161 research outputs found

    Intrinsically conductive polymers for striated cardiac muscle repair

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    One of the most important features of striated cardiac muscle is the excitability that turns on the excitation-contraction coupling cycle, resulting in the heart blood pumping function. The function of the heart pump may be impaired by events such as myocardial infarction, the consequence of coronary artery thrombosis due to blood clots or plaques. This results in the death of billions of cardiomyocytes, the formation of scar tissue, and consequently impaired contractility. A whole heart transplant remains the gold standard so far and the current pharmacological approaches tend to stop further myocardium deterioration, but this is not a long-term solution. Electrically conductive, scaffold-based cardiac tissue engineering provides a promising solution to repair the injured myocardium. The non-conductive component of the scaffold provides a biocompatible microenvironment to the cultured cells while the conductive component improves intercellular coupling as well as electrical signal propagation through the scar tissue when implanted at the infarcted site. The in vivo electrical coupling of the cells leads to a better regeneration of the infarcted myocardium, reducing arrhythmias, QRS/QT intervals, and scar size and promoting cardiac cell maturation. This review presents the emerging applications of intrinsically conductive polymers in cardiac tissue engineering to repair post-ischemic myocardial insult

    Engineering of Electrically Conductive Cardiac Microtissues to Study the Influence of Gold Nanomaterials on Maturation and Electrophysiology of Cardiomyocytes

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    abstract: Myocardial infarction (MI) remains the leading cause of mortality and morbidity in the U.S., accounting for nearly 140,000 deaths per year. Heart transplantation and implantation of mechanical assist devices are the options of last resort for intractable heart failure, but these are limited by lack of organ donors and potential surgical complications. In this regard, there is an urgent need for developing new effective therapeutic strategies to induce regeneration and restore the loss contractility of infarcted myocardium. Over the past decades, regenerative medicine has emerged as a promising strategy to develop scaffold-free cell therapies and scaffold-based cardiac patches as potential approaches for MI treatment. Despite the progress, there are still critical shortcomings associated with these approaches regarding low cell retention, lack of global cardiomyocytes (CMs) synchronicity, as well as poor maturation and engraftment of the transplanted cells within the native myocardium. The overarching objective of this dissertation was to develop two classes of nanoengineered cardiac patches and scaffold-free microtissues with superior electrical, structural, and biological characteristics to address the limitations of previously developed tissue models. An integrated strategy, based on micro- and nanoscale technologies, was utilized to fabricate the proposed tissue models using functionalized gold nanomaterials (GNMs). Furthermore, comprehensive mechanistic studies were carried out to assess the influence of conductive GNMs on the electrophysiology and maturity of the engineered cardiac tissues. Specifically, the role of mechanical stiffness and nano-scale topographies of the scaffold, due to the incorporation of GNMs, on cardiac cells phenotype, contractility, and excitability were dissected from the scaffold’s electrical conductivity. In addition, the influence of GNMs on conduction velocity of CMs was investigated in both coupled and uncoupled gap junctions using microelectrode array technology. Overall, the key contributions of this work were to generate new classes of electrically conductive cardiac patches and scaffold-free microtissues and to mechanistically investigate the influence of conductive GNMs on maturation and electrophysiology of the engineered tissues.Dissertation/ThesisSupplementary VideosDoctoral Dissertation Biomedical Engineering 201

