The roles and therapeutic potentialof mesenchymal stem/stromal cells and their extracellular vesicles in tendinopathies.

Tendinopathies encompass a highly prevalent, multi-faceted spectrum of disorders, characterized by activity-related pain, compromised function, and propensity for an extended absence from sport and the workplace. The pathophysiology of tendinopathy continues to evolve. For decades, it has been related primarily to repetitive overload trauma but more recently, the onset of tendinopathy has been attributed to the tissue's failed attempt to heal after subclinical inflammatory and immune challenges (failed healing model). Conventional tendinopathy management produces only short-term symptomatic relief and often results in incomplete repair or healing leading to compromised tendon function. For this reason, there has been increased effort to develop therapeutics to overcome the tissue's failed healing response by targeting the cellular metaplasia and pro-inflammatory extra-cellular environment. On this basis, stem cell-based therapies have been proposed as an alternative therapeutic approach designed to modify the course of the various tendon pathologies. Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells often referred to as "medicinal signaling cells" due to their immunomodulatory and anti-inflammatory properties that can produce a pro-regenerative microenvironment in pathological tendons. However, the adoption of MSCs into clinical practice has been limited by FDA regulations and perceived risk of adverse events upon infusion in vivo. The introduction of cell-free approaches, such as the extracellular vesicles of MSCs, has encouraged new perspectives for the treatment of tendinopathies, showing promising short-term results. In this article, we review the most recent advances in MSC-based and MSC-derived therapies for tendinopathies. Preclinical and clinical studies are included with comment on future directions of this rapidly developing therapeutic modality, including the importance of understanding tissue loading and its relationship to any treatment regimen.

Cell-based therapies have disease-modifying effects on osteoarthritis in animal models. A systematic review by the ESSKA Orthobiologic Initiative. Part 2: bone marrow-derived cell-based injectable therapies.

PURPOSE: Aim of this systematic review was to determine if bone marrow-derived cell-based injectable therapies induce disease-modifying effects in joints affected by osteoarthritis (OA) in animal models. METHODS: A systematic review was performed on three electronic databases (PubMed, Web of Science, Embase) according to PRISMA guidelines. A synthesis of the results was performed investigating disease-modifying effects in preclinical animal studies comparing injectable bone marrow-derived products with OA controls or other products, different formulations or injection intervals, and the combination with other products. The risk of bias was assessed according to the SYRCLE's tool. RESULTS: Fifty-three studies were included (1819 animals) with an increasing publication trend over time. Expanded cells were used in 48 studies, point-of-care products in 3 studies, and both approaches were investigated in 2 studies. Among the 47 studies presenting results on the disease-modifying effects, 40 studies (85%) reported better results with bone marrow-derived products compared to OA controls, with positive findings evident in 14 out of 20 studies (70%) in macroscopic assessment, in 30 out of 41 studies (73%) in histological assessment, and in 10 out of 13 studies (77%) in immunohistochemical evaluations. Clinical evaluations showed positive results in 7 studies out of 9 (78%), positive imaging results in 11 studies out of 17 (65%), and positive biomarker results in 5 studies out of 10 (50%). While 36 out of 46 studies (78%) reported positive results at the cartilage level, only 3 out of 10 studies (30%) could detect positive changes at the synovial level. The risk of bias was low in 42% of items, unclear in 50%, and high in 8%. CONCLUSION: This systematic review of preclinical studies demonstrated that intra-articular injections of bone marrow-derived products can induce disease-modifying effects in the treatment of OA, slowing down the progression of cartilage damage with benefits at macroscopic, histological, and immunohistochemical levels. Positive results have been also observed in terms of clinical and imaging findings, as well as in the modulation of inflammatory and cartilage biomarkers, while poor effects have been described on the synovial membrane. These findings are important to understand the potential of bone marrow-derived products and to guide further research to optimise their use in the clinical practice. LEVEL OF EVIDENCE: II.

Cell-based therapies have disease-modifying effects on osteoarthritis in animal models. A systematic review by the ESSKA Orthobiologic Initiative. Part 1: adipose tissue-derived cell-based injectable therapies.

