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Over the past three decades, cell therapy development has fallen short of expectations, with many cellular sources demonstrating a 'Janus effect' and raising safety concerns. Extracellular vesicles (EVs), supported by advanced technologies, present a promising avenue in regenerative medicine, offering benefits such as immune tolerance and avoidance of negative aspects associated with cell transplants. Our previous research showcased enhanced and organized subcutaneous vascularization using three-dimensional bioprinted patches containing HUVEC-derived EVs in immunodeficient animal models. In this context, stress conditions on the cells of origin further boosted the EVs' neoangiogenic potential. Since neovascularization is the first regenerative target requiring restoration, the present study aims to complement our previous work by employing an injectable gelatin methacrylate (GelMA) hydrogel functionalized with HUVEC-derived EVs in a pathological condition of acute myocardial infarction. This bioactive hydrogel resulted in reduced fibrosis, improved contractility, and promoted angiogenesis, showing promise in countering tissue deterioration and addressing vascular deficits. Moreover, the molecular characterization of EVs through miRNome and proteomic analyses further supports their potential as bio-additives for hydrogel functionalization. This cell-free approach mitigates immune rejection and oncogenic risks, offering innovative therapeutic advantages., (Creative Commons Attribution license.)

In this work, we present an innovative, high-throughput rotary wet-spinning biofabrication method for manufacturing cellularized constructs composed of highly-aligned hydrogel fibers. The platform is supported by an innovative microfluidic printing head (MPH) bearing a crosslinking bath microtank with a co-axial nozzle placed at the bottom of it for the immediate gelation of extruded core/shell fibers. After a thorough characterization and optimization of the new MPH and the fiber deposition parameters, we demonstrate the suitability of the proposed system for the in vitro engineering of functional myo-substitutes. The samples produced through the described approach were first characterized in vitro and then used as a substrate to ascertain the effects of electro-mechanical stimulation on myogenic maturation. Of note, we found a characteristic gene expression modulation of fast (MyH1), intermediate (MyH2), and slow (MyH7) twitching myosin heavy chain isoforms, depending on the applied stimulation protocol. This feature should be further investigated in the future to biofabricate engineered myo-substitutes with specific functionalities., (Creative Commons Attribution license.)

The cellular heterogeneity of the tumor environment of breast cancer (BC) is extremely complex and includes different actors such as neoplastic, stromal, and immunosuppressive cells, which contribute to the chemical and mechanical modification of the environment surrounding the tumor-exasperating immune-escaping mechanisms. In addition to molecular signals that make the tumor microenvironment (TME) unacceptable for the penetrance of the immune system, the physical properties of tumoral extracellular matrix (tECM) also have carved out a fundamental role in the processes of the protection of the tumor niche. Tumor-associated macrophages (TAMs), with an M2 immunosuppressive phenotype, are important determinants for the establishment of a tumor phenotype excluded from T cells. NF-κB transcription factors orchestrate innate immunity and represent the common thread between inflammation and cancer. Many studies have focused on canonical activation of NF-κB; however, activation of non-canonical signaling predicts poor survival and resistance to therapy. In this scenario, we demonstrated the existence of an unusual association of NF-κB components in TAMs that determines the deposition of HSPG2 that affects the stiffness of tECM. These results highlight a new mechanism counterbalanced between physical factors and a new perspective of mechano-pathology to be targeted to counteract immune evasion in BC.

