Mechanism of Action Behind the Pain-Relief Effects of Extracellular Vesicles in Microfragmented Adipose Tissue: An In vitro and In vivo Study

Mechanism of Action Behind the Pain-Relief Effects of Extracellular Vesicles in Microfragmented Adipose Tissue: An In vitro and In vivo Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanism of Action Behind the Pain-Relief Effects of Extracellular Vesicles in Microfragmented Adipose Tissue: An In vitro and In vivo Study Francesco De Francesco, Letizia Ferroni, Ilaria Zanolla, Lucia Sileo, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6725135/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Aug, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background Trapeziometacarpal (TMC) osteoarthritis is a prevalent and debilitating condition that impairs hand functionality and reduces quality of life. Current treatments—including conservative measures such as splinting and anti-inflammatory medications, as well as surgical interventions—often exhibit limited efficacy or involve invasive procedures. Novel therapeutic approaches are necessary to address the pain and functional limitations experienced by affected patients. Methods This study investigates the potential of extracellular vesicles (EVs) derived from microfragmented adipose tissue (aMAT) as a minimally invasive treatment for TMC osteoarthritis. EVs were characterized using morphological, proteomic, and functional analyses, revealing their ability to modulate cellular processes through proteins associated with extracellular matrix organization, wound healing, and inflammation regulation. Results Functional studies demonstrated that EVs modulate calcium signaling and mitochondrial activity, enhancing cellular bioenergetics and mitigating inflammation-induced dysfunction. Trial registration This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and adhered to all relevant national and institutional ethical guidelines for research involving human participants. Approval for the study was obtained from the Ethics Committee of Marche Region, protocol n. 154/2021. All participants provided written informed consent before enrollment in the study. They were informed about the study’s purpose, procedures, potential risks, and their right to withdraw at any time without consequences. Informed Patient Consent Statement: Informed consent was obtained from all individual participants included in the study, following the guidelines of the Human Research Approval Committee protocol number 2/2019. Adipose stem cells non-enzymatic method Rigenera protocol trapeziometacarpal arthritis microfragmented adipose tissue extracellular vesicles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Background Extracellular vesicles (EVs) have emerged as promising biological agents, capable of delivering active biomolecules to target cells and modulating a range of biological processes [ 1 ] These nano-sized particles, secreted by cells, facilitate intercellular communication and influence tissue repair, inflammation, and immune responses [ 2 ]. Interest in EVs has grown recently due to their therapeutic potential in regenerative medicine and pain management, underscoring the importance of elucidating their precise mechanisms of action [ 3 , 4 ]. Given their ability to deliver proteins, lipids, and genetic material to target cells, EVs represent a promising approach for treating chronic conditions, particularly those involving tissue degeneration and pain, such as musculoskeletal diseases [ 5 – 7 ]. Musculoskeletal diseases (MSDs) are a significant health concern globally, affecting millions and imposing a high socioeconomic burden. Key MSDs include rheumatoid arthritis (RA), osteoarthritis (OA), low-back pain (LBP), neck pain (NP), and gout [ 8 , 9 ], with OA standing out as the most common degenerative joint disease among adults, significantly impacting daily functioning and quality of life. According to the Centers for Disease Control and Prevention, arthritis affects one in four adults, with prevalence surging by over 113% from 1990 to 2020 [ 10 ]. OA frequently affects the hand, knee, and hip joints, leading to substantial healthcare costs [ 11 ]. Notably, hand OA has a high clinical impact due to the extensive loss of functionality in small, complex joints, which contributes to pain and significant disability [ 12 , 13 ]. OA is often diagnosed at an advanced stage, when joint degeneration