PURPOSE: The aim of this systematic review was to determine if adipose tissue-derived cell-based injectable therapies can induce disease-modifying effects in joints affected by osteoarthritis (OA). METHODS: A systematic review was performed on three electronic databases (PubMed, Web of Science, Embase) according to PRISMA guidelines. A synthesis of the results was performed investigating disease-modifying effects in preclinical studies comparing injectable adipose-derived products with OA controls or other products, different formulations or injection intervals, and the combination with other products. The risk of bias was assessed according to the SYRCLE's tool. RESULTS: Seventy-one studies were included (2,086 animals) with an increasing publication trend over time. Expanded cells were used in 65 studies, 3 studies applied point of care products, and 3 studies investigated both approaches. Overall, 48 out of 51 studies (94%) reported better results with adipose-derived products compared to OA controls, with positive findings in 17 out of 20 studies (85%) in macroscopic, in 37 out of 40 studies (93%) in histological, and in 22 out of 23 studies (96%)  in immunohistochemical evaluations. Clinical and biomarker evaluations showed positive results in 14 studies out of 18 (78%) and 12 studies out of 14 (86%), while only 9 studies out of 17 (53%) of the imaging evaluations were able to detect differences versus controls. The risk of bias was low in 38% of items, unclear in 51%, and high in (11%). CONCLUSION: The current preclinical models document consistent evidence of disease-modifying effects of adipose-derived cell-based therapies for the treatment of OA. The high heterogeneity of the published studies highlights the need for further targeted research to provide recommendations on the optimal methodologies for a more effective application of these injective therapies for the treatment of OA in clinical practice. LEVEL OF EVIDENCE: II.

Tissue-Protective and Anti-Inflammatory Landmark of PRP-Treated Mesenchymal Stromal Cells Secretome for Osteoarthritis.

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.

Secreted Factors and Extracellular Vesicles Account for the Immunomodulatory and Tissue Regenerative Properties of Bone-Marrow-Derived Mesenchymal Stromal Cells for Osteoarthritis.

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.

Comparison of EV-free fraction, EVs, and total secretome of amniotic mesenchymal stromal cells for their immunomodulatory potential: a translational perspective.

Amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties demonstrated in vitro and in vivo in various diseases in which the dysregulated immune system plays a major role. The immunomodulatory and pro-regenerative effects of MSCs, among which hAMSCs lie in the bioactive factors they secrete and in their paracrine activity, is well known. The mix of these factors (i.e., secretome) can be either freely secreted or conveyed by extracellular vesicles (EV), thus identifying two components in the cell secretome: EV-free and EV fractions. This study aimed to discern the relative impact of the individual components on the immunomodulatory action of the hAMSC secretome in order to obtain useful information for implementing future therapeutic approaches using immunomodulatory therapies based on the MSC secretome. To this aim, we isolated EVs from the hAMSC secretome (hAMSC-CM) by ultracentrifugation and validated the vesicular product according to the International Society for Extracellular Vesicles (ISEV) criteria. EVs were re-diluted in serum-free medium to maintain the EV concentration initially present in the original CM. We compared the effects of the EV-free and EV fractions with those exerted by hAMSC-CM in toto on the activation and differentiation of immune cell subpopulations belonging to both the innate and adaptive immune systems. We observed that the EV-free fraction, similar to hAMSC-CM in toto, a) decreases the proliferation of activated peripheral blood mononuclear cells (PBMC), b) reduces the polarization of T cells toward inflammatory Th subsets, and induces the induction of regulatory T cells; c) affects monocyte polarization to antigen-presenting cells fostering the acquisition of anti-inflammatory macrophage (M2) markers; and d) reduces the activation of B lymphocytes and their maturation to plasma cells. We observed instead that all investigated EV fractions, when used in the original concentrations, failed to exert any immunomodulatory effect, even though we show that EVs are internalized by various immune cells within PBMC. These findings suggest that the active component able to induce immune regulation, tested at original concentrations, of the hAMSC secretome resides in factors not conveyed in EVs. However, EVs isolated from hAMSC could exert actions on other cell types, as reported by others.

Systematic review and meta-analysis on the use of human platelet lysate for mesenchymal stem cell cultures: comparison with fetal bovine serum and considerations on the production protocol.

