Fast Stereolithography Printing of Large-Scale Biocompatible Hydrogel Models.

Large size cell-laden hydrogel models hold great promise for tissue repair and organ transplantation, but their fabrication using 3D bioprinting is limited by the slow printing speed that can affect the part quality and the biological activity of the encapsulated cells. Here a fast hydrogel stereolithography printing (FLOAT) method is presented that allows the creation of a centimeter-sized, multiscale solid hydrogel model within minutes. Through precisely controlling the photopolymerization condition, low suction force-driven, high-velocity flow of the hydrogel prepolymer is established that supports the continuous replenishment of the prepolymer solution below the curing part and the nonstop part growth. The rapid printing of centimeter-sized hydrogel models using FLOAT is shown to significantly reduce the part deformation and cellular injury caused by the prolonged exposure to the environmental stresses in conventional 3D printing methods. Embedded vessel networks fabricated through multiscale printing allows media perfusion needed to maintain the high cellular viability and metabolic functions in the deep core of the large-sized models. The endothelialization of this vessel network allows the establishment of barrier functions. Together, these studies demonstrate a rapid 3D hydrogel printing method and represent a first step toward the fabrication of large-sized engineered tissue models.

Monocyte recruitment and fate specification after myocardial infarction.

Monocytes are critical mediators of the inflammatory response following myocardial infarction (MI) and ischemia-reperfusion injury. They are involved in both initiation and resolution of inflammation and play an integral role in cardiac repair. The antagonistic nature of their function is dependent on their subset heterogeneity and biphasic response following injury. New advancements in single-cell transcriptomics and mass cytometry have allowed us to identify smaller, transcriptionally distinct clusters that may have functional relevance in disease and homeostasis. Additionally, recent insights into the spatiotemporal dynamics of monocytes following ischemic injury and their subsequent interactions with the endothelium and other immune cells reveal a complex interplay between monocytes and the cardiac milieu. In this review, we highlight recent findings on monocyte functional heterogeneity, present new mechanistic insight into monocyte recruitment and fate specification following MI, and discuss promising therapeutic avenues targeting monocytes for the treatment of ischemic heart disease.

CDC-derived extracellular vesicles reprogram inflammatory macrophages to an arginase 1-dependent proangiogenic phenotype.

Macrophages play a pivotal role in tissue repair following myocardial infarction (MI). In response to injury, they exist along a spectrum of activation states tightly regulated by their microenvironment. Cardiosphere-derived cells (CDCs) have been shown to mediate cardioprotection via modulation of the macrophage response. Our study was designed to gain mechanistic insight into the role of CDC-derived extracellular vesicles (EVs) in modulating macrophage phenotypes and operant signaling pathways to better understand their potential contribution to immunomodulatory cardioprotection. We found that CDC-derived EVs alter the functional phenotype of macrophages, modifying levels of phagocytosis and efferocytosis without changing viability or proliferation. Interestingly, extracellular vesicles differentially regulate several M1/M2 genes dependent on macrophage activation before EV treatment but consistently upregulate arginase 1 regardless of macrophage origin or polarization state. CDC-derived EVs polarize M1 macrophages to a proangiogenic phenotype dependent on arginase 1 upregulation and independent of VEGF-A. In addition, EV-dependent arginase 1 upregulation downregulates nitric oxide (NO) secretion in activated macrophages. These data suggest a novel urea-cycle-dependent mechanism in macrophages that promotes angiogenesis and provides additional mechanistic insight into the potential contribution of CDC-derived extracellular vesicles in immunomodulatory cardioprotection.NEW & NOTEWORTHY We hypothesized that in the window of therapeutic extracellular vesicle (EV) administration, inflammatory M1 macrophages are likely the primary target of cardiosphere-derived cell (CDC)-derived EVs. The effect of CDC-EVs on this population, however, is currently unknown. In this study, we demonstrate that CDC-derived EVs polarize M1 macrophages to a proangiogenic phenotype dependent on arginase 1 upregulation. These results provide insight into an immunomodulatory mechanism of CDC-EVs in a more physiologically relevant model of post-myocardial infarction (post-MI) macrophage polarization.

Exosomes Engineered to Express a Cardiomyocyte Binding Peptide Demonstrate Improved Cardiac Retention in Vivo.

Injury to the heart results in cardiomyocyte cell death and can lead to pathological remodeling of remaining cells, contributing to heart failure. Despite the therapeutic potential of new drugs and small molecules, there remains a gap in the ability to efficiently deliver cardioprotective agents in a cell specific manner while minimizing nonspecific delivery to other organs. Exosomes derived from cardiosphere-derived cells (CDCs) have been shown to stimulate angiogenesis, induce endogenous cardiomyocyte proliferation and modulate cardiomyocyte apoptosis and hypertrophy. While innately cardioprotective at high doses, unmodified CDC-exosomes demonstrate limited cardiac tropism. To generate an efficient exosomal delivery system that can target cardiomyocytes, we engineered CDCs to express Lamp2b, an exosomal membrane protein, fused to a cardiomyocyte specific peptide (CMP), WLSEAGPVVTVRALRGTGSW. Exosomes isolated from engineered CDCs expressed CMP on their surface and retained their native physical properties. Targeted exosomes resulted in increased uptake by cardiomyocytes, decreased cardiomyocyte apoptosis, and higher cardiac retention following intramyocardial injection when compared with non-targeted exosomes. Importantly, we established a novel targeting system to improve exosomal uptake by cardiomyocytes and laid the foundation for cell-specific exosomal delivery of drug and gene therapies to improve the functional capacity of the heart following both ischemic and non-ischemic injury.

Therapeutic Potential of Engineered Extracellular Vesicles.

Extracellular vesicles (EVs) comprise a heterogeneous group of small membrane vesicles, including exosomes, which play a critical role in intracellular communication and regulation of numerous physiological processes in health and disease. Naturally released from virtually all cells, these vesicles contain an array of nucleic acids, lipids and proteins which they transfer to target cells within their local milieu and systemically. They have been proposed as a means of "cell-free, cell therapy" for cancer, immune disorders, and more recently cardiovascular disease. In addition, their unique properties of stability, biocompatibility, and low immunogenicity have prompted research into their potential as therapeutic delivery agents for drugs and small molecules. In this review, we aim to provide a comprehensive overview of the current understanding of extracellular vesicle biology as well as engineering strategies in play to improve their therapeutic potential.