Donor Sites and Harvesting Techniques Affect miRNA Cargos of Extracellular Vesicles Released by Human Adipose-Derived Mesenchymal Stromal Cells.

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.

Cellular and Structural Changes in Achilles and Patellar Tendinopathies: A Pilot In Vivo Study.

Tendinopathies continue to be a challenge for both patients and the medical teams providing care as no universal clinical practice guidelines have been established. In general, tendinopathies are typically characterized by prolonged, localized, activity-related pain with abnormalities in tissue composition, cellularity, and microstructure that may be observed on imaging or histology. In the lower limb, tendinopathies affecting the Achilles and the patellar tendons are the most common, showing a high incidence in athletic populations. Consistent diagnosis and management have been challenged by a lack of universal consensus on the pathophysiology and clinical presentation. Current management is primarily based on symptom relief and often consists of medications such as non-steroidal anti-inflammatories, injectable therapies, and exercise regimens that typically emphasize progressive eccentric loading of the affected structures. Implementing the knowledge of tendon stem/progenitor cells (TSPCs) and assessing their potential in enhancing tendon repair could fill an important gap in this regard. In the present pilot in vivo study, we have characterized the structural and cellular alterations that occur soon after tendon insult in models of both Achilles and patellar tendinopathy. Upon injury, CD146+ TSPCs are recruited from the interfascicular tendon matrix to the vicinity of the paratenon, whereas the observed reduction in M1 macrophage polarization is related to a greater abundance of reparative CD146+ TSPCs in situ. The robust TSPCs' immunomodulatory effects on macrophages were also demonstrated in in vitro settings where TSPCs can effectively polarize M1 macrophages towards an anti-inflammatory therapeutic M2 phenotype. Although preliminary, our findings suggest CD146+ TSPCs as a key phenotype that could be explored in the development of targeted regenerative therapies for tendinopathies.

Stable Housekeeping Genes in Bone Marrow, Adipose Tissue, and Amniotic Membrane-Derived Mesenchymal Stromal Cells for Orthopedic Regenerative Medicine Approaches.

The therapeutic effect of mesenchymal stromal cells (MSCs) has been described for a variety of disorders, including those affecting musculoskeletal tissues. In this context, the literature reports several data about the regenerative effectiveness of MSCs derived from bone marrow, adipose tissue, and an amniotic membrane (BMSCs, ASCs, and hAMSCs, respectively), either when expanded or when acting as clinical-grade biologic pillars of products used at the point of care. To date, there is no evidence about the superiority of one source over the others from a clinical perspective. Therefore, a reliable characterization of the tissue-specific MSC types is mandatory to identify the most effective treatment, especially when tailored to the target disease. Because molecular characterization is a crucial parameter for cell definition, the need for reliable normalizers as housekeeping genes (HKGs) is essential. In this report, the stability levels of five commonly used HKGs (ACTB, EF1A, GAPDH, RPLP0, and TBP) were sifted into BMSCs, ASCs, and hAMSCs. Adult and fetal/neonatal MSCs showed opposite HKG stability rankings. Moreover, by analyzing MSC types side-by-side, comparison-specific HKGs emerged. The effect of less performant HKG normalization was also demonstrated in genes coding for factors potentially involved in and predicting MSC therapeutic activity for osteoarthritis as a model musculoskeletal disorder, where the choice of the most appropriate normalizer had a higher impact on the donors rather than cell populations when compared side-by-side. In conclusion, this work confirms HKG source-specificity for MSCs and suggests the need for cell-type specific normalizers for cell source or condition-tailored gene expression studies.

Housekeeping Gene Stability in Adipose Mesenchymal Stromal Cells Cultivated in Serum/Xeno-Free Media for Osteoarthritis.

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.

miR-877-5p as a Potential Link between Triple-Negative Breast Cancer Development and Metabolic Syndrome.

