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Introduction: Extracellular vesicles from mesenchymal stromal cells (MSC-EVs) are potent stimulators of naïve cartilage and their injection is studied in clinical trials for cartilage lesions, since often cartilage repaired with conventional approaches is incomplete or less performant leading to joint degeneration. The main pitfall of these innovative approaches is the high EVs dispersion into the joint cavity and consequent low concentration at lesion site. Thus, biological scaffolds for concentration of EVs where needed might be a promising option. This work aimed at producing an enhanced platelet-derived fibrin gel loaded with adipose-derived MSCs (ASCs)-EVs. Methods: EVs’ embedment efficiency in platelet gel, their release and incorporation in OA chondrocytes and cartilage explants were monitored by flow cytometry, microfluidic approaches, scansion electron microscopy and real-time quantitative multimodal nonlinear optics imaging. The effect of released EVs was tested in OA chondrocytes by gene expression studies. Results: A protocol ensuring high incorporation EVs efficiency in platelet gels was defined, relying on a one-step modification of the standard procedure used in current clinical practice. Trapped EVs were released continuously for up to 4 weeks and uptaken in pathologic chondrocytes and cartilage explants. The release of the EVs-loaded platelet gel had stronger and synergic anti-inflammatory/matrix remodelling effects with respect to both EVs per se and unloaded gel released products. Conclusions: These results suggest the feasibility of producing a platelet gel loaded with MSC-EVs at high efficiency that can be used as an enhanced tool to foster chondrocyte homeostasis, a key requisite for proper cartilage healing.

Osteoarthritis (OA) is a highly disabling pathology, characterized by synovial inflammation and cartilage degeneration. Orthobiologics have shown promising results in OA treatment thanks to their ability to influence articular cells and modulate the inflammatory OA environment. Considering their complex mechanism of action, the development of reliable and relevant joint models appears as crucial to select the best orthobiologics for each patient.The aim of this study was to establish a microfluidic OA model to test therapies in a personalized human setting. The joint-on-a-chip model included cartilage and synovial compartments, containing hydrogel-embedded chondrocytes and synovial fibroblasts, separated by a channel for synovial fluid. For the cartilage compartment, a Hyaluronic Acid-based matrix was selected to preserve chondrocyte phenotype. Adding OA synovial fluid induced the production of inflammatory cytokines and degradative enzymes, generating an OA microenvironment. Personalized models were generated using patient-matched cells and synovial fluid to test the efficacy of mesenchymal stem cells on OA signatures. The patient-specific models allowed monitoring changes induced by cell injection, highlighting different individual responses to the treatment. Altogether, these results support the use of this joint-on-a-chip model as a prognostic tool to screen the patient-specific efficacy of orthobiologics.

Summary: 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.

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 ​× ​10−8 ​± ​3.59 ​× ​10−8 ​vs. 0.53 ​× ​10−8 ​± ​0.44 ​× ​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.

New Psychoactive Substances (NPS), and particularly synthetic opioids, are constantly growing in the illicit market, and represent a particular danger due to their high potency and the fact that they're often not covered by legislation yet. Thus, there is the constant need for sensitive, reliable and reproducible techniques to detect and identify these substances in a variety of different matrices, both for clinical and forensic purposes. In particular, hair analysis can provide important information on past and/or continuative use of different substances such as psychoactive drugs.In this study, we report a review of the existing literature on synthetic opioids analysis in hair, in order to summarize the analytical methods currently used for synthetic and semi-synthetic opioids and NPS hair testing. Methanol with addition of diluted acidic solution represents the most frequently extraction solution. Purification procedures, either carried out through solid phase or liquid/liquid are still needed in order to achieve an adequate sensitivity; in particular, several methods provided a limit of detection lower than 1 pg/mg for most of the monitored substances. Chromatographic techniques coupled with mass spectrometric systems confirmed to be the best choice among the available analytical techniques for monitoring psychoactive substances and metabolites in keratin matrices. Though high-resolution mass spectrometric instruments, such as Time of Flight, are gaining interest, triple quad still guarantees better sensitivity. Beside common synthetic and semi-synthetic opioids, such as hydromorphone, hydrocodone and tramadol, the most frequently detected compounds in hair samples, among fentanyl derivatives, are furanyl fentanyl, sufentanil and butyryl fentanyl.

Since the association of SARS-Cov-2 infection with Nervous System (NS) manifestations, we performed RNA-sequencing analysis in Frontal Cortex of COVID-19 positive or negative individuals and affected or not by Dementia individuals. We examined gene expression differences in individuals with COVID-19 and Dementia compared to Dementia only patients by collecting transcript counts in each sample and performing Differential Expression analysis. We found eleven genes satisfying our significance criteria, all of them being protein coding genes.These data are suitable for integration with supplemental samples and for analysis according to different individuals’ classification. Also, differential expression evaluation may be implemented with other scientific purposes, such as research of unannotated genes, mRNA splicing and genes isoforms.The analysis of Differential Expressed genes in COVID-19 positive patients compared to non-COVID-19 patients is published in: S. Gagliardi, E.T. Poloni, C. Pandini, M. Garofalo, F. Dragoni, V. Medici, A. Davin, S.D. Visonà, M. Moretti, D. Sproviero, O. Pansarasa, A. Guaita, M. Ceroni, L. Tronconi, C. Cereda, Detection of SARS-CoV-2 genome and whole transcriptome sequencing in frontal cortex of COVID-19 patients., Brain. Behav. Immun. (2021). https://doi.org/10.1016/j.bbi.2021.05.012.

Summary: The integration of vascular structures into in vitro cultured tissues provides realistic models of complex tissue-vascular interactions. Despite the incidence and impact of muscle-wasting disorders, advanced in vitro systems are still far from recapitulating the environmental complexity of skeletal muscle. Our model comprises differentiated human muscle fibers enveloped by a sheath of human muscle-derived fibroblasts and supported by a vascular network with mural-like cells. Here, we demonstrate the induction of muscle-specific endothelium and the self-organization of endomysial muscle fibroblasts mediated by endothelial cells. We use this model to mimic the fibrotic environment characterizing muscular dystrophies and to highlight key signatures of fibrosis that are neglected or underestimated in traditional 2D monocultures. Overall, this vascularized meso-scale cellular construct finely recapitulates the human skeletal muscle environment and provides an advanced solution for in vitro studies of muscle physiology and pathology. : Bersini et al. demonstrate the generation of a mesoscale model of the human muscle environment and prove its application for the study of fibrosis. This engineered muscle environment promotes the organ-specific differentiation of endothelial cells and the self-assembly of myofibers spontaneously wrapped by a continuous endomysium-like structure. Keywords: endothelial specificity, 3D vascularized muscle model, fibrosis, muscle environment