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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.

Due to the scientific success of in vitro and in vivo model studies, the interest in using mesenchymal stromal cells (MSCs) for the treatment of orthopaedic conditions is growing. In the context of osteoarthritis (OA), MSCs, and, in particular, those derived from adipose tissues (ASCs), have found broader access to clinical use as active components of minimally manipulated orthobiologics, as well as clinically expanded cell preparations, or to collect their released factors (secretome) for cell-free approaches. In this regard, while both inflammatory priming and starvation are common strategies used to empower cell potency or collect the secretome, respectively, little is known about the possible influence of these approaches on the stability of housekeeping genes (HKGs) for molecular studies able to fingerprint cell phenotype or potency. In this report, the reliability of five commonly used HKGs (ACTB, B2M, GAPDH, HPRT1 and RPLP0) was tested in ASCs cultured under standard protocol after inflammatory priming or starvation. Gene expression data were computed with four different applets able to rank genes depending on their stability in either single or combined conditions. The obtained final ranking suggests that for each treatment, a specific HKG is needed, and that starvation is the condition with the stronger effect on HKGs’ stability and, therefore, reliability. The normalization effect of proper HKGs’ use was then validated on three genes involved in OA and whose product is released by ASCs. Overall, data presented herein confirm that the choice of the best HKG has to be carefully considered and that each specific condition has to be tested to identify the most reliable candidate.

Osteoarthritis (OA) is a degenerative joint disorder characterized by the progressive deterioration of articular cartilage driven and sustained by catabolic and inflammatory processes that lead to pain and functional impairment. Adipose-derived stem cells (ASCs) have emerged as a promising therapeutic strategy for OA due to their regenerative potential, which mainly relies on the adaptive release of paracrine molecules that are soluble or encapsulated in extracellular vesicles (EVs). The biological effects of EVs specifically depend on their cargo; in particular, microRNAs (miRNAs) can specifically modulate target cell function through gene expression regulation. This study aimed to investigate the impact of collection site (abdominal vs. peri-trochanteric adipose tissue) and collection method (surgical excision vs. lipoaspiration) on the miRNAs profile in ASC-derived EVs and their potential implications for OA therapy. EV-miRNA cargo profiles from ASCs of different origins were compared. An extensive bioinformatics search through experimentally validated and OA-related targets, pathways, and tissues was conducted. Several miRNAs involved in the restoration of cartilage homeostasis and in immunomodulation were identified in all ASC types. However, EV-miRNA expression profiles were affected by both the tissue-harvesting site and procedure, leading to peculiar characteristics for each type. Our results suggest that adipose-tissue-harvesting techniques and the anatomical site of origin influence the therapeutic efficacy of ASC-EVs for tissue-specific regenerative therapies in OA, which warrants further investigation.

Total Ankle Replacement (TAR) could, in theory, partially restore joint sensation and motion, enhancing motor coordination, functional stability, and proprioception in a joint affected by osteoarthritis. The aim of this study is to evaluate ankle proprioception before and after a Total Ankle Replacement via an instrumental objective assessment. The evaluation of proprioceptive stability was carried out using the dedicated Delos Postural Proprioceptive System (DPPS). The clinical evaluation was performed by administering validated questionnaires, namely the American Orthopaedic Foot and Ankle Society (AOFAS) score and Short Form-36 (SF-36). Twenty patients were selected, undergoing a complete procedure with clinical and instrumental pre-operative and post-operative evaluation at the end of the follow-up. AOFAS ankle–hindfoot and SF-36 both showed a statistically significant improvement between the pre-operative and post-operative state. The postural tests performed using the DPPS showed that the affected limb showed results slightly lower than in pre-operative. In conclusion, according to the current data, the TAR does not show an improvement in proprioceptive joint stability, and although the registered mean values do not present a statistical significance, the stabilometry shows data lower than the healthy limb. In this perspective, further studies are recommended to highlight any rehabilitation scheme or protocols, using wider case series.

