Dissertations / Theses on the topic 'Engineered Heart Muscle Tissues'

The skeletal muscle is the largest tissue of the human body. Its main function is to generate contractile forces, essential for locomotion, thermogenesis, and metabolism. Fundamental research on skeletal muscle in health and disease, and preclinical research for new therapies, are currently based on 2D in vitro cell cultures and in vivo animal models. However, these strategies have important shortcomings. For instance, conventional cell culture models cannot emulate the complex 3D architecture of native skeletal muscle, and the species-specific differences in animal models limit their relevance to humans. In contrast, engineered skeletal muscle tissues are emerging as in vitro 3D cell culture models that complement existing 2D strategies. These engineered tissues can offer an improved microenvironment resembling native muscle tissue, comprised of bundles of aligned, multinucleated fibers. Therefore, the main objective of this thesis was to develop 3D skeletal muscle tissues for in vitro studies of muscle metabolism and disease modeling. Skeletal muscle precursor cells were encapsulated in microfabricated hydrogel scaffolds, introducing the appropriate topographical and microenvironmental cues to guide muscle fiber formation. First, photocrosslinkable gelatin methacryloyl (GelMA)-based composite hydrogels were synthesized and evaluated as cell-laden bioinks for 3D bioprinting of murine skeletal muscle tissue. The fabrication conditions were optimized to ensure the biocompatibility of the process and promote in vitro myogenesis. Our results demonstrated that the composite hydrogels have a higher resistance to degradation than GelMA hydrogels. Thus, the bioprinted scaffolds maintained their 3D structure over a prolonged culture period. Furthermore, the shear stress during extrusion bioprinting combined with the appropriate scaffold geometry resulted in highly aligned myoblasts that correctly differentiated into multinucleated myotubes. Considering these results, GelMA-carboxymethylcellulose methacrylate (CMCMA) hydrogels were then used to generate skeletal muscle microtissues in long-lasting cell cultures. Photomold patterning of cell-laden GelMA-CMCMA filaments led to the formation of highly aligned 3D myotubes expressing sarcomeric proteins. Moreover, the presented protocols were highly biocompatible and reproducible. Murine skeletal muscle microtissues were fabricated in a microfluidic platform integrated with an electrical stimulation system and biosensors for monitoring muscle metabolism in situ. Here, we measured the contraction-induced release of muscle-secreted cytokines upon electrical or biological stimulation. The obtained results confirmed the endocrine function of the bioengineered tissues, obtaining in vivo-like responses upon exercise or endotoxin-induced inflammation. Then, the photomold patterning protocol was optimized for human cells to develop the first in vitro 3D model of myotonic dystrophy type 1 (DM1) human skeletal muscle. DM1 is the most prevalent hereditary myopathy in adults, and there is no effective treatment to date. We proved that 3D micropatterning enhances DM1 myotube formation compared to 2D cultures. Furthermore, we detected the reduced thickness of 3D DM1 myotubes compared to healthy controls, which was proposed as a new in vitro structural phenotype. Thus, as a proof-of-concept, we demonstrated that treatment with an antisense oligonucleotide, antagomiR-23b, could rescue both molecular and structural phenotypes in these bioengineered DM1 muscle tissues. Finally, animal-derived components were eliminated to develop in vitro functional tissues in xeno-free cell culture as a next step towards improving bioengineered human skeletal muscle tissues. Cell-laden nanocomposite hydrogels consisting of human platelet lysate and functionalized cellulose nanocrystals (HUgel) were fabricated in hydrogel casting platforms that implemented uniaxial tension during matrix remodeling. We modulated the content of cellulose nanocrystals to tune the mechanical and biological properties of HUgel and favor the formation of long, highly aligned myotube bundles. Additionally, we performed in situ force measurements of electrical stimulation-induced contractions. Altogether, the results presented in this thesis provide promising approaches to advanced cell culture models of skeletal muscle tissue that could be valuable tools for fundamental studies, disease modeling, and future personalized medicine.
