Xolography is a novel linear volumetric manufacturing technique that offers unparalleled precision and speed. Yet, its application to bioprinting remains limited due to insufficient understanding of biocompatibility constraints. Here, this work establishes fundamental design principles for cell-compatible Xolography bioinks by dissecting the effects of extracellular pH, osmolality, and lysosomotropic stress on cell viability and function. By systematically studying the tolerances for these parameters, this work defines a framework for bioink formulations that enables fast, support-free fabrication of complex designs with maintained cell viability and function as validated in different murine and human cell lines, primary human cells and induced pluripotent stem cell (iPSC)-derived cells. These results show that, unlike triethanolamine, BisTris indeed can function as a fully biocompatible co-initiator enabling cell viability beyond 90% as well as uncompromised metabolic activity and differentiation performance when used in a tightly controlled formulation, contrasting previous reports. This work showcases the biomedical potential of the formulation by achieving fibroblast-driven extracellular matrix (ECM) formation, endothelial sprouting from pre-vascularized spheroids, and maintenance of an iPSC-derived hepatocyte differentiation phenotype within Xolography-printed constructs. These advancements transform Xolography into a powerful and foremost reliable bioprinting platform for fabrication of complex, cell-laden structures for versatile applications in tissue engineering, organ-on-a-chip models, and regenerative medicine.

Biofabrication is an emerging research field aimed at recapitulating in vitro the architecture and functionality of basic tissue units of the human body. A key aspect of biofabrication is the combination of 3D tissue models with microfabrication technologies (e.g. 3D printing, microfluidics) that allow to create spatially confined microenvironments (e.g. an interface between vasculature and a tissue-niche), apply electro-mechanical stimulations (e.g. compression, shear) and live track the dynamic interactions among different cell types. The design of biofabricated models allows to complement available pre-clinical assays and partially overcome their limitations, including the limited complexity of 2D cultures and the low-throughput of animal experiments. During the keynote I will present examples from the lab expertise in biofabrication describing different classes of multi-scale human 3D bone models for the study of: 1) basic mechanisms of breast cancer cell (BCC) dissemination to bone through blood vessels; 2) interactions between BCCs and an already established metastatic niche including immune cells (e.g. neutrophils, macrophages); 3) mechanisms of drug resistance mediated by the cross-talk between specific bone niches and BCCs. The common element across these models is the presence of perfusable microvascular networks that are generated by co-culturing endothelial cells (ECs) and mural cells through self-assembly. Importantly, vascular cells acquire an organ-specific signature when placed in the bone microenvironment, mimicking organotypic differences observed across vascular beds in vivo. We designed multi-scale bone models that recapitulated the bone microenvironment by including osteoblasts, osteoclasts, vascular cells and macrophages within fibrin or collagen matrices. We demonstrated that the bone microenvironment promoted the extravasation of circulating BCCs and the formation of micrometastases compared to control conditions (e.g. skeletal muscle microenvironment). Moreover, the presence of a bone microenvironment promoted the onset of drug resistance mechanisms when established metastases were exposed to doxorubicin or rapamycin. In particular, we showed that BCCs progressively skewed the polarization of resident macrophages towards an M2 pro-tumoral phenotype. We also observed that naive neutrophils perfused through vascularized bone models selectively killed BCCs. Finally, we designed a multi-analytical approach combining organs-on-a-chip and multi-omic analyses to identify the signature of ECs during aging, which might ultimately affect tissue functionality during physiology and disease. In particular, we observed an increase in vascular permeability during aging associated with compromised cell-cell junctions, potentially affecting the formation of metastases. Overall, we biofabricated multi-scale models of vascularized bone that allowed to mimic the dynamic, patho-physiological cross-talk among blood vessels, bone-resident cells and BCCs.

Aging is a complex, multifaceted process affecting all organ systems, with vascular aging playing a central role in organismal health decline. Beyond its role in circulation, the vascular system acts as a dynamic interface between tissues, influencing countless physiological functions such as tissue regeneration and repair, immune responses, and metabolic balance. Importantly, age-related vascular impairment-characterized by a peculiar set of endothelial aging hallmarks-exacerbates age-related diseases (ARDs) such as cardiovascular disorders, neurodegeneration, chronic kidney disease, sarcopenia, and osteoporosis. This review combines basic concepts of angioscience and aging biology with translational interventions to devise clinical strategies promoting a functional rejuvenation of old and compromised blood vessels, fostering the prevention, delay or treatment of ARDs. Starting from the description of the cellular and molecular mechanisms driving vascular aging, a cutting-edge perspective on the organ-specific vascular impairment and its impact on tissue function is offered. Given the central role of the vasculature in aging, how targeting vascular aging through pharmacological, genetic, and lifestyle interventions holds promise for mitigating its systemic consequences and improving healthspan is discussed. Finally, how the combination of animal models (e.g., parabiosis) and novel microphysiological systems, coupled with multi-omics and artificial intelligence-driven analyses, is advancing the field toward the identification of strategies that promote vascular resilience and extend healthspan, addressing one of the most pressing biomedical challenges of a worldwide aging population is highlighted.

