Patterned Arteriole-Scale Vessels Enhance Engraftment, Perfusion, and Vessel Branching Hierarchy of Engineered Human Myocardium for Heart Regeneration.

Heart regeneration after myocardial infarction (MI) using human stem cell-derived cardiomyocytes (CMs) is rapidly accelerating with large animal and human clinical trials. However, vascularization methods to support the engraftment, survival, and development of implanted CMs in the ischemic environment of the infarcted heart remain a key and timely challenge. To this end, we developed a dual remuscularization-revascularization therapy that is evaluated in a rat model of ischemia-reperfusion MI. This study details the differentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for engineering cardiac tissue containing patterned engineered vessels 400 µm in diameter. Vascularized engineered human myocardial tissues (vEHMs) are cultured in static conditions or perfused in vitro prior to implantation and evaluated after two weeks. Immunohistochemical staining indicates improved engraftment of hiPSC-CMs in in vitro-perfused vEHMs with greater expression of SMA+ vessels and evidence of inosculation. Three-dimensional vascular reconstructions reveal less tortuous and larger intra-implant vessels, as well as an improved branching hierarchy in in vitro-perfused vEHMs relative to non-perfused controls. Exploratory RNA sequencing of explanted vEHMs supports the hypothesis that co-revascularization impacts hiPSC-CM development in vivo. Our approach provides a strong foundation to enhance vEHM integration, develop hierarchical vascular perfusion, and maximize hiPSC-CM engraftment for future regenerative therapy.

One Billion hiPSC-Cardiomyocytes: Upscaling Engineered Cardiac Tissues to Create High Cell Density Therapies for Clinical Translation in Heart Regeneration.

Despite the overwhelming use of cellularized therapeutics in cardiac regenerative engineering, approaches to biomanufacture engineered cardiac tissues (ECTs) at clinical scale remain limited. This study aims to evaluate the impact of critical biomanufacturing decisions-namely cell dose, hydrogel composition, and size-on ECT formation and function-through the lens of clinical translation. ECTs were fabricated by mixing human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) and human cardiac fibroblasts into a collagen hydrogel to engineer meso-(3 × 9 mm), macro- (8 × 12 mm), and mega-ECTs (65 × 75 mm). Meso-ECTs exhibited a hiPSC-CM dose-dependent response in structure and mechanics, with high-density ECTs displaying reduced elastic modulus, collagen organization, prestrain development, and active stress generation. Scaling up, cell-dense macro-ECTs were able to follow point stimulation pacing without arrhythmogenesis. Finally, we successfully fabricated a mega-ECT at clinical scale containing 1 billion hiPSC-CMs for implantation in a swine model of chronic myocardial ischemia to demonstrate the technical feasibility of biomanufacturing, surgical implantation, and engraftment. Through this iterative process, we define the impact of manufacturing variables on ECT formation and function as well as identify challenges that must still be overcome to successfully accelerate ECT clinical translation.

A predictive in vitro risk assessment platform for pro-arrhythmic toxicity using human 3D cardiac microtissues.

Cardiotoxicity of pharmaceutical drugs, industrial chemicals, and environmental toxicants can be severe, even life threatening, which necessitates a thorough evaluation of the human response to chemical compounds. Predicting risks for arrhythmia and sudden cardiac death accurately is critical for defining safety profiles. Currently available approaches have limitations including a focus on single select ion channels, the use of non-human species in vitro and in vivo, and limited direct physiological translation. We have advanced the robustness and reproducibility of in vitro platforms for assessing pro-arrhythmic cardiotoxicity using human induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts in 3-dimensional microtissues. Using automated algorithms and statistical analyses of eight comprehensive evaluation metrics of cardiac action potentials, we demonstrate that tissue-engineered human cardiac microtissues respond appropriately to physiological stimuli and effectively differentiate between high-risk and low-risk compounds exhibiting blockade of the hERG channel (E4031 and ranolazine, respectively). Further, we show that the environmental endocrine disrupting chemical bisphenol-A (BPA) causes acute and sensitive disruption of human action potentials in the nanomolar range. Thus, this novel human 3D in vitro pro-arrhythmic risk assessment platform addresses critical needs in cardiotoxicity testing for both environmental and pharmaceutical compounds and can be leveraged to establish safe human exposure levels.

Tissues with Patterned Vessels or Protein Release Induce Vascular Chemotaxis in an In Vitro Platform.