Among the available therapeutics for the conservative treatment of osteoarthritis (OA), mesenchymal stromal cells (MSCs)-based products appear to be the most promising. Alongside minimally manipulated cell-based orthobiologics, where MSCs are the engine of the bioactive properties, cell expansion under good manufacturing practice (GMP) settings is actively studied to obtain clinical-grade pure populations able to concentrate the biological activity. One of the main characteristics of GMP protocols is the use of clinical-grade reagents, including the recently released serum-free/xeno-free (SFM/XFM) synthetic media, which differ significantly from the traditional reagents like those based on fetal bovine serum (FBS). As SFM/XFM are still poorly characterized, a main lack is the notion of reliable housekeeping genes (HKGs) for molecular studies, either standalone or in combination with standard conditions. Indeed, the aim of this work was to test the stability of five commonly used HKGs (ACTB, EF1A, GAPDH, RPLP0, and TBP) in adipose-derived MSCs (ASCs) cultivated in two commercially available SFM/XFM and to compare outcomes with those obtained in FBS. Four different applets widely recognized by the scientific community (NormFinder, geNorm, comparative ΔCt method, and BestKeeper) were used and data were merged to obtain a final stability order. The analysis showed that cells cultured in both synthetic media had a similar ranking for HKGs stability (GAPDH being best), albeit divergent from FBS expanded products (EF1A at top). Moreover, it was possible to identify specific HKGs for side by side studies, with EF1A/TBP being the most reliable normalizers for single SFM/XFM vs. FBS cultured cells and TBP the best one for a comprehensive analysis of all samples. In addition, stability of HKGs was donor-dependent. The normalization effect on selected genes coding for factors known to be involved in OA pathology, and whose amount should be carefully considered for the selection of the most appropriate MSC-based treatment, showed how HKGs choice might affect the perceived amount for the different media or donor. Overall, this work confirms the impact of SFM/XFM conditions on HKGs stability performance, which resulted similarly for both synthetic media analyzed in the study.

Osteoarthritis (OA) is a chronic disease characterized by joint tissue disruption and inflammation with a paucity of therapeutic options. Chondrocyte in vitro models are commonly used as the first step in evaluating new approaches and rely on the stimulation of an OA-like phenotype with inflammation often the method of choice. Inflammatory priming is frequently based on cytokines used at concentrations very far from the reality in the patients’ synovial fluid (SF). The aim of this work was to compare the transcriptional response of chondrocytes to different inflammatory conditions: the high levels of IL1β that are used for standardized inflammation protocols, OA-SF, IL1β, IL6 and IFNγ at SF-like concentrations both individually and simultaneously to mimic a simplified “in vitro” SF. Both high IL1β and OA-SF strongly influenced chondrocytes, while SF-like concentrations of cytokines gave weak (IL1β alone or in combination) or no (IL6 and IFNγ alone) outcomes. Chondrocytes under the two most powerful polarizing conditions had a clearly distinct fingerprint, with only a shared albeit molecularly divergent effect on ECM stability, with IL1β mainly acting on ECM degrading enzymes and OA-SF accounting for a higher turnover in favor of fibrous collagens. Moreover, OA-SF did not induce the inflammatory response observed with IL1β. In conclusion, although partially similar in the endpoint phenotype, this work intends to encourage reflection on the robustness of inflammation-based in vitro OA models for molecular studies on chondrocytes.

Abstract 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.

Bone-marrow-mesenchymal-stromal-cells (BMSCs)- and platelet-rich-plasma (PRP)-based therapies have shown potential for treating osteoarthritis (OA). Recently, the combination of these two approaches was proposed, with results that overcame those observed with the separate treatments, indicating a possible role of PRP in ameliorating BMSCs’ regenerative properties. Since a molecular fingerprint of BMSCs cultivated in the presence of PRP is missing, the aim of this study was to characterize the secretome in terms of soluble factors and extracellular-vesicle (EV)-embedded miRNAs from the perspective of tissues, pathways, and molecules which frame OA pathology. One hundred and five soluble factors and one hundred eighty-four EV-miRNAs were identified in the PRP-treated BMSCs’ secretome, respectively. Several soluble factors were related to the migration of OA-related immune cells, suggesting the capacity of BMSCs to attract lympho-, mono-, and granulocytes and modulate their inflammatory status. Accordingly, several EV-miRNAs had an immunomodulating role at both the single-factor and cell level, together with the ability to target OA-characterizing extracellular-matrix-degrading enzymes and cartilage destruction pathways. Overall, anti-inflammatory and protective signals far exceeded inflammation and destruction cues for cartilage, macrophages, and T cells. This study demonstrates that BMSCs cultivated in the presence of PRP release therapeutic molecules and give molecular ground for the use of this combined and innovative therapy for OA treatment.

