin C and T localization in MYBPC3+/- µHM that were entirely absent in 2D culture. Pharmacologic studies suggested that excessive Ca2+ intake through membrane-embedded channels underlie the observed electrophysiological abnormalities. These results illustrate the power of physiologically relevant engineered tissue models to study inherited disease with iPSC technology.

Engineered tissue geometry and Plakophilin-2 regulate electrophysiology of human iPSC-derived cardiomyocytes.

Engineered heart tissues have been created to study cardiac biology and disease in a setting that more closely mimics in vivo heart muscle than 2D monolayer culture. Previously published studies suggest that geometrically anisotropic micro-environments are crucial for inducing "in vivo like" physiology from immature cardiomyocytes. We hypothesized that the degree of cardiomyocyte alignment and prestress within engineered tissues is regulated by tissue geometry and, subsequently, drives electrophysiological development. Thus, we studied the effects of tissue geometry on electrophysiology of micro-heart muscle arrays (µHM) engineered from human induced pluripotent stem cells (iPSCs). Elongated tissue geometries elicited cardiomyocyte shape and electrophysiology changes led to adaptations that yielded increased calcium intake during each contraction cycle. Strikingly, pharmacologic studies revealed that a threshold of prestress and/or cellular alignment is required for sodium channel function, whereas L-type calcium and rapidly rectifying potassium channels were largely insensitive to these changes. Concurrently, tissue elongation upregulated sodium channel (NaV1.5) and gap junction (Connexin 43, Cx43) protein expression. Based on these observations, we leveraged elongated µHM to study the impact of loss-of-function mutation in Plakophilin 2 (PKP2), a desmosome protein implicated in arrhythmogenic disease. Within µHM, PKP2 knockout cardiomyocytes had cellular morphology similar to what was observed in isogenic controls. However, PKP2-/- tissues exhibited lower conduction velocity and no functional sodium current. PKP2 knockout µHM exhibited geometrically linked upregulation of sodium channel but not Cx43, suggesting that post-translational mechanisms, including a lack of ion channel-gap junction communication, may underlie the lower conduction velocity observed in tissues harboring this genetic defect. Altogether, these observations demonstrate that simple, scalable micro-tissue systems can provide the physiologic stresses necessary to induce electrical remodeling of iPS-CM to enable studies on the electrophysiologic consequences of disease-associated genomic variants.

Mechanical Resistance to Micro-Heart Tissue Contractility unveils early Structural and Functional Pathology in iPSC Models of Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy is the most common cause of sudden death in the young. Because the disease exhibits variable penetrance, there are likely nongenetic factors that contribute to the manifestation of the disease phenotype. Clinically, hypertension is a major cause of morbidity and mortality in patients with HCM, suggesting a potential synergistic role for the sarcomeric mutations associated with HCM and mechanical stress on the heart. We developed an in vitro physiological model to investigate how the afterload that the heart muscle works against during contraction acts together with HCM-linked MYBPC3 mutations to trigger a disease phenotype. Micro-heart muscle arrays (µHM) were engineered from iPSC-derived cardiomyocytes bearing MYBPC3 loss-of-function mutations and challenged to contract against mechanical resistance with substrates stiffnesses ranging from the of embryonic hearts (0.4 kPa) up to the stiffness of fibrotic adult hearts (114 kPa). Whereas MYBPC3 +/- iPSC-cardiomyocytes showed little signs of disease pathology in standard 2D culture, µHMs that included components of afterload revealed several hallmarks of HCM, including cellular hypertrophy, impaired contractile energetics, and maladaptive calcium handling. Remarkably, we discovered changes in troponin C and T localization in the MYBPC3 +/- µHM that were entirely absent in 2D culture. Pharmacologic studies suggested that excessive Ca 2+ intake through membrane-embedded channels, rather than sarcoplasmic reticulum Ca 2+ ATPase (SERCA) dysfunction or Ca 2+ buffering at myofilaments underlie the observed electrophysiological abnormalities. These results illustrate the power of physiologically relevant engineered tissue models to study inherited disease mechanisms with iPSC technology.

Hydrogel-Assisted Double Molding Enables Rapid Replication of Stereolithographic 3D Prints for Engineered Tissue Design.