Metabolic syndrome (MS) is a risk factor for breast cancer (BC) that increases its aggressiveness and metastasis. The prevalence of MS is higher in triple-negative breast cancer (TNBC), which is the molecular subtype with the worst prognosis. The molecular mechanisms underlying this association have not been fully elucidated. MiRNAs are small, non-coding RNAs that regulate gene expression. Aberrant expression of miRNAs in both tissues and fluids are linked to several pathologies. The aim of this work was to identify circulating miRNAs in patients with alterations associated with MS (AAMS) that also impact on BC. Using microarray technology, we detected 23 miRNAs altered in the plasma of women with AAMS that modulate processes linked to cancer. We found that let-7b-5p and miR-28-3p were decreased in plasma from patients with AAMS and also in BC tumors, while miR-877-5p was increased. Interestingly, miR-877-5p expression was associated with lower patient survival, and its expression was higher in PAM50 basal-like BC tumors compared to the other molecular subtypes. Analyses from public databases revealed that miR-877-5p was also increased in plasma from BC patients compared to plasma from healthy donors. We identified IGF2 and TIMP3 as validated target genes of miR-877-5p whose expression was decreased in BC tissue and moreover, was negatively correlated with the levels of this miRNA in the tumors. Finally, a miRNA inhibitor against miR-877-5p diminished viability and tumor growth of the TNBC model 4T1. These results reveal that miR-877-5p inhibition could be a therapeutic option for the treatment of TNBC. Further studies are needed to investigate the role of this miRNA in TNBC progression.

Identification of biomarkers in patients with rheumatoid arthritis responsive to DMARDs but with progressive bone erosion.

Introduction: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that may cause joint destruction and disability. The pharmacological treatment of RA aims at obtaining disease remission by effectively ceasing joint inflammation and arresting progressive bone erosions. Some patients present bone lesions accrual even after controlling joint inflammation with current therapies. Our study aimed to analyze lymphocyte subsets and levels of circulating cytokines in patients with RA with progressive bone erosions. Methods: We enrolled 20 patients with a diagnosis of RA and 12 healthy donors (HD). Patients with RA were divided into patients with bone erosions (RA-BE+) and without bone erosions (RA-BE-). Lymphocyte subsets in peripheral blood were evaluated by flow cytometry. Circulating cytokines levels were evaluated by protein array. Results: The distribution of lymphocyte subsets was not able to separate HD from AR patients and RA-BE+ and RA-BE- in cluster analysis. We observed a significant expansion of CXCR5- PD1+ T peripheral helper cells (Tph cells) and a reduction in both total memory B cells and switched memory B cells in RA patients compared to HD. We observed an expansion in the frequency of total B cells in RA-BE+ patients compared to RA-BE- patients. Unsupervised hierarchical clustering analysis of 39 cytokines resulted in a fairly good separation of HD from RA patients but not of RA-BE+ patients from RA-BE- patients. RA-BE+ patients showed significantly higher levels of IL-11 and IL-17A than RA-BE- patients. Conclusion: We show that patients with progressive erosive disease are characterized by abnormalities in B cells and in cytokines with a proven role in bone reabsorption. Understanding the role played by B cells and the cytokine IL-11 and IL-17A in progressive erosive disease can help identify novel biomarkers of erosive disease and design treatment approaches aimed at halting joint damage in RA.

Inflammatory Treatment Used to Mimic Osteoarthritis and Patients' Synovial Fluid Have Divergent Molecular Impact on Chondrocytes In Vitro.

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 IL1ß that are used for standardized inflammation protocols, OA-SF, IL1ß, IL6 and IFNγ at SF-like concentrations both individually and simultaneously to mimic a simplified "in vitro" SF. Both high IL1ß and OA-SF strongly influenced chondrocytes, while SF-like concentrations of cytokines gave weak (IL1ß 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 IL1ß 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 IL1ß. 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.

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.

Inflammatory priming with IL-1ß promotes the immunomodulatory behavior of adipose derived stem cells.