Bone marrow-derived mesenchymal stromal cells (BMSCs)-based therapies show a great potential to manage inflammation and tissue degeneration in osteoarthritis (OA) patients. Clinical trials showed the ability to manage pain and activation of immune cells and allowed restoration of damaged cartilage. To date, a molecular fingerprint of BMSC-secreted molecules in OA joint conditions able to support clinical outcomes is missing; the lack of that molecular bridge between BMSC activity and clinical results hampers clinical awareness and translation into practice. In this study, BMSCs were cultured in synovial fluid (SF) obtained from OA patients and, for the first time, a thorough characterization of soluble factors and extracellular vesicles (EVs)-embedded miRNAs was performed in this condition. Molecular data were sifted through the sieve of molecules and pathways characterizing the OA phenotype in immune cells and joint tissues. One-hundred and twenty-five secreted factors and one-hundred and ninety-two miRNAs were identified. The combined action of both types of molecules was shown to, first, foster BMSCs interaction with the most important OA immune cells, such as macrophages and T cells, driving their switch towards an anti-inflammatory phenotype and, second, promote cartilage homeostasis assisting chondrocyte proliferation and attenuating the imbalance between destructive and protective extracellular matrix-related players. Overall, molecular data give an understanding of the clinical results observed in OA patients and can enable a faster translation of BMSC-based products into everyday clinical practice.

Bone-marrow-derived mesenchymal stromal cells (BMSCs) showed therapeutic potential in the treatment of musculoskeletal diseases, including osteoarthritis (OA). Their soluble mediators and extracellular vesicles (EVs), which make up the secretome, suppress immune response, attenuate inflammation and promote cartilage repair. EVs, as well as the whole secretome, have been investigated as cell free approaches for OA although, to date, a disease-tailored molecular fingerprint is missing. In this study, soluble mediators and miRNAs were sifted in the BMSCs’ secretome and EVs, respectively, and analyzed in the frame of cell types and factors involved in OA. The majority of identified molecules repress the activation of immune cells and the production of OA-related inflammatory mediators, as well as promote cartilage protection by acting on both chondrocytes homeostasis and extracellular matrix-degrading enzymes. These data provide the molecular ground for the therapeutic potential of BMSCs for regenerative applications for OA and support the use of secretome or EVs as cell-free applications in joint diseases.

In regenerative medicine related to orthopedic conditions, mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) have been proposed as innovative clinical options. The definition of EV-shuttled signals and their modulation under orthopedic settings, such as osteoarthritis (OA), is crucial for MSC-related research, both for basic science and for use in clinical settings, either as therapeutics or as producers of cell-free products such as EVs or secretome. The objective of this work is to compare the literature available on high-throughput EV-miRNA data obtained from adipose-derived MSCs (ASCs) in standard conditions or cultured in high levels of IFNγ, low-level inflammatory conditions mimicking OA synovial fluid (SF), and OA-SF. The first result was that both IFNγ and low-level inflammatory treatment led to an increase, whereas SF led to a reduction in EV release. Second, more than 200 EV-miRNAs were found to be shared across the different conditions. After a bioinformatics search through experimentally validated and OA-related targets, pathways and tissues, several miRNAs resulted in the restoration of cartilage and synovium stability and the homeostasis of inflammatory cells, including macrophages, promoting their switch towards an M2 anti-inflammatory phenotype. Third, IFNγ and especially SF culturing were able to modulate the overall EV-miRNA fingerprint, although the main molecular messages related to OA resulted conserved between treatments with the majority of modulations within 2-fold range. In conclusion, ASC EV-miRNAs may be modulated in their overall landscape by OA-related culturing conditions albeit resulted largely stable in their specific OA-protective signals allowing for a faster clinical translation of these new cell-free therapies for joint diseases.