Engineered tissues designed for translational applications in regenerative medicine require vascular networks to deliver oxygen and nutrients rapidly to the implanted cells. A limiting factor of in vivo translation is the rapid and successful inosculation, or connection, of host and implanted vascular networks and subsequent perfusion of the implant. An approach gaining favor in vascular tissue engineering is to provide instructive cues from the engineered tissue to enhance host vascular penetration and connection with the implant. Here, we use a novel in vitro platform based on the aortic ring assay to evaluate the impact of patterned, endothelialized vessels or growth factor release from engineered constructs on preinosculative vascular cell outgrowth from surrogate host tissue in a controlled, defined environment, and introduce robust tools for evaluating vascular morphogenesis and chemotaxis. We demonstrate the creation of engineered vessels at the arteriole scale, which develop basement membrane, exhibit tight junctions, and actively sprout into the surrounding bulk hydrogel. Vessel-containing constructs are co-cultured adjacent to rodent aortic rings, and the resulting heterocellular outgrowth is quantified. Cells originating from the aortic ring migrate preferentially toward constructs containing engineered vessels with 1.5-fold faster outgrowth kinetics, 2.5-fold increased cellular density, and 1.6-fold greater network formation versus control (no endothelial cells and growth factor-reduced culture medium). Growth factor release from constructs with nonendothelialized channels and in reduced factor medium equivalently stimulates sustained vascular outgrowth distance, cellular density, and network formation, akin to engineered vessels in endothelial growth medium 2 (EGM-2) medium. In conclusion, we show that three-dimensional endothelialized patterned vessels or growth factor release stimulate a robust, host-derived vascular cell chemotactic response at early time points critical for instructive angiogenic cues. Further, we developed robust, unbiased tools to quantify metrics of vascular morphogenesis and preinosculative heterocellular outgrowth from rat aortic rings and demonstrated the utility of our complex, controlled environment, heterocellular in vitro platform. Impact statement Using a novel in vitro platform, we show that engineered constructs with patterned vessels or angiogenic growth factor release, two methods of instructing host revascularization responses, equivalently improve early host-derived vascular outgrowth. Our platform leverages the aortic ring assay in a tissue engineering context to study preinosculative vascular cell chemotaxis from surrogate host vascular cells in response to paracrine cues from co-cultured engineered tissues using robust, open-source quantification tools. Our accessible and flexible platform enables translationally focused studies in revascularization using implantable therapeutics containing prepatterned vessels with greater environmental control than in vivo studies to advance vascular tissue engineering.

Engineered human myocardium with local release of angiogenic proteins improves vascularization and cardiac function in injured rat hearts.

Heart regeneration after myocardial infarction requires new cardiomyocytes and a supportive vascular network. Here, we evaluate the efficacy of localized delivery of angiogenic factors from biomaterials within the implanted muscle tissue to guide growth of a more dense, organized, and perfused vascular supply into implanted engineered human cardiac tissue on an ischemia/reperfusion injured rat heart. We use large, aligned 3-dimensional engineered tissue with cardiomyocytes derived from human induced pluripotent stem cells in a collagen matrix that contains dispersed alginate microspheres as local protein depots. Release of angiogenic growth factors VEGF and bFGF in combination with morphogen sonic hedgehog from the microspheres into the local microenvironment occurs from the epicardial implant site. Analysis of the 3D vascular network in the engineered tissue via Microfil® perfusion and microCT imaging at 30 days shows increased volumetric network density with a wider distribution of vessel diameters, proportionally increased branching and length, and reduced tortuosity. Global heart function is increased in the angiogenic factor-loaded cardiac implants versus sham. These findings demonstrate for the first time the efficacy of a combined remuscularization and revascularization therapy for heart regeneration after myocardial infarction.

Digital Design and Automated Fabrication of Bespoke Collagen Microfiber Scaffolds.

A great variety of natural and synthetic polymer materials have been utilized in soft tissue engineering as extracellular matrix (ECM) materials. Natural polymers, such as collagen and fibrin hydrogels, have experienced especially broad adoption due to the high density of cell adhesion sites compared to their synthetic counterparts, ready availability, and ease of use. However, these and other hydrogels lack the structural and mechanical anisotropy that define the ECM in many tissues, such as skeletal and cardiac muscle, tendon, and cartilage. Herein, we present a facile, low-cost, and automated method of preparing collagen microfibers, organizing these fibers into precisely controlled mesh designs, and embedding these meshes in a bulk hydrogel, creating a composite biomaterial suitable for a wide variety of tissue engineering and regenerative medicine applications. With the assistance of custom software tools described herein, mesh patterns are designed by a digital graphical user interface and translated into protocols that are executed by a custom mesh collection and organization device. We demonstrate a high degree of precision and reproducibility in both fiber and mesh fabrication, evaluate single fiber mechanical properties, and provide evidence of collagen self-assembly in the microfibers under standard cell culture conditions. This work offers a powerful, flexible platform for the study of tissue engineering and cell material interactions, as well as the development of therapeutic biomaterials in the form of custom collagen microfiber patterns that will be accessible to all through the methods and techniques described here. Impact Statement Collagen microfiber meshes have immediate and broad applications in tissue engineering research and show high potential for later use in clinical therapeutics due to their compositional similarities to native extracellular matrix and tunable structural and mechanical characteristics. Physical and biological characterizations of these meshes demonstrate physiologically relevant mechanical properties, native-like collagen structure, and cytocompatibility. The methods presented herein not only describe a process through which custom collagen microfiber meshes can be fabricated but also provide the reader with detailed device plans and software tools to produce their own bespoke meshes through a precise, consistent, and automated process.

Optimizing Blended Collagen-Fibrin Hydrogels for Cardiac Tissue Engineering with Human iPSC-derived Cardiomyocytes.