Inflammatory processes contribute to osteoarthritis (OA) severity and progression. Mesenchymal stem cells, particularly those derived from adipose tissue (ASCs), are able to sense and control the inflammatory environment. This immunomodulatory potential can be boosted by different priming strategies based on inflammatory stimulation. The aim of the present study is to investigate the transcriptional modulation of a huge panel of genes and functionally verify the predicted immunomodulatory ability of ASCs after interleukin one beta (IL-1ß) priming. ASCs were isolated from adipose tissue obtained from three donors and expanded. After stimulation with 1 ng/ml of IL-1ß for 48 h, cells were collected for gene array and functional tests. Pooled cells from three donors were used for RNA extraction and gene array analysis. Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA) were performed to assess the involvement of the modulated genes after priming in specific biological processes and pathways. Functional co-culture tests of ASCs with T cells and macrophages were performed to assess the ability of primed ASCs to modulate immune cell phenotype. Among the overall genes analyzed in the gene array, about the 18% were up- or down-regulated in ASCs after IL-1ß priming. GO enrichment analysis of up- or down-regulated genes in ASCs after IL-1ß priming allowed identifying specific pathways involved in the modulation of inflammation and extracellular matrix remodeling. The main processes enriched according to the GSEA are related to the inflammatory response and cell proliferative processes. Functional tests on immune cells showed that primed and non-primed ASCs induced a decrease in the CD3+ T lymphocytes survival rate and an anti-inflammatory macrophage polarization. In conclusion, IL-1ß priming represents a tailored strategy to enhance the ability of ASCs to direct macrophages towards an anti-inflammatory phenotype and, consequently, improve the efficacy of ASCs in counteracting the OA inflammatory component.

Immunomodulatory potential of secretome from cartilage cells and mesenchymal stromal cells in an arthritic context: From predictive fiction toward reality.

The purpose of the present study is to predict by bioinformatics the activity of the extracellular vesicle (EV)-embedded micro RNA (miRNAs) secreted by cartilage cells (CCs), adipose tissue-derived- (ASCs), and bone marrow-derived stem cells (BMSCs) and verify their immunomodulatory potential supporting our bioinformatics findings to optimize the autologous cell-based therapeutic strategies for osteoarthritis (OA) management. Cells were isolated from surgical waste tissues of three patients who underwent total hip replacement, expanded and the EVs were collected. The expression of EV-embedded miRNA was evaluated with the QuantStudio 12 K Flex OpenArray® platform. Mientournet and ingenuity pathway analysis (IPA) were used for validated target prediction analysis and to identify miRNAs involved in OA and inflammation. Cells shared the expression of 325 miRNAs embedded in EVs and differed for the expression of a small number of them. Mienturnet revealed no results for miRNAs selectively expressed by ASCs, whereas miRNA expressed by CCs and BMSCs were putatively involved in the modulation of cell cycle, senescence, apoptosis, Wingless and Int-1 (Wnt), transforming growth factor beta (TGFß), vascular endothelial growth factor (VEGF), Notch, Hippo, tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1ß), insulin like growth factor 1 (IGF-1), RUNX family transcription factor 2 (RUNX2), and endochondral ossification pathways. Cartilage homeostasis, macrophages and T cells activity and inflammatory mediators were identified by IPA as targets of the miRNAs found in all the cell populations. Co-culture tests on macrophages and T cells confirmed the immuno-modulatory ability of CCs, ASCs, and BMSCs. The study findings support the rationale behind the use of cell-based therapy for the treatment of OA.

Integrated lung ultrasound score for early clinical decision-making in patients with COVID-19: results and implications.