In the attempt to overcome the increasingly recognized shortcomings of existing in vitro and in vivo models, researchers have started to implement alternative models, including microphysiological systems. First examples were represented by 2.5D systems, such as microfluidic channels covered by cell monolayers as blood vessel replicates. In recent years, increasingly complex microphysiological systems have been developed, up to multi-organ on chip systems, connecting different 3D tissues in the same device. However, such an increase in model complexity raises several questions about their exploitation and implementation into industrial and clinical applications, ranging from how to improve their reproducibility, robustness, and reliability to how to meaningfully and efficiently analyze the huge amount of heterogeneous datasets emerging from these devices. Considering the multitude of envisaged applications for microphysiological systems, it appears now necessary to tailor their complexity on the intended purpose, being academic or industrial, and possibly combine results deriving from differently complex stages to increase their predictive power.

Fibrosis is a pathological condition that in the muscular context is linked to primary diseases such as dystrophies, laminopathies, neuromuscular disorders, and volumetric muscle loss following traumas, accidents, and surgeries. Although some basic mechanisms regarding the role of myofibroblasts in the progression of muscle fibrosis have been discovered, our knowledge of the complex cell-cell, and cell-matrix interactions occurring in the fibrotic microenvironment is still rudimentary. Recently, vascular dysfunction has been emerging as a key hallmark of fibrosis through a process called endothelial-mesenchymal transition (EndoMT). Nevertheless, no effective therapeutic options are currently available for the treatment of muscle fibrosis. This lack is partially due to the absence of advanced in vitro models that can recapitulate the 3D architecture and functionality of a vascularized muscle microenvironment in a human context. These models could be employed for the identification of novel targets and for the screening of potential drugs blocking the progression of the disease. In this review, we explore the potential of 3D human muscle models in studying the role of endothelial cells and EndoMT in muscle fibrotic tissues and identify limitations and opportunities for optimizing the next generation of these microphysiological systems. Starting from the biology of muscle fibrosis and EndoMT, we highlight the synergistic links between different cell populations of the fibrotic microenvironment and how to recapitulate them through microphysiological systems.

Different tissues have different endothelial features, however, the implications of this heterogeneity in pathological responses are not clear yet. “Inflamm-aging” has been hypothesized as a possible trigger of diseases, including osteoarthritis (OA) and sarcopenia, often present in the same patient. To highlight a possible contribution of organ-specific endothelial cells (ECs), we compare ECs derived from bone and skeletal muscle of the same OA patients. OA bone ECs show a pro-inflammatory signature and higher angiogenic sprouting as compared to muscle ECs, in control conditions and stimulated with TNFα. Furthermore, growth of muscle but not bone ECs decreases with increasing patient age and systemic inflammation. Overall, our data demonstrate that inflammatory conditions in OA patients differently affect bone and muscle ECs, suggesting that inflammatory processes increase angiogenesis in subchondral bone while associated systemic low-grade inflammation impairs angiogenesis in muscle, possibly highlighting a vascular trigger linking OA and sarcopenia.

Even though breast cancers usually have a good outcome compared to other tumors, the cancer can progress and create metastases in different parts of the organism, the bone being a predilection locus. These metastases are usually the cause of death, as they are mostly resistant to treatments. This resistance can be caused by intrinsic properties of the tumor, such as its heterogeneity, but it can also be due to the protective role of the microenvironment. By activating signaling pathways protecting cancer cells when exposed to chemotherapy, contributing to their ability to reach dormancy, or even reducing the amount of drug able to reach the metastases, among other mechanisms, the specificities of the bone tissue are being investigated as important players of drug resistance. To this date, most mechanisms of this resistance are yet to be discovered, and many researchers are implementing in vitro models to study the interaction between the tumor cells and their microenvironment. Here, we will review what is known about breast cancer drug resistance in bone metastasis due to the microenvironment and we will use those observations to highlight which features in vitro models should include to properly recapitulate these biological aspects in vitro. We will also detail which elements advanced in vitro models should implement in order to better recapitulate in vivo physiopathology and drug resistance.

The vascular system is a key player for the maintenance of healthy tissues, suggesting how the physiological decline of blood vessel functionality during aging could be a major contributor of organ degeneration. While basic research studies have begun to pinpoint potential mechanisms of vascular aging, it is now critical to translate them into therapeutically relevant options. Microphysiological systems represent a powerful tool to precisely control which combinations of stimuli are provided to in vitro reconstructed blood vessels and to analyze their functional consequences. After highlighting key aspects of vascular aging, this review discusses in vitro models that are able to recapitulate relevant features of blood vessel damage during aging. Strategies to improve current in vitro systems so that they will more faithfully recapitulate vascular aging are proposed, emphasizing the importance of combining in vivo models with microphysiological systems for an effective translation of vascular aging biomarkers and therapies to the clinical level.