Abstract Background 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. Methods 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. Results 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. Conclusions 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.

The use of microfragmented adipose tissue (µFAT) for the treatment of musculoskeletal disorders, especially osteoarthritis (OA), is gaining popularity, following positive results reported in recent case series and clinical trials. Although these outcomes were postulated to rely on paracrine signals, to date, a thorough fingerprint of released molecules is largely missing. The purpose of this study was to first characterize both structure and cell content of unprocessed lipoaspirate (LA) and µFAT, and further identify and frame the array of signaling factors in the context of OA disease, by means of high throughput qRT-PCR for extracellular-vesicle (EV) embedded miRNAs and proteomics for tissue and secreted factors. Cell count showed reduction of blood cells in µFAT, confirmed by histological and flow cytometry analyses, that also showed a conserved presence of structural, endothelial and stromal components and pericytes. In the secretome, 376 and 381 EV-miRNAs in LA and µFAT, respectively, were identified. In particular, most abundant and µFAT upregulated EV-miRNAs were mainly recapitulating those already reported as ASC-EVs-specific, with crucial roles in cartilage protection and M2 macrophage polarization, while only a scarce presence of those related to blood cells emerged. Furthermore, secretome proteomic analysis revealed reduction in µFAT of acute phase factors driving OA progression. Taken together, these results suggest that processing of LA into µFAT allows for removal of blood elements and maintenance of tissue structure and stromal cell populations, and possibly the increase of OA-protective molecular features. Thus, microfragmentation represents a safe and efficient method for the application of adipose tissue properties in the frame of musculoskeletal disorders.

Bone-marrow-derived stromal cells (BMSCs) have emerged as promising therapeutic option for the treatment of osteoarthritis (OA) due to their tissue regenerative and anti-inflammatory features. BMSCs’ clinical potential is mainly ascribed to their released factors and extracellular vesicles (EVs), whose therapeutic portfolio may be modulated by the environment in vivo or specific priming in vitro. Within the array of molecules shaping EVs’ power, miRNAs are considered privileged players. In this frame, a correct EV-miRNA detection and quantification is mandatory to understand and possibly boost BMSCs potential, either when envisioned as cell therapeutics or when proposed as producer of cell-free and clinical grade EVs. The aim of this study is to identify reliable reference genes (RGs) to study miRNAs in BMSC-EVs cultivated under standard or OA synovial fluid (OA-SF). miR-23a-3p and miR-221-3p emerged as the best candidates, respectively. Moreover, when both conditions were analyzed together, miR-24-3p resulted the most stable RGs, allowing for a sharper comparison of EVs content, further validated on the OA-related miRNA-193b-5p. The different RG stability ranking depending on the culturing conditions, as well as its divergence with respect to adipose (ASCs) and amniotic (hAMSCs) MSCs, confirm that miRNA RG selection in EVs is a mandatory step and that the identification of the most reliable candidate is greatly depending on the cell type and culturing/environmental conditions.

The purpose of the present study was to assess, prospectively, the safety, clinical effectiveness, and feasibility of a single intra-articular injection of microfragmented adipose tissue in different stages of knee osteoarthritis (OA). The study included patients (aged 18–70 years), affected by OA (Kellgren–Lawrence I-IV). Unselected patients were evaluated before and prospectively after 6, 12, and 24 months from the injection. Visual analog scale (VAS) and knee injury and osteoarthritis outcome score (KOOS) were used for clinical evaluations. A total of 202 patients were eligible. The mean follow-up time in the cohort of patients was 24.5 ± 9.6 months. Total KOOS significantly improved from pre-operative baseline levels to 6-month follow-up (p < 0.001), and again between 6- and 12-month follow-ups (p < 0.001). The VAS showed a prompt reduction at 6 months (p < 0.001 vs. baseline), but then it increased again at 12 months compared to the 6-month assessment (p < 0.001), even though it remained lower than baseline (p < 0.001). At 24 months, patients with KL-IV demonstrated a lower improvement compared to baseline; patients that had undergone previous corticosteroid injections had a greater risk to further injection treatment. The collected clinical results suggest that MFAT may represent a safe and effective treatment for OA symptoms, offering a low-demanding and minimally invasive treatment.