El músculo esquelético tiene funciones esenciales para la salud que pueden verse afectadas por enfermedades neuromusculares o metabólicas. Actualmente, la investigación fundamental y preclínica se basa en cultivos celulares en 2D y modelos animales. Sin embargo, estos ensayos tienen relevancia limitada para la salud humana. En cambio, modelos in vitro de tejidos 3D que mimeticen la arquitectura y funcionalidad del músculo esquelético, podrían complementar las estrategias 2D tradicionales. Por lo tanto, el objetivo principal de esta tesis fue desarrollar tejidos de músculo esquelético en 3D para estudios sobre el metabolismo muscular y modelos de enfermedades in vitro. Los tejidos fueron desarrollados mediante diferentes técnicas de microfabricación de hidrogeles, en los que se encapsularon células precursoras del músculo esquelético introduciendo las señales topográficas adecuadas para guiar la formación de fibras musculares. Las propiedades de estos biomateriales fueron optimizadas para garantizar su biocompatibilidad y promover la miogénesis. Estos biomateriales mantienen su estructura durante periodos de cultivo prolongados, permitiendo la formación y diferenciación de miotubos 3D altamente alineados. La función endócrina de los tejidos fue evaluada utilizando un dispositivo músculo-en-un-chip, con el que fue posible medir la liberación de citoquinas secretadas tras estimulación eléctrica o biológica. Posteriormente, se desarrolló el primer modelo 3D de músculo esquelético humano para la distrofia miotónica tipo 1. Como prueba de concepto, demostramos que el tratamiento con un oligonucleótido antisentido, antagomiR-23b, podría rescatar fenotipos moleculares y estructurales en los tejidos fabricados a partir de células de pacientes. Finalmente, se desarrollaron tejidos funcionales en cultivos celulares xeno-free, con el objetivo de incrementar la relevancia de modelos humanos en los que fue posible medir las fuerzas generada por tejidos contráctiles. En conjunto, los resultados de esta tesis proporcionan enfoques prometedores para modelos avanzados de músculo esquelético que podrían ser herramientas valiosas para estudios fundamentales, modelos de enfermedades y medicina personalizada.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 101-112).
Engineered skeletal muscle tissue is a three-dimensional contractile tissue made from muscle cells and the extracellular matrix (ECM). It can be used as a drug testing platform or an implantable tissue, but its practical use has been limited by inferior contractile performance and small size compared to natural muscles. This thesis aims to implement environmental cues and essential elements of natural muscles to improve the contractile performance and increase its size beyond the diffusion limit. Firstly, inspired by the observation that the natural muscles are exposed to electric potentials from neurons in combination with mechanical stretching from surrounding muscles, a new muscle training system was developed to apply coordinated electrical and mechanical stimulation.
Both the experimental results and the mechanistic model suggest the combined stimulation reorients the ECM fibers in such a way that the parallel ECM stiffness is reduced, while the serial ECM stiffness is increased, which reduces resistance to muscle contraction and increases force transmission in the engineered muscles, respectively. Secondly, large-sized natural muscles are fully vascularized so that oxygen and nutrients can be supplied. However, vascularization of the engineered skeletal muscle has been challenging because the microenvironmental requirement for differentiating myoblasts is incompatible with the one for culturing endothelial cells. In contrast, the natural muscle tissue has a compartment structure, where endothelial cells are exposed to blood plasma, while myoblasts are surrounded by interstitial fluid.
In this thesis, we modeled the natural fluid compartments by creating an in vitro perfusable vasculature running through a skeletal muscle tissue with physiologic cell density. The tissue is designed to have a coaxial tubular shape with a perfusable vasculature at the center. Through the in vitro fluid compartments, endothelial cells are exposed to endothelial cell growth medium running through the vascular channel, and the skeletal muscle cells are surrounded by muscle differentiation medium. By using this platform, engineered muscle tissue was successfully scaled up from microscale to subcentimeter scale. This platform also enabled to show that coculturing with the two separate media from an early stage of muscle differentiation leads to increased contractile force, thicker myotubes, and more muscle differentiation compared to using a single coculture medium.