Mesenchymal stem cell (MSC) culturing for cell therapies needs a step forward to be routinely used in clinical settings. Main concerns regard the use of animal origin reagents, in particular supplementing the culture medium with FBS. Lately, Human Platelet Lysate (HPL) has been proposed as animal-free alternative, described as an excellent supplement for culturing MSCs. The aim of this systematic review was to analyze the current literature on the effect of HPL and FBS on ASCs and BMSCs. The primary outcome was the proliferation rate of cells cultured with FBS and HPL. Differences in terms of doubling time (DT) and population doubling (PD) were evaluated by meta-analysis, subgrouping data according to the cell type. A total of 35 articles were included. BMSCs and ASCs were used in 65.7% (23) and 28.6% (10) studies, respectively. Only two studies included both cell types. Overall, 22 studies were eligible for the meta-analysis. Among them, 9 articles described ASCs and 13 BMSCs. The results showed that BMSCs and ASCs cultured with 10% HPL and 5% HPL have lower DT and higher PD compared to cells cultured with 10% FBS. A possible correlation between the DT decrease and the application of at least 3 freeze/thaw cycles to induce platelet lysis was found. Additionally, HPL increased VEGF secretion and maintained the immuno-modulatory abilities for both cell types. The clarification reported here of the higher efficiency of HPL compared to FBS can help the transition of the scientific community towards clinical-related procedures. 1. The meta-analysis shows that HPL induces a population doubling increase and a doubling time decrease of both ASCs and BMSCs compared to FBS. 2. When at least 3 freeze/thaw cycles are applied to induce platelet lysis, the doubling time of HPL-cultured cells is lower than FBS-cultured cells (Created with BioRender.com).

Endogenous Controls for the Evaluation of Osteoarthritis-Related miRNAs in Extracellular Vesicles from Bone-Marrow-Derived Mesenchymal Stromal Cells and the Impact of Osteoarthritis Synovial Fluid.

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.

A single step, centrifuge-free method to harvest bone marrow highly concentrated in mesenchymal stem cells: results of a pilot trial.

PURPOSE: The aims of the present study were: (1) to characterize the bone-marrow aspirate (BMA) obtained with a centrifuge-free process, employing a dedicated aspiration device; (2) to test the in vitro efficacy of BMA in a model of cartilage inflammation; and (3) to report the preliminary clinical results in a small cohort of patients affected by knee OA. METHODS: Ten patients (4 M, 6 W; mean age: 51.9 ± 9.2 yy) affected by mild to moderate unicompartmental knee OA (KL grade 2-3) were treated by intra-articular and subchondral injections of BMA obtained by a centrifuge-free process. To evaluate the effectiveness of the device in harvesting mesenchymal stem cells (MSCs), samples of the obtained BMA were tested by flow cytometry before and after subculture; BMA ability to counteract inflammation was also tested in an in vitro model of cartilage cell inflammation, evaluating the expression of MMP1, MMP3, TGFß and TIMP-1 by real-time PCR. Patients were also evaluated up to two years' follow-up by using: VAS for pain, IKDC-subjective and KOOS scores. RESULTS: The laboratory analysis showed that BMSCs accounted for 0.011% of BMA cells, similar to what had been expected in native bone marrow. The paracrine activity of BMA was able to reduce in vitro the catabolic response of human chondrocyte, as shown by the decrease in metalloproteases concentration and increase in anti-inflammatory mediators. Moreover, the clinical evaluation showed significant improvements in all scores adopted, with stable results up to two years. CONCLUSION: The present data showed the effectiveness of the study device to harvest pure bone marrow with minimal peripheral blood contamination. The relevant content of MSCs resulted in the ability to counteract the catabolic cascade through a paracrine action. The clinical outcomes in patients affected by unicompartmental knee OA were encouraging in terms of pain reduction and functional improvement up to mid-term evaluation.

Adipose-Derived Mesenchymal Stromal Cells Treated with Interleukin 1 Beta Produced Chondro-Protective Vesicles Able to Fast Penetrate in Cartilage.

The study of the miRNA cargo embedded in extracellular vesicles (EVs) released from adipose-derived mesenchymal stromal cells (ASC) preconditioned with IL-1ß, 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-1ß 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, TGFß 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-1ß 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.

Cartilage Protective and Immunomodulatory Features of Osteoarthritis Synovial Fluid-Treated Adipose-Derived Mesenchymal Stem Cells Secreted Factors and Extracellular Vesicles-Embedded miRNAs.

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.

Superior Osteo-Inductive and Osteo-Conductive Properties of Trabecular Titanium vs. PEEK Scaffolds on Human Mesenchymal Stem Cells: A Proof of Concept for the Use of Fusion Cages.