The study of the miRNA cargo embedded in extracellular vesicles (EVs) released from adipose-derived mesenchymal stromal cells (ASC) preconditioned with IL-1β, an inflammatory stimulus driving osteoarthritis (OA), along with EVs-cartilage dynamic interaction represent poorly explored fields and are the purpose of the present research. ASCs were isolated from subcutaneous adipose tissue and EVs collected by ultracentrifugation. Shuttled miRNAs were scored by high-throughput screening and analyzed through bioinformatics approach that predicted the potentially modulated OA-related pathways. Fluorescently labeled EVs incorporation into OA cartilage explants was followed in vitro by time-lapse coherent anti-Stokes Raman scattering; second harmonic generation and two-photon excited fluorescence. After IL-1β preconditioning, 7 miRNA were up-regulated, 4 down-regulated, 37 activated and 17 silenced. Bioinformatics allowed to identify miRNAs and target genes mainly involved in Wnt, Notch, TGFβ and Indian hedgehog (IHH) pathways, cartilage homeostasis, immune/inflammatory responses, cell senescence and autophagy. As well, ASC-EVs steadily diffuse in cartilage cells and matrix, reaching a plateau 16 h after administration. Overall, ASCs preconditioned with IL-1β allows secretion of EVs embedded with a chondro-protective miRNA cargo, able to fast penetrate in collagen-rich areas of cartilage with tissue saturation in a day. Further functional studies exploring the EVs dose-effects are needed to achieve clinical relevance.

Intra-articular administration of adipose-derived mesenchymal stem cells (ASCs), either in vitro expanded or within adipose tissue-based products obtained at point-of-care, has gained popularity as innovative regenerative medicine approach for osteoarthritis (OA) treatment. ASCs can stimulate tissue repair and immunomodulation through paracrine factors, both soluble and extracellular vesicles (EV) embedded, collectively defining the secretome. Interaction with the degenerative/inflamed environment is a crucial factor in understanding the finely tuned molecular message but, to date, the majority of reports have described ASC-secretome features in resting conditions or under chemical stimuli far from the in vivo environment of degenerated OA joints. In this report, the secretory profile of ASCs treated with native synovial fluid from OA patients was evaluated, sifting 200 soluble factors and 754 EV-embedded miRNAs. Fifty-eight factors and 223 EV-miRNAs were identified, and discussed in the frame of cartilage and immune cell homeostasis. Bioinformatics gave a molecular basis for M2 macrophage polarization, T cell proliferation inhibition and T reg expansion enhancement, as well as cartilage protection, further confirmed in an in vitro model of OA chondrocytes. Moreover, a strong influence on immune cell chemotaxis emerged. In conclusion, obtained molecular data support the regenerative and immunomodulatory properties of ASCs when interacting with osteoarthritic joint environment.

Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) are promising therapeutic nano-carriers for the treatment of osteoarthritis (OA). The assessment of their uptake in tissues is mandatory but, to date, available technology does not allow to track and quantify incorporation in real-time. To fill this knowledge gap, the present study was intended to develop an innovative technology to determine kinetics of fluorescent MSC-EV uptake by means of time-lapse quantitative microscopy techniques. Adipose-derived mesenchymal stromal cells (ASCs)-EVs were fluorescently labeled and tracked during their uptake into chondrocytes micromasses or cartilage explants, both derived from OA patients. Immunofluorescence and time-lapse coherent anti-Stokes Raman scattering, second harmonic generation and two-photon excited fluorescence were used to follow and quantify incorporation. EVs penetration appeared quickly after few minutes and reached 30–40 μm depth after 5 h in both explants and micromasses. In explants, uptake was slightly faster, with EVs signal overlapping both extracellular matrix and chondrocytes, whereas in micromasses a more homogenous diffusion was observed. The finding of this study demonstrates that this innovative technology is a powerful tool to monitor EVs migration in tissues characterized by a complex extracellular network, and to obtain data resembling in vivo conditions.