BACKGROUND AND OBJECTIVES: Lung Ultrasound Score (LUS) identifies and monitors pneumonia by assigning increasing scores. However, it does not include parameters, such as inferior vena cava (IVC) diameter and index of collapse, diaphragmatic excursions and search for pleural and pericardial effusions. Therefore, we propose a new improved scoring system, termed "integrated" lung ultrasound score (i-LUS) which incorporates previously mentioned parameters that can help in prediction of disease severity and survival, choice of oxygenation mode/ventilation and assignment to subsequent areas of care in patients with COVID-19 pneumonia. METHODS: Upon admission at the sub-intensive section of the emergency medical department (SEMD), 143 consecutively examined COVID-19 patients underwent i-LUS together with all other routine analysis. A database for anamnestic information, laboratory data, gas analysis and i-LUS parameters was created and analyzed. RESULTS: Of 143 enrolled patients, 59.4% were male (mean age 71 years) and 40.6% female. (mean age 79 years: p = 0.005). Patients that survived at 1 month had i-LUS score of 16, which was lower than that of non-survivors (median 20; p = 0.005). Survivors had a higher PaO2/FiO2 (median 321.5) compared to non-survivors (median 229, p < 0.001). There was a correlation between i-LUS and PaO2/FiO2 ratio (rho:-0.4452; p < 0.001), PaO2/FiO2 and survival status (rho:-0.3452; p < 0.001), as well as i-LUS score and disease outcome (rho:0.24; p = 0.005). In non-survivors, the serum values of different significant COVID indicators were severely expressed. The i-LUS score was higher (median 20) in patients who required non-invasive ventilation (NIV) than in those treated only by oxygen therapy (median 15.42; p = 0.003). The odds ratio for death outcome was 1.08 (confidence interval 1.02-1.15) for each point increased. At 1-month follow-up, 65 patients (45.5%) died and 78 (54.5%) survived. Patients admitted to the high critical ward had higher i-LUS score than those admitted to the low critical one (p < 0.003). CONCLUSIONS: i-LUS could be used as a helpful clinical tool for early decision-making in patients with COVID-19 pneumonia.

A simple prognostic score based on troponin and presepsin for COVID-19 patients admitted to the emergency department: a single-center pilot study.

BACKGROUND: The need to determine prognostic factors that can predict a particularly severe or, conversely, the benign course of COVID-19 is particularly perceived in the Emergency Department (ED), considering the scarcity of resources for a conspicuous mass of patients. The aim of our study was to identify some predictors for 30-day mortality among some clinical, laboratory, and ultrasound variables in a COVID-19 patients population. METHODS: Prospective single-center pilot study conducted in an ED of a University Hospital. A consecutive sample of confirmed COVID-19 patients with acute respiratory failure was enrolled from March 8th to April 15th, 2020. RESULTS: 143 patients were enrolled. Deceased patients (n = 65) were older (81 vs. 61 years, p <0.001), and they had more frequently a history of heart disease, neurological disease, or chronic obstructive pulmonary disease (p-values = 0.026, 0.025, and 0.034, respectively) than survived patients. Troponin I and presepsin had a significant correlation with a worse outcome. Troponin achieved a sensitivity of 77% and a specificity of 82% for a cut-off value of 27.6 ng/L. The presepsin achieved a sensitivity of 54% and a specificity of 92% for a cut-off value of 871 pg/mL. CONCLUSION: In a population of COVID-19 patients with acute respiratory failure in an ED, presepsin and troponin I are accurate predictors of 30-day mortality. Presepsin is highly specific and could permit the early identification of patients who could benefit from more intensive care as soon as they enter the ED. Further validation studies are needed to confirm this result.

High-Throughput Gene and Protein Analysis Revealed the Response of Disc Cells to Vitamin D, Depending on the VDR FokI Variants.

Vitamin D showed a protective effect on intervertebral disc degeneration (IDD) although conflicting evidence is reported. An explanation could be due to the presence of the FokI functional variant in the vitamin D receptor (VDR), observed as associated with spine pathologies. The present study was aimed at investigating-through high-throughput gene and protein analysis-the response of human disc cells to vitamin D, depending on the VDR FokI variants. The presence of FokI VDR polymorphism was determined in disc cells from patients with discopathy. 1,25(OH)2D3 was administered to the cells with or without interleukin 1 beta (IL-1ß). Microarray, protein arrays, and multiplex protein analysis were performed. In both FokI genotypes (FF and Ff), vitamin D upregulated metabolic genes of collagen. In FF cells, the hormone promoted the matrix proteins synthesis and a downregulation of enzymes involved in matrix catabolism, whereas Ff cells behaved oppositely. In FF cells, inflammation seems to hamper the synthetic activity mediated by vitamin D. Angiogenic markers were upregulated in FF cells, along with hypertrophic markers, some of them upregulated also in Ff cells after vitamin D treatment. Higher inflammatory protein modulation after vitamin D treatment was observed in inflammatory condition. These findings would help to clarify the clinical potential of vitamin D supplementation in patients affected by IDD.