Fusion cages composed of titanium and its alloys are emerging as valuable alternative to standard polyetheretherketone (PEEK) ones routinely used in cervical and lumbar spine surgery. Aim of this study was to evaluate osteo-inductive and osteo-conductive ability of an innovative trabecular titanium (T-Ti) scaffold on human mesenchymal stem cells (hMSCs), in both absence and presence of biochemical osteogenic stimuli. Same abilities were assessed on PEEK and standard 2D plastic surface, the latter meant as gold-standard for in vitro differentiation studies. hMSCs adhered and colonized both T-Ti and PEEK scaffolds. In absence of osteogenic factors, T-Ti triggered osteogenic induction of MSCs, as demonstrated by alkaline phosphatase activity and calcium deposition increments, while PEEK and standard 2D did not. Addition of osteogenic stimuli reinforced osteogenic differentiation of hMSCs cultured on T-Ti in a significantly higher manner with respect to standard 2D plastic culture surfaces, whereas PEEK almost completely abolished the process. T-Ti driven differentiation towards osteoblasts was confirmed by gene and marker expression analyses, even in absence of osteogenic stimuli. These results clearly indicate superior in vitro osteo-inductive and osteo-conductive capacity of T-Ti compared to PEEK, and make ground for further studies supporting the use of T-Ti cages to improve bone fusion.

Amniotic membrane-mesenchymal stromal cells secreted factors and extracellular vesicle-miRNAs: Anti-inflammatory and regenerative features for musculoskeletal tissues.

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-1ß 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.

Pain and Functional Scores in Patients Affected by Knee OA after Treatment with Pulsed Electromagnetic and Magnetic Fields: A Meta-Analysis.

OBJECTIVE: The purpose of this systematic review and meta-analysis was to evaluate the effect of electromagnetic field treatment on the symptoms of knee osteoarthritis (OA). In addition, the influence of the type of control group and other covariates have been investigated to identify the sources of heterogeneity in the results of the available clinical trials. METHODS: Randomized controlled trials reporting pulsed electromagnetic field-based therapies for the treatment of knee OA have been included. Main outcomes were self-reported pain and activity scores collected by Visual Analogue Scale (VAS) and/or Western Ontario McMaster Universities Osteoarthritis Index (WOMAC) at short term after treatment. RESULTS: Thirteen studies comprising 914 unique patients were included in the analysis. Overall reduction in pain score was observed after treatment (standardized mean difference -0.4059, P = 0.0091), while improvement in the activity score was not significant (standardized mean difference -0.4452, P = 0.0859). Type of control (i.e., placebo or alternative therapies) and time of follow-up resulted as the two major elements influencing the outcomes. Indeed, the restriction of the analysis to placebo-controlled trials demonstrated higher standardized mean differences between treatment and control groups, with lower P value for pain, while statistical significance became evident also for the activity score. On the contrary, no differences were observed pooling only studies comparing pulsed electromagnetic or magnetic fields to alternative treatments. In addition, longer follow-up correlated with lower differences between treated and control patients. CONCLUSIONS: Pulsed electromagnetic field therapy effectively relieves knee OA symptoms at short term, but it is not superior to other conservative therapies such as physiotherapy.

Pulsed electromagnetic fields improve the healing process of Achilles tendinopathy: a pilot study in a rat model.

AIMS: In the context of tendon degenerative disorders, the need for innovative conservative treatments that can improve the intrinsic healing potential of tendon tissue is progressively increasing. In this study, the role of pulsed electromagnetic fields (PEMFs) in improving the tendon healing process was evaluated in a rat model of collagenase-induced Achilles tendinopathy. METHODS: A total of 68 Sprague Dawley rats received a single injection of type I collagenase in Achilles tendons to induce the tendinopathy and then were daily exposed to PEMFs (1.5 mT and 75 Hz) for up to 14 days - starting 1, 7, or 15 days after the injection - to identify the best treatment option with respect to the phase of the disease. Then, 7 and 14 days of PEMF exposure were compared to identify the most effective protocol. RESULTS: The daily exposure to PEMFs generally provided an improvement in the fibre organization, a decrease in cell density, vascularity, and fat deposition, and a restoration of the physiological cell morphology compared to untreated tendons. These improvements were more evident when the tendons were exposed to PEMFs during the mid-acute phase of the pathology (7 days after induction) rather than during the early (1 day after induction) or the late acute phase (15 days after induction). Moreover, the exposure to PEMFs for 14 days during the mid-acute phase was more effective than for 7 days. CONCLUSION: PEMFs exerted a positive role in the tendon healing process, thus representing a promising conservative treatment for tendinopathy, although further investigations regarding the clinical evaluation are needed.Cite this article: Bone Joint Res 2020;9(9):613-622.