is irreversible, necessitating surgical intervention [ 14 , 15 ], especially in joints like the thumb's trapeziometacarpal (TMC) joint [ 16 ]. Trapeziometacarpal osteoarthritis (TMC OA) is a multifactorial degenerative disease that affects not only the articular cartilage, but also the synovial membrane, joint capsule, and periarticular soft tissues. Within this microenvironment, fibroblasts play a central role by producing extracellular matrix components, modulating local inflammation, and influencing tissue remodeling [ 17 ]. Emerging evidence has implicated synovial fibroblasts in the pathogenesis of OA through their secretion of pro-inflammatory cytokines, matrix-degrading enzymes, and their ability to respond to mechanical and biochemical stress [ 18 ]. Given their functional relevance and accessibility for in vitro modeling, fibroblasts represent a suitable cellular system to investigate the mechanistic effects of EV-based therapies in OA. In earlier stages of joint degeneration, conservative treatments such as splinting and the use of analgesics or NSAIDs are typically prescribed [ 19 , 20 ]. Corticosteroid and hyaluronic acid injections have also been applied, though their efficacy remains inconclusive, with guidelines recommending these treatments mainly for mildly symptomatic cases [ 21 , 22 ]. When conservative measures fail, surgical options such as joint arthroplasty or ligament repair become necessary, particularly in cases like TMC joint OA hands [ 23 , 24 ]. Emerging therapies, including the use of platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs), offer promising alternatives. MSCs from sources like bone marrow, umbilical cord, synovium, and adipose tissue have demonstrated potential in reducing inflammation and alleviating symptoms through the secretion of paracrine factors [ 25 , 26 ]. Among these sources, adipose tissue offers distinct advantages that make it particularly suitable for clinical use. Compared to bone marrow or umbilical cord-derived MSCs [ 27 ], adipose-derived MSCs (AD-MSCs) are more abundant, easier to harvest via minimally invasive procedures, and exhibit a strong paracrine secretory profile. Moreover, the use of autologous adipose tissue reduces immunogenicity risks and complies more readily with current regulatory frameworks, especially when processed via minimal manipulation methods such as microfragmentation. These characteristics support the rationale for selecting microfragmented adipose tissue (aMAT) as the tissue source in this study and highlight its translational potential in regenerative approaches for osteoarthritis and pain. In particular, micro-fragmented adipose tissue has gained attention for its regenerative potential in joint pathologies [ 28 – 30 ]. This tissue is commonly prepared using non-enzymatic methods, resulting in minimally manipulated products that comply with regulatory standards [ 31 ]. aMAT contains a heterogeneous population of cells, including MSCs, pericytes, preadipocytes, and immune cells [ 32 ]. Among them, AD-MSCs are particularly abundant and play a crucial role in tissue repair due to their potent paracrine activity. AD-MSCs are known to secrete EVs enriched in cytokines, growth factors, and microRNAs that can modulate inflammation, promote matrix remodeling, and influence pain signaling [ 33 , 34 ]. These properties make them a promising candidate for regenerative approaches in osteoarthritis and pain management. Despite advancements in MSC-based therapies, significant gaps remain in understanding the mechanisms through which EVs modulate nociceptive pathways. EVs derived from aMAT-EVs may bridge this gap by providing a minimally invasive therapeutic option. Their ability to influence inflammation, extracellular matrix organization, and cellular bioenergetics highlights their potential not only for joint preservation but also for effective pain management. Given the limitations of conventional therapies, exploring EV-mediated mechanisms in pain modulation could revolutionize treatment paradigms for OA and other chronic conditions. Considering such consideration in this study, we focus on characterizing EVs derived from treated adipose grafts, specifically analyzing their potential to alleviate osteoarticular pain. Building on our expertise in EV isolation and characterization, we aim to identify the mechanisms by which these vesicles interact with nociceptive receptors to modulate pain. Our approach includes morphological, proteomic, and functional analyses of these vesicles, with the goal of elucidating pathways involved in pain perception and assessing their efficacy in clinical pain. Through both in vitro and in vivo on human studies, this investigation aims to advance our understanding of EV-mediated pain modulation, potentially paving the way for innovative, minimally invasive treatments for OA and related chronic pain conditions. 