Furthermore, the engineered skeletal muscles were further vascularized by inducing angiogenic sprouting from the vascular channel penetrating into the muscle tissue. This thesis will contribute to utilizing engineered skeletal muscles in practical applications with improved functionalities and provide a new model to study heterotypic cell-cell interactions in skeletal muscle tissues.
by Hyeon Yu Kim.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Mechanical Engineering

Myocardial infarction is one of the most severe acute pathologies of the cardiovascular system. The adult mammalian heart is indeed unable to regenerate most of the lost cardiomyocytes (CMs) after cardiac injury. The loss of cardiomyocytes and the myocardial scarring after myocardial infarction eventually compromise contractility of the remaining myocardium, leading to heart failure. Therefore, promoting heart regeneration is one of the most crucial therapeutic targets in cardiovascular medicine. The lack of regenerative response is due to the loss of proliferative capacity of adult CMs which in mice occurs seven days after birth. One of the events which occur at birth in neonatal hearts is a sudden increase in mechanical loading that may contribute to switching mammal CMs phenotype from neonatal proliferative to adult postmitotic. Therefore, understanding the role of mechanotransduction in regulating the balance between CM proliferation and maturation may bring us to the identification of unknown mediators and new potential strategies to induce cardiac regeneration. Regulation of mechanical load in bi-dimensional cultures of CMs can be achieved in different ways, however, the poor degree of CM maturation that can be reached in a culture dish together with the lack of a tridimensional structure represent a major limitation to performing mechanotransduction studies. In our work we developed a novel system to study mechanotransduction of CMs based on 3D culture of cardiac cells, called engineered heart tissues (EHTs), that allow us to reduce or increase mechanical loading easily. We show that the three-dimensional setting of the culture leads to an improvement of CM maturation that may be reversed by mechanical unloading inducing cell proliferation. On the other hand, a persisting overload stimulus eventually induces CM switch to a more mature phenotype with a low degree of proliferation. Also, we have focused our work on developing an EHT-based model able to recapitulate the adult infarct injury in order to investigate the biology of cardiac regeneration in this setting. Specifically, we set up a cryoinjury protocol that is relatively easy and reproducible. Cryoinjury produces a localized injury without compromising EHT’s structural integrity. Indeed, all the EHTs subjected to cryoinjury preserved their contractile activity and did not show any significant change in shape. Considering that EHTs are unpurified cardiac culture rich in fibroblast and endothelial cells, we observed that cryoinjury induce fibroblast proliferation and activation together with a lack of proliferative response of the cardiomyocytes which is, on the other hand, present in the early phase of EHT’s development, similarly to what has been shown in mice and rats after myocardial infarction, highlighting the robustness of our cryoinjury approach as a model to investigate cardiac regeneration.

The human heart has poor endogenous regeneration. If myocytes are lost due to injury, the myocardium is unable to restore its myocyte content and instead undergoes compensatory hypertrophy and remodeling. Cardiac tissue engineering aims to recreate and provide functional myocardium that replaces the injured myocardium. In this study, human engineered heart muscle (EHM) from cardiomyogenically differentiated human embryonic stem cells was generated. EHMs consisted of elongated, anisotropically organized cardiomyocyte bundles and responded “physiologically” to increasing calcium concentrations. To generate large myocardium capable of encompassing the ventricles, a novel process to systematically upscale the dimensions of engineered myocardium to a humanized Biological Ventricular Assisted Device (hBioVAD) was introduced. The hBioVADs formed a “pouch-like” myocardium at rabbit heart dimensions and were beating spontaneously. Further enhancement by biomimetic pulsatile loading generated “more mature” myocardium. Additional paracrine functionality was integrated by generating insulin-like growth factor-1 (IGF-1) secreting fibroblasts for tissue engineering applications. IGF-1 release induced higher levels of Akt phosphorylation and hypertrophy in cardiomyocytes resulting in increased force generation of EHM. Finally, feasibility of “paraBioVAD” (IGF-1 cell line and cardiomyocytes) implantation was demonstrated in a healthy rat model. Histological observations demonstrated engraftment on the heart and the presence of vascular structures. In conclusion, a humanized “paraBioVAD” technology for mechanic and paracrine heart support was developed. Future studies will assess its therapeutic utility in heart failure