    Nanowired Human Cardiac Spheroids for Cardiac Regenerative Medicine

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    3D scaffold-free spherical micro-tissue (spheroids) holds great potential in tissue engineering as building blocks to fabricate the functional tissues or organs in vitro. To date, agarose based hydrogel molds have been extensively used to facilitate fusion process of tissue spheroids. As a molding material, agarose typically requires low temperature plates for gelation and/or heated dispenser units. Here, we developed an alginate-based, direct 3D mold-printing technology: 3D printing micro-droplets of alginate solution into biocompatible, bio-inert alginate hydrogel molds for the fabrication of scaffold-free tissue engineering constructs. Specifically, we developed a 3D printing technology to deposit micro-droplets of alginate solution on calcium containing substrates in a layer-by-layer fashion to prepare ring-shaped 3D agarose hydrogel molds. Tissue spheroids composed of 50% human endothelial cells and 50% human smooth muscle cells were robotically dispensed into the 3D printed alginate molds using a 3D printer, and were found to rapidly fuse into toroid-shaped tissue units. Histological and immunofluorescence analysis indicated that the cells secreted collagen type I playing a critical role in promoting cell-cell adhesion, tissue formation and maturation. The current inability to derive mature cardiomyocytes (CMs) from human pluripotent stem cells (hiPSC) has been the limiting step for transitioning this powerful technology into clinical therapies. To address this, scaffold-based tissue engineering approaches have been utilized to mimic heart development in vitro and promote maturation of CMs derived from hiPSC. While scaffolds can provide 3D microenvironments, current scaffolds lack the matched physical/chemical/biological properties of native extracellular environments. On the other hand, scaffold-free, 3D cardiac spheroids prepared by seeding CMs into agarose microwells were shown to improve cardiac functions. However, CMs within the spheroids could not assemble in a controlled manner and led to compromised, unsynchronized contractions. Here we show, for the first time, that incorporation of a trace amount (i.e., ~0.004% w/v) of electrically conductive silicon nanowires (e-SiNWs) in otherwise scaffold-free cardiac spheroids can form an electrically conductive network, leading to synchronized and significantly enhanced contraction (i.e., \u3e55% increase in average contraction amplitude), resulting in significantly more advanced cellular structural and contractile maturation. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. Here, we examined two important factors that can affect functions of the nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. To examine the first factor, we prepared hiPSC cardiac spheroids with four different sizes by varying cell number per spheroid (~0.5k, ~1k, ~3k, ~7k cells/spheroid). Spheroids with ~3k cells/spheroid was found to maximize the beneficial effects of the 3D spheroid microenvironment. This result was explained with a semi-quantitative theory that considers two competing factors: 1) the improved 3D cell-cell adhesion, and 2) the reduced oxygen supply to the center of spheroids with the increase of cell number. Also, the critical role of electrical conductivity of silicon nanowires has been confirmed in improving tissue function of hiPSC cardiac spheroids. These results lay down a solid foundation to develop suitable nanowired hiPSC cardiac spheroids as an innovative cell delivery system to treat cardiovascular diseases. We reasoned that the presence of e-SiNWs in the injectable spheroids improves their ability to receive exogenous electromechanical pacing from the host myocardium to enhance their integration with host tissue post-transplantation. In this study, we examined the cardiac biocompatibility of the e-SiNWs and cell retention, engraftment and integration after injection of the nanowired hiPSC cardiac spheroids into adult rat hearts. Our results showed that the e-SiNWs caused minimal toxicity to rat adult hearts after intramyocardial injection. Further, the nanowired spheroids were shown to significantly improve cell retention and engraftment, when compared to dissociated hiPSC-CMs and unwired spheroids. The 7-days-old nanowired spheroid grafts showed alignment with the host myocardium and development of sarcomere structures. The 28-days-old nanowired spheroid grafts showed gap junctions, mechanical junctions and vascular integration with host myocardium. Together, our results clearly demonstrate the remarkable potential of the nanowired spheroids as cell delivery vehicles to treat cardiovascular diseases