Tissue-engineered in vitro models are an essential tool in biomedical research. Tissue geometry is a key determinant of function, but controlling the geometry of microscale tissues remains challenging. Additive manufacturing approaches have emerged as a promising means for rapid and iterative changes in the geometry of microdevices. However, it has been shown that poly(dimethylsiloxane) (PDMS) cross-linking is often inhibited at the interface of materials printed with stereolithography. While approaches to replica mold stereolithographic three-dimensional (3D) prints have been described, these methods are inconsistent and often lead to print destruction when unsuccessful. Additionally, 3D-printed materials often leach toxic chemicals into directly molded PDMS. Here, we developed a double molding approach that allows precise replication of high-resolution stereolithographic prints into poly(dimethylsiloxane) (PDMS) elastomer, facilitating rapid design iterations and highly parallelized sample production. Inspired by lost wax casting, we used hydrogels as intermediary molds to transfer high-resolution features from high-resolution 3D prints into PDMS, while previously published work focused on enabling direct molding of PDMS onto 3D prints through the use of coatings and post-cross-linking treatments of the 3D print itself. Hydrogel mechanical properties, including cross-link density, predict replication fidelity. We demonstrate the ability of this approach to replicate a variety of shapes that would be impossible to create using photolithography techniques traditionally used to create engineered tissue designs. This method also enabled the replication of 3D-printed features into PDMS that would not be possible with direct molding as the stiffness of these materials leads to material fracture when unmolding, while the increased toughness in the hydrogels can elastically deform around complex features and maintain replication fidelity. Finally, we highlight the ability of this method to minimize the potential for toxic materials to transfer from the original 3D print into the PDMS replica, enhancing its use for biological applications. This minimization of the transfer of toxic materials has not been reported in other previously reported methods describing replication of 3D prints into PDMS, and we demonstrate its use through the creation of stem cell-derived microheart muscles. This method can also be used in future studies to understand the effects of geometry on engineered tissues and their constitutive cells.

Dynamic mechanobiology of cardiac cells and tissues: Current status and future perspective.

Mechanical forces impact cardiac cells and tissues over their entire lifespan, from development to growth and eventually to pathophysiology. However, the mechanobiological pathways that drive cell and tissue responses to mechanical forces are only now beginning to be understood, due in part to the challenges in replicating the evolving dynamic microenvironments of cardiac cells and tissues in a laboratory setting. Although many in vitro cardiac models have been established to provide specific stiffness, topography, or viscoelasticity to cardiac cells and tissues via biomaterial scaffolds or external stimuli, technologies for presenting time-evolving mechanical microenvironments have only recently been developed. In this review, we summarize the range of in vitro platforms that have been used for cardiac mechanobiological studies. We provide a comprehensive review on phenotypic and molecular changes of cardiomyocytes in response to these environments, with a focus on how dynamic mechanical cues are transduced and deciphered. We conclude with our vision of how these findings will help to define the baseline of heart pathology and of how these in vitro systems will potentially serve to improve the development of therapies for heart diseases.

The Use of 3D Polylactic Acid Scaffolds with Hydroxyapatite/Alginate Composite Injection and Mesenchymal Stem Cells as Laminoplasty Spacers in Rabbits.

Several types of laminoplasty spacer have been used to fill bone gaps and maintain a widened canal. A 3D scaffold can be used as an alternative spacer to minimize the risk observed in allografts or autografts. This study aims to evaluate the in vivo biocompatibility and tissue−scaffold integration of a polylactic acid (PLA) scaffold with the addition of alginate/hydroxyapatite (HA) and mesenchymal stem cell (MSc) injections. This is an experimental study with a pretest and post-test control group design. A total of 15 laminoplasty rabbit models were divided into five groups with variations in the autograft, PLA, HA/alginate, and MSc scaffold. In general, there were no signs of inflammation in most samples (47%), and there were no samples with areas of necrosis. There were no significant differences in the histopathological results and microstructural assessment between the five groups. This demonstrates that the synthetic scaffolds that we used had a similar tissue reaction and tissue integration profile as the autograft (p > 0.05). We recommend further translational studies in humans so that this biocompatible fabricated scaffold can be used to fill bone defects.

Elastomer-Grafted iPSC-Derived Micro Heart Muscles to Investigate Effects of Mechanical Loading on Physiology.

Mechanical loading plays a critical role in cardiac pathophysiology. Engineered heart tissues derived from human induced pluripotent stem cells (iPSCs) allow rigorous investigations of the molecular and pathophysiological consequences of mechanical cues. However, many engineered heart muscle models have complex fabrication processes and require large cell numbers, making it difficult to use them together with iPSC-derived cardiomyocytes to study the influence of mechanical loading on pharmacology and genotype-phenotype relationships. To address this challenge, simple and scalable iPSC-derived micro-heart-muscle arrays (µHM) have been developed. "Dog-bone-shaped" molds define the boundary conditions for tissue formation. Here, we extend the µHM model by forming these tissues on elastomeric substrates with stiffnesses spanning from 5 to 30 kPa. Tissue assembly was achieved by covalently grafting fibronectin to the substrate. Compared to µHM formed on plastic, elastomer-grafted µHM exhibited a similar gross morphology, sarcomere assembly, and tissue alignment. When these tissues were formed on substrates with different elasticity, we observed marked shifts in contractility. Increased contractility was correlated with increases in calcium flux and a slight increase in cell size. This afterload-enhanced µHM system enables mechanical control of µHM and real-time tissue traction force microscopy for cardiac physiology measurements, providing a dynamic tool for studying pathophysiology and pharmacology.