2. Methods 2.1. Surgical procedure to obtain adipose micro-grafts tissue (aMAT) Patients were positioned supine, and local anesthesia was administered. A small incision was made to introduce a blunt cannula attached to a Luer-lock 60-cc syringe. Klein sterile solution, containing saline and lidocaine, was injected into the subcutaneous fat layer of either the abdominal or thigh region. Subsequently, approximately 30 mL of adipose tissue was extracted. The collected lipoaspirate was processed under sterile conditions in a closed system using Rigenera® technology (HBW, Turin, Italy). This non-enzymatic approach is specifically designed to disaggregate human tissues such as adipose tissue, dental pulp, cartilage, dermis, and bone for reapplication. The process has been documented in multiple studies [ 35 – 39 ]. This disposable device progressively reduces the size of adipose tissue clusters from their initial dimensions of 1–3.5 mm to finer fragments measuring 0.2–0.8 mm, resulting in an adipose micro-graft. The surgical workflow consists of two stages: liposuction followed by tissue disaggregation to create micro-grafts. In this procedure, 4 mL of lipoaspirate was mechanically fragmented using Rigeneracons® (Human Brain Wave S.r.L., Turin, Italy) while mixing with 4 mL of saline solution. This process yielded approximately 6–7 mL of micro-fragmented adipose tissue, ready for direct application. 2.2. Isolation and Characterization of aMAT contained Extracellular Vesicles (aMAT-EVs) aMAT was resuspended in 10 mL of PBS and applied to Amicon Ultra-15 100 kDa centrifugal devices (Millipore, MA, USA), which had been pre-sterilized with 70% ethyl alcohol (Sigma-Aldrich, Saint Louis, MA, USA). The devices were centrifuged at 2000xg for 20 minutes at 4°C. The resulting filtrate was then washed with sterile PBS (Euroclone, Milan, Italy) via another centrifugation step at 2000xg for 20 minutes at + 4°C. The isolated aMAT-EVs were recovered from the filtering unit, quantified using the Pierce™ BCA protein assay kit (Thermo Fisher Scientific), and promptly stored in small aliquots at -80°C. 2.3. Nanoparticle tracking analysis (NTA) Nanoparticle tracking analysis (NTA) employs laser light scattering and Brownian motion to assess the size and concentration of EVs. Using the NanoSight NS300 instrument (Malvern, UK) equipped with a 488 nm laser, particle size and distribution were measured. Samples were diluted in filtered PBS to a final volume of one mL. Optimal measurement concentrations were determined by pre-testing the ideal particle per frame value (20–100 particles/frame). Each measurement involved capturing five 1-minute videos at a temperature of 25°C and a syringe pump speed of 30. The data are presented as averaged finite track length adjustment (FTLA) concentration/size. 2.4. Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) was utilized for imaging. The sEVs were first fixed in a 2% glutaraldehyde solution in phosphate buffer (1:1 ratio), following a previously outlined protocol [ 40 ]. Subsequently, the fixed sEVs were deposited, rinsed, and stained with heavy metal compounds on a gridded slide using standard procedures. Imaging was performed using a TEM Zeiss EM 910 instrument (Zeiss, Oberkochen, Germany). 2.5. aMAT-EVs markers analysis Superficial Extracellular Vescicles markers were detected with Exosome-human CD81 Flow Detection Reagent (Thermo Fisher Scientific) as described elsewhere [ 41 ]. PE-conjugated anti-human CD81 monoclonal antibody or PE-conjugated anti-human CD63 monoclonal antibody (Thermo Fisher Scientific) were used to label exosome captured by beads. Negative control was performed by staining EV resuspension medium (without EVs). Data collection and analysis were performed with Attune™ NxT Acoustic Focusing Cytometer (Life Technologies, Carlsbad, California, USA) and Attune NxT Software version 2.5 data, respectively. 