Indiana University-Purdue University Indianapolis (IUPUI)
Factors released from coronary perivascular adipose tissue (PVAT), which surrounds large coronary arteries, have been implicated in the development of coronary disease. However, the precise contribution of coronary PVAT-derived factors to the initiation and progression of coronary vascular dysfunction remains ill defined. Accordingly, this investigation was designed to delineate the mechanisms by which PVAT-derived factors influence obesity-induced coronary smooth muscle dysfunction. Isometric tension studies of coronary arteries from lean and obese swine demonstrated that both lean and obese coronary PVAT attenuate vasodilation via inhibitory effects on smooth muscle K+ channels. Specifically, lean coronary PVAT attenuated KCa and KV7 channel-mediated dilation, whereas obese coronary PVAT impaired KATP channel-mediated dilation. Importantly, these effects were independent of alterations in underlying smooth muscle function in obese arteries. The PVAT-derived factor calpastatin impaired adenosine dilation in lean but not obese arteries, suggesting that alterations in specific factors may contribute to the development of smooth muscle dysfunction. Further studies tested the hypothesis that leptin, which is expressed in coronary PVAT and is upregulated in obesity, acts as an upstream mediator of coronary smooth muscle dysfunction. Long-term administration (3 day culture) of obese concentrations of leptin markedly altered the coronary artery proteome, favoring pathways associated with calcium signaling and cellular proliferation. Isometric tension studies demonstrated that short-term (30 min) exposure to leptin potentiated depolarization-induced contraction of coronary arteries and that this effect was augmented following longer-term leptin administration (3 days). Inhibition of Rho kinase reduced leptin-mediated increases in coronary artery contractions. Acute treatment was associated with increased Rho kinase activity, whereas longer-term exposure was associated with increases in Rho kinase protein abundance. Alterations in Rho kinase signaling were also associated with leptin-mediated increases in coronary vascular smooth muscle proliferation. These findings provide novel mechanistic evidence linking coronary PVAT with vascular dysfunction and further support a role for coronary PVAT in the pathogenesis of coronary disease.

Indiana University-Purdue University Indianapolis (IUPUI)
Obesity increases cardiovascular disease risk and is associated with factors of the “metabolic syndrome” (MetS), a disorder including hypertension, hypercholesterolemia and/or impaired glucose tolerance. Expanding adipose and subsequent inflammation is implicated in vascular dysfunction in MetS. Perivascular adipose tissue (PVAT) surrounds virtually every artery and is capable of releasing factors that influence vascular reactivity, but the effects of PVAT in the coronary circulation are unknown. Accordingly, the goal of this investigation was to delineate mechanisms by which lean vs. MetS coronary PVAT influences vasomotor tone and the coronary PVAT proteome. We tested the hypothesis that MetS alters the functional expression and vascular contractile effects of coronary PVAT in an Ossabaw swine model of the MetS. Utilizing isometric tension measurements of coronary arteries in the absence and presence of PVAT, we revealed the vascular effects of PVAT vary according to anatomical location as coronary and mesenteric, but not subcutaneous adipose tissue augmented coronary artery contractions to KCl. Factors released from coronary PVAT increase baseline tension and potentiate constriction of isolated coronary arteries relative to the amount of adipose tissue present. The effects of coronary PVAT are elevated in the setting of MetS and occur independent of endothelial function. MetS is also associated with substantial alterations in the coronary PVAT proteome and underlying increases in vascular smooth muscle Ca2+ handling via CaV1.2 channels, H2O2-sensitive K+ channels and/or upstream mediators of these ion channels. Rho-kinase signaling participates in the increase in coronary artery contractions to PVAT in lean, but not MetS swine. These data provide novel evidence that the vascular effects of PVAT vary according to anatomic location and are influenced by the MetS phenotype.