    나노입자 기반, 세포 거동조절을 통한

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    학위논문 (박사)-- 서울대학교 대학원 공과대학 화학생물공학부, 2017. 8. 김병수.Myocardial infarction (MI) is one of the leading causes of death worldwide, and accounts for majority of cardiac-associated disorders. MI originates from reduced blood supply to the heart and subsequent cardiac necrosis, hence, tissue engineering approaches are required for successful cardiac repair. Recently, various types of cells and nanoparticles drew significant attention as efficient therapeutics for cardiac repair, and combinatorial treatments between cells and nanoparticles have been introduced. Even so, majority of nanomaterials have been mostly utilized as delivery carriers, and studies regarding how nanoparticles actively modulate cell behaviors and potentiate the therapeutic efficacy of these cells remain unexplored. Current dissertation presents the integration of stem or immune cells with most widely used nanoparticles, such as iron oxide nanoparticles or graphene oxide, for the treatment of MI. More specifically, biological role of these nanoparticles and how innate chemical properties of nanoparticles mediate cell behaviors is elucidated. Major goals of dissertation are summarized as follows1) Elucidation of metal ion-delivering capability of iron oxide nanoparticles, and investigation on the modulation of cell signaling transduction and development of intercellular gap junction crosstalk 2) Elucidation of sp2 chemistry-based intracellular antioxidant chemistry of graphene oxide, and its immune modulatory function for therapeutic polarization of macrophages in MI treatment. First, we showed that iron oxide nanoparticles can modulate intracellular signaling transduction in cardiac cells, and improve intercellular gap junction formation in stem cell co-culture. Co-culture of stem cells with cardiac cells has windowed a platform for cardiac priming of MSCs prior to in vivo transplantation, and active gap junctional crosstalk between stem cells and cardiac cells are crucial in stem cell modification. In this study, we report that iron oxide nanoparticles can augment the expression of gap junction protein connexin 43 in cardiac cells to better form gap junction channels with stem cells. Stem cells co-cultured with nanoparticle-harboring cardiac cells exhibited active biomolecule transfer and showed increased level of electrophysiological cardiac biomarkers and cardiac repair-favorable paracrine secretion. Implanted in rat MI models, cardiac-primed stem cells significantly reduced cardiac fibrosis, promoted cardiac tissue regeneration and function. Secondly, we exhibited that graphene oxide with carbon-based sp2 chemistry can function as reactive oxygen species scavenger within the cells and prevent inflammatory activation of macrophages. Furthermore, we functionalized graphene oxide nanoparticles with plasmid DNA to better polarize inflammatory cells at cardiac infarction area into tissue regenerative macrophages. After the onset of MI, excessive amount of inflammatory macrophages propagates at the peri-infarct to exacerbate tissue necrosis, suggesting that uncontrolled differentiation and activation of inflammatory macrophages greatly hamper proper tissue regeneration. In this study, we demonstrated that graphene oxides can act as an antioxidant to prevent inflammatory activation of macrophages, and further DNA functionalization significantly improved therapeutic polarization of these macrophages. Furthermore, injection of DNA-functionalized graphene oxides in mouse MI models notably reduced immune cell infiltration and mitigated cardiac fibrosis for cardiac performance improvement.Chapter 1. Research backgrounds and objectives 1 1.1. Myocardial infarction (MI) and current