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.

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.

Casein phosphopeptides (CPPs), derived by casein proteolysis, can bind calcium ions and keep them in solution. In vitro studies have demonstrated CPP-induced cell calcium uptake, depending on the formation of (CPP + calcium) complexes and on the degree of differentiation of the intestinal cells. With the present study, we address the persistence of the complexes and of the CPP-induced calcium uptake in intestinal like cells after the digestion process, thus examining their eligibility to serve as nutraceuticals. A calcium-preloaded CPP preparation of commercial origin (Ca–CPPs) was subjected to in vitro digestion. The evolution of the supramolecular structure of the Ca–CPP complexes was studied using laser-light and X-ray scattering. The bioactivity of the pre- and post-digestion Ca–CPPs was determined in differentiated Caco2 and HT-29 cells by video imaging experiments using Fura-2. We found that Ca–CPP aggregates keep a complex supramolecular organization upon digestion, despite getting smaller in size and increasing internal calcium dispersion. Concomitantly and most interestingly, digested Ca–CPPs clearly enhance the uptake of calcium ions, especially in Caco2 cells. In contrast, digestion depletes the ability of post-loaded decalcified-CPPs (Ca–dekCPPs), with a weaker internal structure, to induce calcium uptake. The enhanced bioactivity reached upon digestion strongly suggests a recognized role of Ca–CPPs, in the form used here, as nutraceuticals.

Mesenchymal stem cells (MSCs) derived from adipose tissue and used either as expanded cells or minimally manipulated cell preparations showed positive clinical outcomes in regenerative medicine approaches based on tissue restoration and inflammation control, like in osteoarthritis (OA). Recently, MSCs’ healing capacity has been ascribed to the large array of soluble factors, including soluble cytokines/chemokines and miRNAs conveyed within extracellular vesicles (EVs). Therefore, in this study, 200 secreted cytokines, chemokines and growth factors via ELISA, together with EV-embedded miRNAs via high-throughput techniques, were scored in adipose-derived MSCs (ASCs) cultivated under inflammatory conditions, mimicking OA synovial fluid. Both factors (through most abundantly expressed TIMP1, TIMP2, PLG and CTSS) and miRNAs (miR-24-3p, miR-222-3p and miR-193b-3p) suggested a strong capacity for ASCs to reduce matrix degradation activities, as those activated in OA cartilage, and switch synovial macrophages, often characterized by an M1 inflammatory polarization, towards an M2 phenotype. Moreover, the crucial importance of selecting the target tissue is discussed, showing how a focused search may greatly improve potency prediction and explain clinical outcomes. In conclusion, herein presented data shed light about the way ASCs regulate cell homeostasis and regenerative pathways in an OA-resembling environment, therefore suggesting a rationale for the use of MSC-enriched clinical products, such as stromal vascular fraction and microfragmented adipose tissue, in joint pathologies.

Background: This study aims to investigate the clinical and radiological efficacy of three-dimensional acellular scaffolds (MaioRegen) in restoring osteochondral knee defects. Methods: MEDLINE, Scopus, CINAHL, Embase, and Cochrane Databases were searched for articles in which patients were treated with MaioRegen for osteochondral knee defects. Results: A total of 471 patients were included in the study (mean age 34.07 ± 5.28 years). The treatment involved 500 lesions divided as follows: 202 (40.4%) medial femoral condyles, 107 (21.4%) lateral femoral condyles, 28 (5.6%) tibial plateaus, 46 (9.2%) trochleas, 74 (14.8%) patellas, and 43 (8.6%) unspecified femoral condyles. Mean lesion size was 3.6 ± 0.85 cm². Only four studies reported a follow-up longer than 24 months. Significant clinical improvement has been reported in almost all studies with further improvement up to 5 years after surgery. A total of 59 complications were reported of which 52 (11.1%) experienced minor complications and 7 (1.48%) major complications. A total of 16 (3.39%) failures were reported. Conclusion: This systematic review describes the current available evidence for the treatment of osteochondral knee defects with MaioRegen Osteochondral substitute reporting promising satisfactory and reliable results at mid-term follow-up. A low rate of complications and failure was reported, confirming the safety of this scaffold. Considering the low level of evidence of the study included in the review, this data does not support the superiority of the Maioregen in terms of clinical improvement at follow-up compared to conservative treatment or other cartilage techniques.