miR-103a-3p and miR-22-5p Are Reliable Reference Genes in Extracellular Vesicles From Cartilage, Adipose Tissue, and Bone Marrow Cells.

Cartilage cells (CCs), adipose tissue (ASC)- and bone marrow (BMSC)-derived mesenchymal stromal cells (MSCs) have been shown as promising candidates for the treatment of osteoarthritis (OA). Despite their adaptive ability, exposure to chronic catabolic and inflammatory processes can limit their survival and healing potential. An attractive cell-free alternative or complementary strategy is represented by their secreted extracellular vesicles (EVs), having homeostatic properties on OA chondrocytes and synovial cells. In view of clinical translation, a thorough characterization of the shuttled therapeutic molecules, like miRNAs, is greatly needed to fingerprint and develop the most effective EV formulation for OA treatment. To date, a crucial pitfall is given by the lack of EV-miRNA-associated reference genes (RGs) for the reliable quantification and comparison among different therapeutic EV-based therapeutic products. In this study, the stability of 12 putative miRNA RGs (let-7a-5p, miR-16-5p, miR-22-5p, miR-23a-3p, miR-26a-5p, miR-29a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p and miR-660-5p), already proposed by literature in EV products from alternative sources, was assessed in EVs isolated from three donor-matched ASCs, BMSCs, and CCs through geNorm, NormFinder, BestKeeper, and ΔCt algorithms and the geometric mean of rankings. ASC-EVs and BMSC-EVs shared more similar molecular signatures than cartilage-derived EVs, although overall miR-103a-3p consistently ranked as the first and miR-22-5p as the second most stable EV-miRNA RG, whereas miR-221-3p behaved poorly. Further, to emphasize the impact of incorrect RG choice, the abundance of four OA-therapeutic miRNAs (miR-93-5p, miR-125b-5p, miR-455-3p, and miR-27b-3p) was compared. The use of miR-221-3p led to less accurate EV fingerprinting and, when applied to sift therapeutic potency prediction, to misleading indication of the most appropriate clinical product. In conclusion, miR-103a-3p and miR-22-5p will represent reliable RGs for the quantification of miRNAs embedded in MSC- and CC-EVs, a mandatory step for the molecular definition and comparison of the clinical potency of these innovative cell-free-based therapeutic products for OA in particular, as well as for a wider array of regenerative-medicine-based approaches.

Tendon Cells Derived From The Long Head Of The Biceps And The Supraspinatus Tendons Of Patients Affected By Rotator Cuff Tears Show Different Expression Of Inflammatory Markers.