Natural polymer hydrogels are used ubiquitously as scaffold materials for cardiac tissue engineering as well as for soft tissue engineering more broadly because of FDA approval, minimal immunogenicity, and well-defined physiological clearance pathways. However, the relationships between natural polymer hydrogels and resident cell populations in directing the development of engineered tissues are poorly defined. This interaction is of particular concern for tissues prepared with iPSC-derived cell populations, in which population purity and batch-to-batch variability become additional critical factors to consider. Herein, the design space for a blended fibrin and collagen scaffold is characterized for applications in creating engineered myocardium with human iPSC-derived cardiomyocytes. Stiffness values of the acellular hydrogel formulations approach those of native myocardium in compression, but deviate significantly in tension when compared to rat myocardium in both transverse and longitudinal fiber orientations. A response surface methodology approach to understanding the relationship between collagen concentration, fibrin concentration, seeding density, and cardiac purity found a statistically significant predictive model across three repeated studies that confirms that all of these factors contribute to tissue compaction. In these constructs, increased fibrin concentration and seeding density were each associated with increased compaction, while increased collagen concentration was associated with decreased compaction. Both the lowest (24.4% cTnT+) and highest (60.2% cTnT+) cardiomyocyte purities evaluated were associated with decreased compaction, whereas the greatest compaction was predicted to occur in constructs prepared with a 40-50% cTnT+ population. Constructs prepared with purified cardiomyocytes (≥75.5% cTnT+) compacted and formed syncytia well, although increased fibrin concentration in these groups was associated with decreased compaction, a reversal of the trend observed in unpurified cardiomyocytes. This study demonstrates an analytical approach to understanding cell-scaffold interactions in engineered tissues and provides a foundation for the development of more sophisticated and customized scaffold platforms for human cardiac tissue engineering.

Integrated approaches to spatiotemporally directing angiogenesis in host and engineered tissues.

The field of tissue engineering has turned towards biomimicry to solve the problem of tissue oxygenation and nutrient/waste exchange through the development of vasculature. Induction of angiogenesis and subsequent development of a vascular bed in engineered tissues is actively being pursued through combinations of physical and chemical cues, notably through the presentation of topographies and growth factors. Presenting angiogenic signals in a spatiotemporal fashion is beginning to generate improved vascular networks, which will allow for the creation of large and dense engineered tissues. This review provides a brief background on the cells, mechanisms, and molecules driving vascular development (including angiogenesis), followed by how biomaterials and growth factors can be used to direct vessel formation and maturation. Techniques to accomplish spatiotemporal control of vascularization include incorporation or encapsulation of growth factors, topographical engineering, and 3D bioprinting. The vascularization of engineered tissues and their application in angiogenic therapy in vivo is reviewed herein with an emphasis on the most densely vascularized tissue of the human body - the heart. STATEMENT OF SIGNIFICANCE: Vascularization is vital to wound healing and tissue regeneration, and development of hierarchical networks enables efficient nutrient transfer. In tissue engineering, vascularization is necessary to support physiologically dense engineered tissues, and thus the field seeks to induce vascular formation using biomaterials and chemical signals to provide appropriate, pro-angiogenic signals for cells. This review critically examines the materials and techniques used to generate scaffolds with spatiotemporal cues to direct vascularization in engineered and host tissues in vitro and in vivo. Assessment of the field's progress is intended to inspire vascular applications across all forms of tissue engineering with a specific focus on highlighting the nuances of cardiac tissue engineering for the greater regenerative medicine community.

Paramagnetic Properties of Metal-Free Boron-Doped Graphene Quantum Dots and Their Application for Safe Magnetic Resonance Imaging.

A boron-doped graphene quantum dot (B-GQD) as a metal-free multimodal contrast agent (CA) for safe magnetic resonance imaging and fluorescence imaging is reported. In vivo T1 -weighted magnetic resonance images show that B-GQDs induce significant contrast enhancement on the heart, liver, spleen, and kidney, and sustain for more than 1 h, about 10 times longer than Gd-based CAs currently used in clinic.

Anti-HER2/neu peptide-conjugated iron oxide nanoparticles for targeted delivery of paclitaxel to breast cancer cells.

Nanoparticles (NPs) for targeted therapy are required to have appropriate size, stability, drug loading and release profiles, and efficient targeting ligands. However, many of the existing NPs such as albumin, liposomes, polymers, gold NPs, etc. encounter size limit, toxicity and stability issues when loaded with drugs, fluorophores, and targeting ligands. Furthermore, antibodies are bulky and this can greatly affect the physicochemical properties of the NPs, whereas many small molecule-based targeting ligands lack specificity. Here, we report the utilization of biocompatible, biodegradable, small (∼30 nm) and stable iron oxide NPs (IONPs) for targeted delivery of paclitaxel (PTX) to HER2/neu positive breast cancer cells using an anti-HER2/neu peptide (AHNP) targeting ligand. We demonstrate the uniform size and high stability of these NPs in biological medium, their effective tumour targeting in live mice, as well as their efficient cellular targeting and selective killing in human HER2/neu-positive breast cancer cells.