This protocol describes the biofabrication of 3D millimeter-scale human muscle units, embedding non-planar muscle fibers wrapped by fibroblasts-rich endomysium and intertwined with microvascular networks. Suspended muscle fibers are formed through the self-assembly of human myoblasts within cylindrical cavities generated in a sacrificial gelatin template cast in a 3D printed frame. Following myotube differentiation, muscle fibers are embedded in a 3D matrix containing endothelial cells and muscle-derived fibroblasts. The cellular complexity of the environment is instrumental to drive fibroblast migration towards muscle fibers and to induce the organ-specific differentiation of endothelial cells. This advanced 3D muscle model can be applied to analyze the biological mechanisms underlying specific muscle diseases which involve a complex remodeling of the muscle environment (e.g., muscular dystrophies and fibrosis) whereby the pathological interplay among different cell populations drives the onset and progression of the disease.

The organ-specific metastatization of breast cancer to bone is driven by specific interactions between the host microenvironment and cancer cells (CCs). However, it is still unclear the role that circulating immune cells, including neutrophils, play during bone colonization (i.e. pro-tumoral vs. anti-tumoral). Here, we aimed at analyzing the migratory behavior of neutrophils when exposed to breast CCs colonizing the bone and their contribution to the growth of breast cancer micrometastases. Based on our previous bone metastasis models, we designed a microfluidic system that allows to independently introduce human vascularized breast cancer metastatic seeds within a bone-mimicking microenvironment containing osteo-differentiated mesenchymal stromal cells and endothelial cells (ECs). ECs self-assembled into microvascular networks and connected the bone-mimicking microenvironment with the metastatic seed. Compared to controls without CCs, metastatic seeds compromised the architecture of microvascular networks resulting in a lower number of junctions (5.7 +/- 1.2 vs. 18.8 +/- 4.5, p = 0.025) and shorter network length (10.5 +/- 1.0 vs. 13.4 +/- 0.8 [mm], p = 0.042). Further, vascular permeability was significantly higher with CCs (2.60 x 10(-8) +/- 3.59 x 10(-8) vs. 0.53 x 10-8 +/- 0.44 x 10(-8) [cm/s], p = 0.05). Following metastatic seed maturation, neutrophils were injected into microvascular networks resulting in a higher extravasation rate when CCs were present (27.9 +/- 13.7 vs. 14.7 +/- 12.4 [%], p = 0.01). Strikingly, the percentage of dying CCs increased in presence of neutrophils, as confirmed by confocal imaging and flow cytometry on isolated cells from the metastatic seeds. The biofabricated metastatic niche represents a powerful tool to analyze the mechanisms of interaction between circulating immune cells and organ-specific micrometastases and to test novel drug combinations targeting the metastatic microenvironment.

This protocol describes a comprehensive practical guide for the biofabrication of 3D in vitro models of vascularized and mineralized bone Minitissues. These models give the possibility to study the contribution of physical and biochemical parameters on bone vascularization, as well as the osteoblast/osteoclast mediated matrix remodeling. Based on the specific pathophysiological processes to be investigated, the 3D bone Minitissues allow to select the most suitable cell composition, by coculturing up to four cell types, and to customize the material properties of the hydrogel matrix. Considering their versatility, these 3D bone Minitissues could be relevant for the recapitulation of bone pathologies such as bone tumors and metastases and could be and used as screening platforms to test antimetastatic drugs.

ABSTRACT Identification of cancer stemness genes is crucial to understanding the underlying biology of therapy resistance, relapse, and metastasis. Ewing sarcoma (EwS) is the second most common bone tumor in children and adolescents. It is a highly aggressive cancer associated with a dismal survival rate (<30%) for patients with metastatic disease at diagnosis (∼25% of cases). Hence, deciphering the underlying mechanisms of metastasis is imperative. EwS tumors are characterized by a remarkably ‘silent’ genome with a single driver mutation generating an oncogenic fusion transcription factor ( EWSR1-ETS ). Thus, EwS constitutes an ideal model to study how perturbation of a transcriptional network by a dominant oncogene can mediate metastasis, even though canonical metastasis-associated genes are not mutated. Here, through the implementation of an integrative systems biology approach, we identified transcription factor 7 like 1 ( TCF7L1 , alias TCF3 ) as a prognostically-relevant and EWSR1-ETS suppressed determinant of metastasis in EwS. We demonstrated that conditional TCF7L1 re-expression significantly reduces EwS single-cell migration, invasion and anchorage-independent growth in 3D assays in vitro , and tumorigenesis in vivo mediated by its DNA binding domain. In primary EwS tumors as well as in functional orthotopic in vivo models, low TCF7L1 expression was associated with pro-metastatic gene signatures and a much higher migratory and metastatic capacity of EwS cells, which correlated with poor outcome of EwS patients. Collectively, our findings establish TCF7L1 as a major regulator of metastasis in EwS, which may be utilized as a prognostic biomarker and open inroads to future therapeutic intervention.