Extracellular vesicles (EVs) showed therapeutic properties in several applications, many in regenerative medicine. A clear example is in the treatment of osteoarthritis (OA), where adipose-derived mesenchymal stem cells (ASCs)-EVs were able to promote regeneration and reduce inflammation in both synovia and cartilage. A still obscure issue is the effective ability of EVs to be internalized by target cells, rather than simply bound to the extracellular matrix (ECM) or plasma membrane, since the current detection or imaging technologies cannot fully decipher it due to technical limitations. In the present study, human articular chondrocytes (ACHs) and fibroblast-like synoviocytes (FLSs) isolated from the same OA patients were cocultured in 2D as well as in 3D conditions with fluorescently labeled ASC-EVs, and analyzed by flow cytometry or confocal microscopy, respectively. In contrast with conventional 2D, in 3D cultures, confocal microscopy allowed a clear detection of the tridimensional morphology of the cells and thus an accurate discrimination of EV interaction with the external and/or internal cell environment. In both 2D and 3D conditions, FLSs were more efficient in interacting with ASC-EVs and 3D imaging demonstrated a faster uptake process. The removal of the hyaluronic acid component from the ECM of both cell types reduced their interaction with ASC-EVs only in the 2D system, showing that 2D and 3D conditions can yield different outcomes when investigating events where ECM plays a key role. These results indicate that studying EVs binding and uptake both in 2D and 3D guarantees a more precise and complementary characterization of the molecular mechanisms involved in the process. The implementation of this strategy can become a valuable tool not only for basic research, but also for release assays and potency prediction for clinical EV batches.

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.

Cell-based therapies have recently been proposed for the treatment of degenerative articular pathologies, such as early osteoarthritis, with an emphasis on autologous mesenchymal stem cells (MSCs), as an alternative to terminally differentiated cells. In this study, we performed a donor-matched comparison between infrapatellar fat pad MSCs (IFP-MSCs) and knee subcutaneous adipose tissue stem cells (ASCs), as appealing candidates for cell-based therapies that are easily accessible during surgery. IFP-MSCs and ASCs were obtained from 25 osteoarthritic patients undergoing total knee replacement and compared for their immunophenotype and differentiative potential. Undifferentiated IFP-MSCs and ASCs displayed the same immunophenotype, typical of MSCs (CD13+/CD29+/CD44+/CD73+/CD90+/CD105+/CD166+/CD31-/CD45-). IFP-MSCs and ASCs showed similar adipogenic potential, though undifferentiated ASCs had higher LEP expression compared to IFP-MSCs (p < 0.01). Higher levels of calcified matrix (p < 0.05) and alkaline phosphatase (p < 0.05) in ASCs highlighted their superior osteogenic commitment compared to IFP-MSCs. Conversely, IFP-MSCs pellets showed greater amounts of glycosaminoglycans (p < 0.01) and superior expression of ACAN (p < 0.001), SOX9, COMP (p < 0.001) and COL2A1 (p < 0.05) compared to ASCs pellets, revealing a superior chondrogenic potential. This was also supported by lower COL10A1 (p < 0.05) and COL1A1 (p < 0.01) expression and lower alkaline phosphatase release (p < 0.05) by IFP-MSCs compared to ASCs. The observed dissimilarities between IFP-MSCs and ASCs show that, despite expressing similar surface markers, MSCs deriving from different fat depots in the same surgical site possess specific features. Furthermore, the in vitro peculiar commitment of IFP-MSCs and ASCs from osteoarthritic donors towards the chondrogenic or osteogenic lineage may suggest a preferential use for cartilage and bone cell-based treatments, respectively.