Alterations in the DMD gene, which codes for the protein dystrophin, cause forms of dystrophinopathies such as Duchenne muscular dystrophy, an X-linked disease. Cardiomyopathy linked to DMD mutations is becoming the leading cause of death in patients with dystrophinopathy. Since phenotypic pathophysiological mechanisms are not fully understood, the improvement and development of new disease models, considering their relative advantages and disadvantages, is essential. The application of genetic engineering approaches on induced pluripotent stem cells, such as gene-editing technology, enables the development of physiologically relevant human cell models for in vitro dystrophinopathy studies. The combination of induced pluripotent stem cells-derived cardiovascular cell types and 3D bioprinting technologies hold great promise for the study of dystrophin-linked cardiomyopathy. This combined approach enables the assessment of responses to physical or chemical stimuli, and the influence of pharmaceutical approaches. The critical objective of in vitro microphysiological systems is to more accurately reproduce the microenvironment observed in vivo. Ground-breaking methodology involving the connection of multiple microphysiological systems comprised of different tissues would represent a move toward precision body-on-chip disease modelling could lead to a critical expansion in what is known about inter-organ responses to disease and novel therapies that have the potential to replace animal models. In this review, we will focus on the generation, development, and application of current cellular, animal, and potential for bio-printed models, in the study of the pathophysiological mechanisms underlying dystrophin-linked cardiomyopathy in the direction of personalized medicine., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

In the last decades, biomedical research has significantly boomed in the academia and industrial sectors, and it is expected to continue to grow at a rapid pace in the future. An in-depth analysis of such growth is not trivial, given the intrinsic multidisciplinary nature of biomedical research. Nevertheless, technological advances are among the main factors which have enabled such progress. In this review, we discuss the contribution of two state-of-the-art technologies-namely biofabrication and organ-on-a-chip-in a selection of biomedical research areas. We start by providing an overview of these technologies and their capacities in fabricating advanced in vitro tissue/organ models. We then analyze their impact on addressing a range of current biomedical challenges. Ultimately, we speculate about their future developments by integrating these technologies with other cutting-edge research fields such as artificial intelligence and big data analysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Celikkin, Presutti, Maiullari, Fornetti, Agarwal, Paradiso, Volpi, Święszkowski, Bearzi, Barbetta, Zhang, Gargioli, Rizzi and Costantini.)

The immune system is a fine modulator of the tumor biology supporting or inhibiting its progression, growth, invasion and conveys the pharmacological treatment effect. Tumors, on their side, have developed escaping mechanisms from the immune system action ranging from the direct secretion of biochemical signals to an indirect reaction, in which the cellular actors of the tumor microenvironment (TME) collaborate to mechanically condition the extracellular matrix (ECM) making it inhospitable to immune cells. TME is composed of several cell lines besides cancer cells, including tumor-associated macrophages, cancer-associated fibroblasts, CD4 + and CD8 + lymphocytes, and innate immunity cells. These populations interface with each other to prepare a conservative response, capable of evading the defense mechanisms implemented by the host's immune system. The presence or absence, in particular, of cytotoxic CD8 + cells in the vicinity of the main tumor mass, is able to predict, respectively, the success or failure of drug therapy. Among various mechanisms of immunescaping, in this study, we characterized the modulation of the phenotypic profile of CD4 + and CD8 + cells in resting and activated states, in response to the mechanical pressure exerted by a three-dimensional in vitro system, able to recapitulate the rheological and stiffness properties of the tumor ECM.

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease with a high death rate mainly due to the metastatic spread. The expression of MDM4, a well-known p53-inhibitor, is positively associated with chemotherapy response and overall survival (OS) in EOC. However, the basis of this association remains elusive. We show that in vivo MDM4 reduces intraperitoneal dissemination of EOC cells, independently of p53 and an immune-competent background. By 2D and 3D assays, MDM4 impairs the early steps of the metastatic process. A 3D-bioprinting system, ad hoc developed by co-culturing EOC spheroids and endothelial cells, showed reduced dissemination and intravasation into vessel-like structures of MDM4-expressing cells. Consistent with these data, high MDM4 levels protect mice from ovarian cancer-related death and, importantly, correlate with increased 15 y OS probability in large data set analysis of 1656 patients. Proteomic analysis of EOC 3D-spheroids revealed decreased protein synthesis and mTOR signaling, upon MDM4 expression. Accordingly, MDM4 does not further inhibit cell migration when its activity towards mTOR is blocked by genetic or pharmacological approaches. Importantly, high levels of MDM4 reduced the efficacy of mTOR inhibitors in constraining cell migration. Overall, these data demonstrate that MDM4 impairs EOC metastatic process by inhibiting mTOR activity and suggest the usefulness of MDM4 assessment for the tailored application of mTOR-targeted therapy.