therapeutics 2 1.2. Cell therapy for MI 5 1.2.1. MSC-mediated therapy for MI 5 1.2.2. Macrophage therapy for MI 9 1.3. Nanomaterial-mediated cell therapy for MI 12 1.3.1. Nanomaterial-mediated stem cell delivery 14 1.3.2. Topographical cues of nanomaterials for stem cell and macrophage behavior modulation 16 1.3.3. Electrical properties of nanomaterials for stem cell behavior modulation 18 1.3.4. Intrinsic properties of nanomaterials for stem cell and macrophage behavior control 20 1.4. Limitations of previous cell or nanomaterial-mediated therapy 21 1.5. Iron oxide nanoparticles (IONPs) and graphene oxide (GO) for cell modulation and tissue engineering 22 1.6. Research objectives 23 Chapter 2. Experimental procedures 24 2.1. Preparation of iron oxide nanoparticle (IONP) 25 2.2. Characterization of IONP 26 2.3. Cell preparation and IONP-based cell culture 27 2.3.1. MSC and H9C2 culture using IONP and nanoparticle toxicity 27 2.3.2. IONP-based MSC co-culture 28 2.3.3. IONP-mediated cell sorting after co-culture 29 2.4. In vitro analysis 30 2.4.1. TEM analysis 30 2.4.2. Fluorescent images 31 2.4.3. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) 32 2.4.4. Western blot assay 33 2.4.5. Calcein-AM dye transfer assay 34 2.4.6. Immunocytochemistry 35 2.4.7. Paracrine profile analysis 36 2.5. Rat MI model and cMSC treatment 37 2.6. In vivo assessment 38 2.6.1. Histological and immunohistochemical assessment 38 2.6.2. Evaluation of cardiac performance 39 2.7. Preparation of graphene oxide (GO) and GO derivatives 40 2.8. Characterization and DNA conjugation of GO derivatives 42 2.9. Cell preparation and uptake of GO derivatives 43 2.9.1. Culture of mouse bone marrow-derived macrophages 43 2.9.2. Uptake and cellular affinity of GO derivatives 44 2.10. In vitro analysis 45 2.10.1. Metal chelating assay 45 2.10.2. Intracellular reactive oxygen species (ROS) generation 46 2.10.3. qRT-PCR and western blot assay 47 2.10.4. Paracrine secretion analysis 48 2.10.5. Fluorescent imaging 49 2.10.6. Co-culture of macrophages and cardiomyocytes 50 2.11. Mouse MI model and MGC injection 51 2.12. In vivo assessment 52 2.12.1. Histological assessment and evaluation of genes and proteins 52 2.12.2. Evaluation of cardiac performance 54 2.13. Statistical analysis 55 Chapter 3. Iron oxide nanoparticle-mediated development of cellular gap junction crosstalk to improve mesenchymal stem cells therapeutic efficacy for myocardial infarction 56 3.1. Introduction 57 3.2. Results and discussion 61 3.2.1. Internalization of IONP and H9C2 behavior modulation 61 3.2.2. IONP-based magnetic H9C2 sorting post co-culture 66 3.2.3. Cardiac phenotype development in MSCs after co-culture 69 3.2.4. Cardiac repair-favorable paracrine profile in MSCs after co-culture 72 3.2.5. Attenuation of left ventricular remodeling 75 3.2.6. Improved vessel density 77 3.2.7. Enhancement in animal survival and cardiac function 79 Chapter 4. Intracellular antioxidant function development via DNA-functionalized graphene oxide to modulate inflammation and repolarize macrophages for the treatment of myocardial infarction 82 4.1. Introduction 83 4.2. Results and discussion 88 4.2.1. Preparation and characterization of macrophage-targeting/polarizing graphene oxide complex (MGC) 88 4.2.2. Selective cellular uptake and cytotoxicity of MGC 91 4.2.3. Reactive oxygen species scavenging and inflammation modulation by MGC 95 4.2.4. Polarization of M1 macrophages to M2 macrophages 99 4.2.5. Attenuation of inflammation and early shift to reparative M2 phase after MGC/IL-4 pDNA injection in vivo 106 4.2.6. Improved left ventricular remodeling and increased vessel density in vivo 111 4.2.7. Improved recovery of cardiac function 116 Chapter 5. Conclusions 119 References 122 요약 (국문 초록) 156Docto