Among all joints affected, knee osteoarthritis has a prevalence of about 10% in men and 13% in women over 60 years old. Knee osteoarthritis has high economic and social costs and may have a devastating impact on patient quality of life. Treatment of symptomatic knee Osteoarthritis may involve oral or topical administration of non-steroidal anti-inflammatory drugs or intra-articular injection of corticosteroids. Recently, a novel injectable collagen formulation (ChondroGrid) consisting of bovine hydrolyzed MMP1/MMP3 and TIMP1/TIMP3 was then assessed through RT-PCR and that of TGFβ1, IGF-I, and VEGF using ELISA assays. Shape and ECM deposition were assessed using the Bern score after a 28-day ChondroGrid exposure, and collagen deposition was assessed using immunostaining. Records of 20 patients affected by Kellgren Lawrence grade 1 to 4 knee osteoarthritis who received three 4 mg/2 mL ChondroGrid injections 2 weeks apart were then retrospectively assessed to compare VAS, Lequesne, and WOMAC scores collected before and 15, 45, and 225 days after the first injection. ChondroGrid had no effects on the markers under consideration, but induced type-II and inhibited type-I collagen deposition; the Bern score was higher when cells were cultured with ChondroGrid. Patients experienced a 44% Lequesne score and a 55% VAS at moving score reduction. All other scores decreased >70%. ChondroGrid may prompt chondrocytes to produce hyaline cartilage, prevent fibrous tissue formation, and be a safe and effective adjuvant to treat symptomatic knee osteoarthritis.

Mesenchymal stem cells (MSCs) are promising tools for cell-based therapies due to their homing to injury sites, where they secrete bioactive factors such as cytokines, lipids, and nucleic acids, either free or conveyed within extracellular vesicles (EVs). Depending on the local environment, MSCs’ therapeutic value may be modulated, determining their fate and cell behavior. Inflammatory signals may induce critical changes on both the phenotype and secretory portfolio. Intriguingly, in animal models resembling joint diseases as osteoarthritis (OA), inflammatory priming enhanced the healing capacity of MSC-derived EVs. In this work, we selected miRNA reference genes (RGs) from the literature (let-7a-5p, miR-16-5p, miR-23a-3p, miR-26a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, U6 snRNA), using EVs isolated from adipose-derived MSCs (ASCs) primed with IFNγ (iASCs). geNorm, NormFinder, BestKeeper, and ΔCt methods identified miR-26a-5p/16-5p as the most stable, while miR-103a-rp/425-5p performed poorly. Our results were validated on miRNAs involved in OA cartilage trophism. Only a proper normalization strategy reliably identified the differences between donors, a critical factor to empower the therapeutic value of future off-the-shelf MSC-EV isolates. In conclusion, the proposed pipeline increases the accuracy of MSC-EVs embedded miRNAs assessment, and help predicting donor variability for precision medicine approaches.

Osteoarthritis (OA) leads to chronic pain and disability, and traditional conservative treatments are not effective in the long term. The intra-articular injection of mesenchymal stem cells (MSCs) is considered a novel therapy for OA whose efficacy mainly relies on the adaptive release of paracrine molecules which are either soluble or extracellular vesicles (EVs) embedded. The correct quantification of EV-miRNAs using reliable reference genes (RGs) is a crucial step in optimizing this future therapeutic cell-free approach. The purpose of this study is to rate the stabilities of literature-selected proposed RGs for EV-miRNAs in adipose derived-MSCs (ASCs). EVs were isolated by ultracentrifugation from ASCs cultured with or without inflammatory priming mimicking OA synovial fluid condition. Expression of putative RGs (let-7a-5p, miR-16-5p, miR-23a-3p, miR-26a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, U6 snRNA) was scored by using the algorithms geNorm, NormFinder, BestKeeper and ΔCt method. miR-16a-5p/miR-23a-3p yielded the most stable RGs, whereas let-7a-5p/miR-425-5p performed poorly. Outcomes were validated by qRT-PCR on miR-146a-5p, reported to be ASC-EVs enriched and involved in OA. Incorrect RG selection affected the evaluation of miR-146a-5p abundance and modulation by inflammation, with both values resulting strongly donor-dependent. Our findings demonstrated that an integrated approach of multiple algorithms is necessary to identify reliable, stable RGs for ASC-EVs miRNAs evaluation. A correct approach would increase the accuracy of embedded molecule assessments aimed to develop therapeutic strategies for the treatment of OA based on EVs.