Extracellular vesicles (EVs) have become a key tool in the biotechnological landscape due to their well-documented ability to mediate intercellular communication. This feature has been explored and is under constant investigation by researchers, who have demonstrated the important role of EVs in several research fields ranging from oncology to immunology and diagnostics to regenerative medicine. Unfortunately, there are still some limitations to overcome before clinical application, including the inability to confine the EVs to strategically defined sites of interest to avoid side effects. In this study, for the first time, EV application is supported by 3D bioprinting technology to develop a new strategy for applying the angiogenic cargo of human umbilical vein endothelial cell-derived EVs in regenerative medicine. EVs, derived from human endothelial cells and grown under different stressed conditions, were collected and used as bioadditives for the formulation of advanced bioinks. After in vivo subcutaneous implantation, we demonstrated that the bioprinted 3D structures, loaded with EVs, supported the formation of a new functional vasculature in situ , consisting of blood-perfused microvessels recapitulating the printed pattern. The results obtained in this study favour the development of new therapeutic approaches for critical clinical conditions, such as the need for prompt revascularization of ischaemic tissues, which represent the fundamental substrate for advanced regenerative medicine applications., (Creative Commons Attribution license.)

The recent advances, offered by cell therapy in the regenerative medicine field, offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors, such as myoblasts or satellite cells, possess a marked regenerative capacity, but the exploitation of this potential still encounters significant challenges in clinical application, due to low rate of proliferation in vitro, as well as a reduced self-renewal capacity. In this scenario, induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches, but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs, generated from pericytes and fibroblasts, exploiting skeletal muscle-derived extracellular vesicles (EVs), in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological "shuttles" to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches.

The myocardium behaves like a sophisticated orchestra that expresses its true potential only if each member performs the correct task harmonically. Recapitulating its complexity within engineered 3D functional constructs with tailored biological and mechanical properties, is one of the current scientific priorities in the field of regenerative medicine and tissue engineering. In this study, driven by the necessity of fabricating advanced model of cardiac tissue, we present an innovative approach consisting of heterogeneous, multi-cellular constructs composed of Human Umbilical Vein Endothelial Cells (HUVECs) and induced pluripotent cell-derived cardiomyocytes (iPSC-CMs). Cells were encapsulated within hydrogel strands containing alginate and PEG-Fibrinogen (PF) and extruded through a custom microfluidic printing head (MPH) that allows to precisely tailor their 3D spatial deposition, guaranteeing a high printing fidelity and resolution. We obtained a 3D cardiac tissue compose of iPSC-derived CMs with a high orientation index imposed by the different defined geometries and blood vessel-like shapes generated by HUVECs which, as demonstrated by in vivo grafting, better support the integration of the engineered cardiac tissue with host's vasculature.

Cardiovascular diseases (CVDs) are a major burden on the healthcare system: indeed, over two million new cases are diagnosed every year worldwide. Unfortunately, important drawbacks for the treatment of these patients derive from our current inability to stop the structural alterations that lead to heart failure, the common endpoint of many CVDs. In this scenario, a better understanding of the role of epigenetics - hereditable changes of chromatin that do not alter the DNA sequence itself - is warranted. To date, hyperacetylation of histones has been reported in hypertension and myocardial infarction, but the use of inhibitors for treating CVDs remains limited. Here, we studied the effect of the histone deacetylase inhibitor Givinostat on a mouse model of acute myocardial infarction. We found that it contributes to decrease endothelial-to-mesenchymal transition and inflammation, reducing cardiac fibrosis and improving heart performance and protecting the blood vessels from apoptosis through the modulatory effect of cardiac fibroblasts on endothelial cells. Therefore, Givinostat may have potential for the treatment of CVDs.