    Ultra-sensitive bioelectronic transducers for extracellular electrophysiological studies

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    Extracellular electrical activity of cells is commonly recorded using microelectrode arrays (MEA) with planar electrodes. MEA technology has been optimized to record signals generated by excitable cells such as neurons. These cells produce spikes referred to as action potentials. However, all cells produce membrane potentials. In contrast to action potentials, electrical signals produced by non-excitable or non-electrogenic cells, do not exhibit spikes, rather smooth potentials that can change over periods of several minutes with amplitudes of only a few microvolts. These bioelectric signals serve functional roles in signalling pathways that control cell proliferation, differentiation and migration. Measuring and understanding these signals is of high priority in developmental biology, regenerative medicine and cancer research. The objective of this thesis is to fabricate and characterise bioelectronic transducers to measure in vitro the bioelectrical activity of non-electrogenic cells. Since these signals are in the order of few microvolts the electrodes must have an unrivaled low detection limit in the order of hundreds of nanovolts. To meet this challenge a methodology to analyze how bioelectrical signals are coupled into sensing surfaces was developed. The methodology relies on a description of the sensing interface by an equivalent circuit. Procedures for circuit parameter extraction are presented. Relation between circuit parameters, material properties and geometrical design was established. This knowledge was used to establish guidelines for device optimization. The methodology was first used to interpret recordings using gold electrodes, later it as extended to conducting polymers surfaces (PEDOT:PSS ) and finally to graphene electrolyte-gated transistors. The results of this thesis have contributed to the advance of the knowledge in bioelectronic transducers in the following aspects: (i) Detection of signals produced by an important class of neural cells, astrocyte and glioma that thus far had remained inaccessible using conventional extracellular electrodes. (ii) Development of an electrophysiological quantitative method for in vitro monitoring of cancer cell migration and cell-to-cell connections. (iii)An understanding of the limitations of electrolyte-gated transistors to record high frequency signals.A atividade elétrica extracelular das células é geralmente medida usando matrizes de micro-elétrodos (MEA) planares. A tecnologia MEA foi otimizada para medir sinais gerados por células excitáveis, como os neurónios. Essas células produzem sinais conhecidos como potenciais de ação. No entanto, todas as células produzem potenciais de membrana. Em contraste com os potenciais de ação, os sinais elétricos gerados por células não excitáveis ou não eletrogénicas, não são “spikes”, mas sinais que variam lentamente e que podem mudar ao longo de períodos de vários minutos com amplitudes de apenas alguns microvolts. Estes sinais desempenham funções importantes nos mecanismos de sinalização que controlam a proliferação, a diferenciação e a migração celular. Medir e entender esses sinais é importante na biologia do desenvolvimento, na medicina regenerativa e no desenvolvimento de novas terapias para combater células cancerosas. O objetivo desta tese é fabricar e caracterizar transdutores para medir in vitro a atividade de células não eletrogénicas. Como esses sinais são da ordem de alguns microvolts, os elétrodos devem ter um limite de detecção na ordem de centenas de nanovolts. Para enfrentar este desafio, foi desenvolvida uma metodologia para analisar a forma como os sinais se acoplam à superfície do sensor. A metodologia baseia-se na descrição da interface de detecção por um circuito eléctrico equivalente. Procedimentos para extração dos parâmetros de circuito e a relação com as propriedades do material e o desenho geométrico foi estabelecida. Este conhecimento foi usado para estabelecer diretrizes para otimização dos transdutores. Em primeiro lugar a metodologia foi usada para interpretar as medidas de sinais usando elétrodos de ouro, posteriormente estendida para analisar superfícies de polímeros condutores (PEDOT: PSS) e, finalmente, para compreender o funcionamento de transístores. Os resultados desta tese contribuíram para o avanço do conhecimento em transdutores bioeletrónicos nos seguintes aspectos: (i) Detecção de sinais produzidos por uma importante classe de células neurais, astrócitos e gliomas, que tem permanecido inacessíveis usando elétrodos extracelulares. (ii) Desenvolvimento de um método eletrofisiológico para medir a migração de células cancerosas e o estabelecimento de conexões entre células. (ii) Estudo das limitações dos transístores para medir sinais eletrofisiológicos rápidos.The work developed in this thesis was carried out within the framework of the project entitled: “Implantable Organic Devices for Advanced Therapies (INNOVATE)”, ref. PTDC/EEI-AUT/5442/2014, financed by Fundação para a Ciência e Tecnologia (FCT).This project was carried out at the laboratories of the “ Instituto de Telecomunicações (IT) UID/Multi/04326/2013” at the University of the Algarve. The PhD study period received full scholarship under European EM program, “Erasmus Mundus Action 2 (EMA2)” coordinated by University of Warsaw