A2A adenosine receptors are involved in the reparative response of tendon cells to pulsed electromagnetic fields.

Tendinopathy is a degenerative disease in which inflammatory mediators have been found to be sometimes present. The interaction between inflammation and matrix remodeling in human tendon cells (TCs) is supported by the secretion of cytokines such as IL-1ß, IL-6 and IL-33. In this context, it has been demonstrated that pulsed electromagnetic fields (PEMFs) were able to reduce inflammation and promote tendon marker synthesis. The aim of this study was to evaluate the anabolic and anti-inflammatory PEMF-mediated response on TCs in an in vitro model of inflammation. Moreover, since PEMFs enhance the anti-inflammatory efficacy of adenosine through the adenosine receptors (ARs), the study also focused on the role of A2AARs. Human TCs were exposed to PEMFs for 48 hours. After stimulation, A2AAR saturation binding experiments were performed. Along with 48 hours PEMF stimulation, TCs were treated with IL-1ß and A2AAR agonist CGS-21680. IL-1Ra, IL-6, IL-8, IL-10, IL-33, VEGF, TGF-ß1, PGE2 release and SCX, COL1A1, COL3A1, ADORA2A expression were quantified. PEMFs exerted A2AAR modulation on TCs and promoted COL3A1 upregulation and IL-33 secretion. In presence of IL-1ß, TCs showed an upregulation of ADORA2A, SCX and COL3A1 expression and an increase of IL-6, IL-8, PGE2 and VEGF secretion. After PEMF and IL-1ß exposure, IL-33 was upregulated, whereas IL-6, PGE2 and ADORA2A were downregulated. These findings demonstrated that A2AARs have a role in the promotion of the TC anabolic/reparative response to PEMFs and to IL-1ß.

Innovative Visualization and Quantification of Extracellular Vesicles Interaction with and Incorporation in Target Cells in 3D Microenvironments.

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.

Signature quality attributes of CD146+ mesenchymal stem/stromal cells correlate with high therapeutic and secretory potency.

CD146+ bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) play key roles in the perivascular niche, skeletogenesis, and hematopoietic support; however, comprehensive evaluation of therapeutic potency has yet to be determined. In this study, in vitro inflammatory priming to crude human BM-MSCs (n = 8) captured a baseline of signature responses, including enriched CD146+ with coexpression of CD107aHigh , CXCR4High , and LepRHigh , transcriptional profile, enhanced secretory capacity, and robust immunomodulatory secretome and function, including immunopotency assays (IPAs) with stimulated immune cells. These signatures were significantly more pronounced in CD146+ (POS)-sorted subpopulation than in the CD146- (NEG). Mechanistically, POS BM-MSCs showed a markedly higher secretory capacity with significantly greater immunomodulatory and anti-inflammatory protein production upon inflammatory priming compared with the NEG BM-MSCs. Moreover, IPAs with stimulated peripheral blood mononuclear cells and T lymphocytes demonstrated robust immunosuppression mediated by POS BM-MSC while inducing significant frequencies of regulatory T cells. in vivo evidence showed that POS BM-MSC treatment promoted pronounced M1-to-M2 macrophage polarization, ameliorating inflammation/fibrosis of knee synovium and fat pad, unlike treatment with NEG BM-MSCs. These data correlate the expression of CD146 with innately higher immunomodulatory and secretory capacity, and thus therapeutic potency. This high-content, reproducible evidence suggests that the CD146+ (POS) MSC subpopulation are the mediators of the beneficial effects achieved using crude BM-MSCs, leading to translational implications for improving cell therapy and manufacturing.

Inflammatory priming enhances mesenchymal stromal cell secretome potential as a clinical product for regenerative medicine approaches through secreted factors and EV-miRNAs: the example of joint disease.

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.

Secreted Factors and EV-miRNAs Orchestrate the Healing Capacity of Adipose Mesenchymal Stem Cells for the Treatment of Knee Osteoarthritis.

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.