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.

Human amniotic membrane-derived mesenchymal stromal cells (hAMSCs) are easily obtained in large quantities and free from ethical concerns. Promising therapeutic results for both hAMSCs and their secreted factors (secretome) were described by several in vitro and preclinical studies, often for treatment of orthopedic disorders such as osteoarthritis (OA) and tendinopathy. For clinical translation of the hAMSC secretome as cell-free therapy, a detailed characterization of hAMSC-secreted factors is mandatory. Herein, we tested the presence of 200 secreted factors and 754 miRNAs in extracellular vesicles (EVs). Thirty-seven cytokines/chemokines were identified at varying abundance, some of which involved in both chemotaxis and homeostasis of inflammatory cells and in positive remodeling of extracellular matrix, often damaged in tendinopathy and OA. We also found 336 EV-miRNAs, 51 of which accounted for more than 95% of the genetic message. A focused analysis based on miRNAs related to OA and tendinopathy showed that most abundant EV-miRNAs are teno- and chondro-protective, able to induce M2 macrophage polarization, inhibit inflammatory T cells, and promote Treg. Functional analysis on IL-1β treated tenocytes and chondrocytes resulted in downregulation of inflammation-associated genes. Overall, presence of key regulatory molecules and miRNAs explain the promising therapeutic results of hAMSCs and their secretome for treatment of musculoskeletal conditions and are a groundwork for similar studies in other pathologies. Furthermore, identified molecules will pave the way for future studies aimed at more sharply predicting disease-targeted clinical efficacy, as well as setting up potency and release assays to fingerprint clinical-grade batches of whole secretome or purified components.

The aims of the study were to evaluate long-term outcomes after autologous matrix-induced chondrogenesis (AMIC) in the treatment of focal chondral lesions and to assess the possible improvements given by the combination of this technique with bone marrow aspirate concentrate (BMAC). Twenty-four patients (age range 18–55 years) affected by focal knee chondral lesions were treated with standard AMIC or AMIC enhanced by BMAC (AMIC+). Pain (Visual Analogue Scale (VAS)) and functional scores (Lysholm, International Knee Documentation Committee (IKDC), Tegner, Knee injury and Osteoarthritis Outcome Score (KOOS)) were collected pre-operatively and then at 6, 12, 24, 60, and 100 months after treatment. Magnetic resonance imaging (MRI) evaluation was performed pre-operatively and at 6, 12, and 24 months follow-ups. Patients treated with AMIC+ showed higher Lysholm scores (p = 0.015) and lower VAS (p = 0.011) in comparison with patients in the standard AMIC group at the 12 months follow-up. Both treatments allowed for functional and pain improvements with respect to pre-operative levels lasting up to 100 months. MRI revealed consistent cartilage repair at 24 months in both groups. This study shows that AMIC and AMIC+ are effective treatments for focal chondral lesions with beneficial effect lasting up to 9 years. AMIC+ allows for faster recovery from injury, and is thus more indicated for patients requiring a prompt return to activity. Level of evidence: II, randomized controlled trial in an explorative cohort.

Osteoarthritis (OA) is the most prevalent joint disease, and to date, no options for effective tissue repair and restoration are available. With the aim of developing new therapies, the impact of mesenchymal stem cells (MSCs) has been explored, and the efficacy of MSCs started to be deciphered. A strong paracrine capacity relying on both secreted and vesicle-embedded (EVs) protein or nucleic acid-based factors has been proposed as the principal mechanism that contributes to tissue repair. This work investigated the mechanism of internalization of extracellular vesicles (EVs) released by adipose-derived MSCs (ASCs) and the role of shuttled miRNAs in the restoration of homeostasis in an in vitro model of human fibroblast-like synoviocytes (FLSs) from OA patients. ASC-EVs were isolated by differential centrifugation and validated by flow cytometry and nanoparticle tracking analysis. ASC-EVs with increased hyaluronan (HA) receptor CD44 levels were obtained culturing ASCs on HA-coated plastic surfaces. OA FLSs with intact or digested HA matrix were co-cultured with fluorescent ASC-EVs, and incorporation scored by flow cytometry and ELISA. ASC-EV complete miRNome was deciphered by high-throughput screening. In inflamed OA FLSs, genes and pathways potentially regulated by ASC-EV miRNA were predicted by bioinformatics. OA FLSs stimulated with IL-1β at physiological levels (25 pg/mL) were treated with ASC-EVs, and expression of inflammation and OA-related genes was measured by qRT-PCR over a 10-day time frame with modulated candidates verified by ELISA. The data showed that HA is involved in ASC-EV internalization in FLSs. Indeed, both removal of HA matrix presence on FLSs and modulation of CD44 levels on EVs affected their recruitment. Bioinformatics analysis of EV-embedded miRNAs showed their ability to potentially regulate the main pathways strictly associated with synovial inflammation in OA. In this frame, ASC-EVs reduced the expression of pro-inflammatory cytokines and chemokines in a chronic model of FLS inflammation. Given their ability to affect FLS behavior in a model of chronic inflammation through direct interaction with HA matrix and miRNA release, ASC-EVs confirm their role as a novel therapeutic option for osteoarthritic joints.