    Conductive Polymeric-Based Electroactive Scaffolds for Tissue Engineering Applications: Current Progress and Challenges from Biomaterials and Manufacturing Perspectives

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    From MDPI via Jisc Publications RouterHistory: accepted 2021-10-25, pub-electronic 2021-10-26Publication status: PublishedThe practice of combining external stimulation therapy alongside stimuli-responsive bio-scaffolds has shown massive potential for tissue engineering applications. One promising example is the combination of electrical stimulation (ES) and electroactive scaffolds because ES could enhance cell adhesion and proliferation as well as modulating cellular specialization. Even though electroactive scaffolds have the potential to revolutionize the field of tissue engineering due to their ability to distribute ES directly to the target tissues, the development of effective electroactive scaffolds with specific properties remains a major issue in their practical uses. Conductive polymers (CPs) offer ease of modification that allows for tailoring the scaffold’s various properties, making them an attractive option for conductive component in electroactive scaffolds. This review provides an up-to-date narrative of the progress of CPs-based electroactive scaffolds and the challenge of their use in various tissue engineering applications from biomaterials perspectives. The general issues with CP-based scaffolds relevant to its application as electroactive scaffolds were discussed, followed by a more specific discussion in their applications for specific tissues, including bone, nerve, skin, skeletal muscle and cardiac muscle scaffolds. Furthermore, this review also highlighted the importance of the manufacturing process relative to the scaffold’s performance, with particular emphasis on additive manufacturing, and various strategies to overcome the CPs’ limitations in the development of electroactive scaffolds

    Aerospace medicine and biology: A continuing bibliography with indexes (supplement 386)

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    This bibliography lists 117 reports, articles and other documents introduced into the NASA Scientific and Technical Information System during Mar. 1994. Subject coverage includes: aerospace medicine and physiology, life support systems and man/system technology, protective clothing, exobiology and extraterrestrial life, planetary biology, and flight crew behavior and performance

    Development of Biomimetic Models of Human Cardiac Tissue

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    The leading cause of death worldwide is cardiovascular disease (CVD). Myocardial infarction (MI) (i.e., heart attack) makes up ~8.5% of CVD and is a common cause of heart failure with a 40% five-year mortality after the first MI. This highlights a substantial patient population and an urgent need to develop new therapeutic strategies (e.g., regenerative cell therapies). Moreover, this also indicates that current models may not sufficiently recapitulate human cardiac tissue. To date, drug development strategies have largely depended on high throughput 2D cell models and pre-clinical testing in animal models of MI leading to minimal improvements in the heart failure treatment paradigm over the past 20 years. Relevant human cardiac models would provide insight into human cardiac tissue physiology and maturation while also providing an advanced in vitro screening tool to explore heart failure pathogenesis. Cardiac tissue engineering has allowed for advances in the development of cardiac constructs by combining developments in biomaterials, 3D microtissue culture, and human induced pluripotent stem cells (hiPSC) technology. Notably, approaches that mimic the natural processes in the body (i.e., biomimetic) have led to further insight into cardiac physiology. Here, I have pursued biomimetic strategies to create a biomimetic model of human cardiac tissue using hiPSC-derived cardiomyocytes (hiPSC-CMs). Throughout this development, I explored the role of the matrix microenvironment on cell behavior using functionalized alginate, the influence of pacemaker-like exogenous electrical stimulation on the maturation of hiPSC-CM spheroids with endogenous electrically conductive nanomaterials, and the development of vascularized, functional cardiac organoids by mimicking the coronary vasculogenesis stage of cardiac development. The research established here provided a biomimetic groundwork for future development into in vitro human cardiac tissue models for applications in basic research, drug discovery, and cell therapy
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