AIM OF THE STUDY: Tendons are exposed to mechanical stress constantly during movements and thus they are frequently subjected to injuries. Rotator cuff tears are common musculoskeletal disorders, mainly involving the supraspinatus tendon. The characterization of the tenocytes derived from this tendon and the comparison to cells isolated from the long head of the biceps tendon obtained from donors affected by rotator cuff disease may improve the knowledge of the cellular mechanisms involved in the initiation and progression of the pathology. Thus, the aim of the present study was to characterize and compare donor-matched human tendon cells (TCs) isolated from the long head of the biceps (LHB-TCs) and the supraspinatus tendons (SSP-TCs) of patients affected by rotator cuff tears. METHODS: donor-matched LHB-TCs and SSP-TCs were isolated and cultured up to passage 3. Phenotypic appearance, metabolic activity, DNA content, production of soluble mediators (IL-1Ra, IL-1ß, IL-6, and VEGF) and gene expression of tendon markers (SCX, COL1A1, COL3A1), inflammatory (PTGS2), and catabolic enzymes (MMP-1, MMP-3) were evaluated. RESULTS: LHB-TCs showed an elongated fibroblast-like shape, while SSP-TCs appeared irregular with jagged membrane. SSP-TCs gene expression revealed an augmented production of PTGS2, a marker of inflammation, whereas they produced a reduced amount of IL-6, in respect to LHB-TCs. CONCLUSION: SSP-TCs showed higher cellular stress and expression of inflammatory markers with respect to donor-matched LHB-TCs, suggesting that addressing the physio-pathological state of supraspinatus tendon cells during treatment of rotator cuff tears could favor tissue healing and possibly prevent relapses.

Autologous microfragmented adipose tissue reduces inflammatory and catabolic markers in supraspinatus tendon cells derived from patients affected by rotator cuff tears.

PURPOSE: Rotator cuff tears are common musculoskeletal disorders, and surgical repair is characterized by a high rate of re-tear. Regenerative medicine strategies, in particular mesenchymal stem cell-based therapies, have been proposed to enhance tendon healing and reduce the re-tear rate. Autologous microfragmented adipose tissue (µFAT) allows for the clinical application of cell therapies and showed the ability to improve tenocyte proliferation and viability in previous in vitro assessments. The hypothesis of this study is that µFAT paracrine action would reduce the catabolic and inflammatory marker expression in tendon cells (TCs) derived from injured supraspinatus tendon (SST). METHODS: TCs derived from injured SST were co-cultured with autologous µFAT in transwell for 48 h. Metabolic activity, DNA content, the content of soluble mediators in the media, and the gene expression of tendon-specific, inflammatory, and catabolic markers were analyzed. RESULTS: µFAT-treated TCs showed a reduced expression of PTGS2 and MMP-3 with respect to untreated controls. Increased IL-1Ra, VEGF, and IL-6 content were observed in the media of µFAT-treated samples, in comparison with untreated TCs. CONCLUSION: µFAT exerted an anti-inflammatory action on supraspinatus tendon cells in vitro through paracrine action, resulting in the reduction of catabolic and inflammatory marker expression. These observations potentially support the use of µFAT as adjuvant therapy in the treatment of rotator cuff disease.

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.

AMIC-Autologous Matrix-Induced Chondrogenesis Technique in Patellar Cartilage Defects Treatment: A Retrospective Study with a Mid-Term Follow-Up.

BACKGROUND: Knee cartilage defects can be retrieved in 60% of patients undergoing knee arthroscopy, especially in the patellofemoral joint. Different techniques have been proposed to treat patellar defects, although most of them are associated with short-term results. In this study Autologous Matrix Induced Chondrogenesis (AMIC), combining subchondral microfractures with a collagen membrane (type I and III collagen), was used in the treatment of isolated patellar cartilage defects. METHODS: Twenty-four patients were enrolled in this retrospective study. Subjective-International Knee Documentation Committee (IKDC), Visual Analog Scale for Pain (VAS), and Kujala score were collected at 1, 3, 6, and 12 months after surgery, whereas the Tegner Activity Level Scale was determined preoperatively and at final follow-up (final-FU). The same postoperative management and rehabilitation protocol was adopted for all the patients. RESULTS: Fourteen patients met the inclusion-exclusion criteria and were evaluated at a mean final-FU of 68.2 months (range 25.4-111.2). At 12 months, Kujala, IKDC, and VAS scores significantly increased in comparison to the preoperative assessment, whereas no statistically significant differences were reported between 12 months and final follow-up. CONCLUSION: This study demonstrated very good results throughout the follow-up, also in sports patients. The AMIC technique, together with an adequate rehabilitation protocol, can be considered as a reliable one-step alternative for the treatment of large isolated patellar cartilage defects.