Background Mesenchymal stromal cell (MSC)-enriched products showed positive clinical outcomes in regenerative medicine, where tissue restoration and inflammation control are needed. GMP-expanded MSCs displayed an even higher potential due to exclusive secretion of therapeutic factors, both free and conveyed within extracellular vesicles (EVs), collectively termed secretome. Moreover, priming with biochemical cues may influence the portfolio and biological activities of MSC-derived factors. For these reasons, the use of naive or primed secretome gained attention as a cell-free therapeutic option. Albeit, at present, a homogenous and comprehensive secretome fingerprint is still missing. Therefore, the aim of this work was to deeply characterize adipose-derived MSC (ASC)-secreted factors and EV-miRNAs, and their modulation after IFNγ preconditioning. The crucial influence of the target pathology or cell type was also scored in osteoarthritis to evaluate disease-driven potency. Methods ASCs were isolated from four donors and cultured with and without IFNγ. Two-hundred secreted factors were assayed by ELISA. ASC-EVs were isolated by ultracentrifugation and validated by flow cytometry, transmission electron microscopy, and nanoparticle tracking analysis. miRNome was deciphered by high-throughput screening. Bioinformatics was used to predict the modulatory effect of secreted molecules on pathologic cartilage and synovial macrophages based on public datasets. Models of inflammation for both macrophages and chondrocytes were used to test by flow cytometry the secretome anti-inflammatory potency. Results Data showed that more than 60 cytokines/chemokines could be identified at varying levels of intensity in all samples. The vast majority of factors are involved in extracellular matrix remodeling, and chemotaxis or motility of inflammatory cells. IFNγ is able to further increase the capacity of the secretome to stimulate cell migration signals. Moreover, more than 240 miRNAs were found in ASC-EVs. Sixty miRNAs accounted for > 95% of the genetic message that resulted to be chondro-protective and M2 macrophage polarizing. Inflammation tipped the balance towards a more pronounced tissue regenerative and anti-inflammatory phenotype. In silico data were confirmed on inflamed macrophages and chondrocytes, with secretome being able to increase M2 phenotype marker CD163 and reduce the chondrocyte inflammation marker VCAM1, respectively. IFNγ priming further enhanced secretome anti-inflammatory potency. Conclusions Given the portfolio of soluble factors and EV-miRNAs, ASC secretome showed a marked capacity to stimulate cell motility and modulate inflammatory and degenerative processes. Preconditioning is able to increase this ability, suggesting inflammatory priming as an effective strategy to obtain a more potent clinical product which use should always be driven by the molecular mark of the target pathology.

The synovium of osteoarthritis (OA) patients can be characterized by an abnormal accumulation of macrophages originating from extravasated monocytes. Since targeting monocyte extravasation may represent a promising therapeutic strategy, our aim was to develop an organotypic microfluidic model recapitulating this process. Synovium and cartilage were modeled by hydrogel-embedded OA synovial fibroblasts and articular chondrocytes separated by a synovial fluid channel. The synovium compartment included a perfusable endothelialized channel dedicated to monocyte injection. Monocyte extravasation in response to chemokines and OA synovial fluid was quantified. The efficacy of chemokine receptor antagonists, RS-504393 (CCR2 antagonist) and Cenicriviroc (CCR2/CCR5 antagonist) in inhibiting extravasation was tested pre-incubating monocytes with the antagonists before injection. After designing and fabricating the chip, culture conditions were optimized to achieve an organotypic model including synovial fibroblasts, articular chondrocytes, and a continuous endothelial monolayer expressing intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. A significantly higher number of monocytes extravasated in response to the chemokine mix (p < 0.01) and OA synovial fluid (p < 0.01), compared to a control condition. In both cases, endothelium pre-activation enhanced monocyte extravasation. The simultaneous blocking of CCR2 and CCR5 proved to be more effective (p < 0.001) in inhibiting monocyte extravasation in response to OA synovial fluid than blocking of CCR2 only (p < 0.01). The study of extravasation in the model provided direct evidence that OA synovial fluid induces monocytes to cross the endothelium and invade the synovial compartment. The model can be exploited either to test molecules antagonizing this process or to investigate the effect of extravasated monocytes on synovium and cartilage cells.

Extravasation is a multi-step process implicated in many physiological and pathological events. This process is essential to get leukocytes to the site of injury or infection but is also one of the main steps in the metastatic cascade in which cancer cells leave the primary tumor and migrate to target sites through the vascular route. In this perspective, extravasation is a double-edged sword. This systematic review analyzes microfluidic 3D models that have been designed to investigate the extravasation of cancer and immune cells. The purpose of this systematic review is to provide an exhaustive summary of the advanced microfluidic 3D models that have been designed to study the extravasation of cancer and immune cells, offering a perspective on the current state-of-the-art. To this end, we set the literature search cross-examining PUBMED and EMBASE databases up to January 2020 and further included non-indexed references reported in relevant reviews. The inclusion criteria were defined in agreement between all the investigators, aimed at identifying studies which investigate the extravasation process of cancer cells and/or leukocytes in microfluidic platforms. Twenty seven studies among 174 examined each step of the extravasation process exploiting 3D microfluidic devices and hence were included in our review. The analysis of the results obtained with the use of microfluidic models allowed highlighting shared features and differences in the extravasation of immune and cancer cells, in view of the setup of a common framework, that could be beneficial for the development of therapeutic approaches fostering or hindering the extravasation process.

Cartilage defects can impair the most elementary daily activities and, if not properly treated, can lead to the complete loss of articular function. The limitations of standard treatments for cartilage repair have triggered the development of stem cell-based therapies. In this scenario, the development of efficient cell differentiation protocols and the design of proper biomaterial-based supports to deliver cells to the injury site need to be addressed through basic and applied research to fully exploit the potential of stem cells. Here, we discuss the use of microfluidics and bioprinting approaches for the translation of stem cell-based therapy for cartilage repair in clinics. In particular, we will focus on the optimization of hydrogel-based materials to mimic the articular cartilage triggered by their use as bioinks in 3D bioprinting applications, on the screening of biochemical and biophysical factors through microfluidic devices to enhance stem cell chondrogenesis, and on the use of microfluidic technology to generate implantable constructs with a complex geometry. Finally, we will describe some new bioprinting applications that pave the way to the clinical use of stem cell-based therapies, such as scaffold-free bioprinting and the development of a 3D handheld device for the in situ repair of cartilage defects.

No abstract

p¼0.0014, respectively) (Fig 1A). Receiver operating characteristics (ROC) analysis of SF TGF-b1 (adjusting for age, BMI, gender, baseline OST and JSN) for the determination of any knee OA progression (either OST, JSN or TKR progression vs. non-progression) yielded an area under the curve (AUC) of 0.95 (95% CI [0.87, 0.98]) (Fig 1B). ROC analysis of elastase yielded an AUC of 0.90 (CI [0.80, 0.95]) (Fig 1B). Applying the Youden index as the cutoff value, for TGF-b1 the sensitivity was 97% (CI [89, 99]), the specificity 85% (CI [62, 97]), and the positive predictive value (PPV) 95% (CI [88, 98]). For elastase the sensitivity was 85% (CI [74, 0.92]), the specificity 86% (CI [65, 97]), and the PPV 95% (CI [86, 98]). Conclusions: We have identified activated macrophages and neutrophils as the functional folate receptor bearing cells in knee OA. The correlation of SF TGF-b1 and elastase with the EC20 imaging phenotype confirmed the pro-inflammatory nature of the FRþ cells in vivo. We further confirmed the pro-inflammatory nature of these cells based on the association of these cell markers with knee OA progression. Taken together, these results suggest that these cells could be potential therapeutic targets in OA. The promising AUC scores for these markers also suggest that SF TGF-b1 and elastase could be the novel biomarkers of risk of progression in knee OA.

No abstract