2.6. Proteomic analyses Proteomic sample preparation and analysis of extracellular vesicles followed established protocols. Protein digestion and clean-up were conducted as described earlier [ 42 ]. The proteomic profiling was performed using an Ultimate 3000 nanoLC system coupled to an Orbitrap Lumos tribrid mass spectrometer, both from Thermo Fisher Scientific. Peptides were trapped using a PepMap trap-cartridge and separated on a C18 reversed-phase column with a 90-minute linear gradient. The mass spectrometry (MS) analysis employed a data-dependent acquisition (DDA) approach, with a mass range of 400–1500 m/z, using HCD fragmentation at 27 normalized collision energy. The resolution was set to 120,000 for MS1 and 15,000 for MS/MS scans. Peptides with single or unassigned charges were excluded, and a quadrupole isolation width of 1.6 Da was used. Data analysis was performed in Proteome Discoverer v2.5, applying search parameters including trypsin as the enzyme, a maximum of one missed cleavage, and mass tolerances of 10 ppm for precursors and 0.6 Da for fragments. Proteins were identified based on at least one unique peptide, with a false discovery rate (FDR) below 0.1. All analyses were carried out in triplicate. Pathway enrichment of the proteomic data was conducted using the STRING database ( https://string-db.org/ ). Protein identifiers were entered, and the analysis was focused on Homo sapiens, with k-means clustering (k = 8) applied to organize functional protein networks. Enriched Gene Ontology (GO) terms for biological processes, cellular components, and molecular functions were prioritized. Visualization and data integration were done using the STRING database and SR plot web server ( https://www.bioinformatics.com.cn/srplot ). 2.7. Fura-2 AM calcium measurements The intracellular calcium indicator Fura-2 AM was used to ratiometrically evaluate the change of intracellular Ca 2+ levels upon EV stimulation. Fibroblasts were grown on 24 mm Ø cover glasses and then incubated for 30 min at 37°C in Krebs-Ringer modified buffer (KRB: 125mM NaCl, 5mM KCl, 1mM Na 3 PO 4 , 1mM MgSO 4 , 5.5mM glucose, and 20mM HEPES, pH 7.4, at 37°C) supplemented with 1 mM CaCl 2 or 100 µM EGTA, supplemented with 2.5 mM Fura-2 AM (Thermo Fisher Scientific), 0.02% Pluronic F-68 (Sigma-Aldrich), and 0.1 mM sulfinpyrazone (Sigma-Aldrich). Cells were washed and the saline was replaced accordingly. Next, the cells were placed in an open Leyden chamber on a 37°C thermostated stage and exposed to 340/380 wavelength light using the Olympus xcellence (Olympus, Shinjuku, Tokyo, Japan) multiple wavelength high-resolution fluorescence microscopy system equipped with a Hamamatsu ORCA ER CCD camera (Hamamatsu Photonics) and a 40x UPLXAPO40XO oil immersion objective (n.a. 1.4) to determine the cytosolic Ca 2+ response. To test mitochondrial Ca 2+ levels, fibroblasts were grown on 24 mm Ø coverslips and transfected with a last generation mitochondrial mt-GCaMP6-encoding plasmid. Calcium Imaging was performed on Olympus excellence multiple wavelength high-resolution fluorescence microscopy system. Cells were alternatively illuminated at 474 and 410 nm and fluorescence was collected through a 515/30‐nm band‐pass filter. Exposure time was set to 200 ms at 474 nm and to 400 ms at 410 nm, to account for the low quantum yield at the latter wavelength. Images were analyzed with ImageJ software. Mitochondrial membrane potential (ΔΨm) measurements Fibroblasts were grown on 24 mm Ø cover glasses and treated for 48 hours with 1 mg/mL MSC-EVs. To measure mitochondrial membrane potential (ΔΨm), fibroblasts were incubated for 30 min at 37°C in KRB/Ca 2+ saline supplemented with 2 nM Tetramethylrhodamine methyl ester (TMRM; Thermo Fisher Scientific) and 10 µM Verapamil (Thermo Fisher Scientific) at 37°C for 30 minutes. After 60 seconds of baseline recording, fibroblasts were stimulated with 1 µM carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; Thermo Fisher Scientific) to dissipate ΔΨm. Measurements were performed by using the Olympus xcellence workstation equipped with an Olympus 40x UPLXAPO40XO oil immersion objective (n.a. 1.4) and a temperature-controlled stage set at 37°C. Images were analysed with ImageJ software. 2.8. Cell treatments with inhibitors DS16570511, mitochondrial calcium uniporter (MCU) complex inhibitor: fibroblasts were treated with 3 µM DS16570511 (Thermo Fisher Scientific) for 48 hours at 37°C in cell culture medium supplemented with exosome-depleted Fetal Bovine Serum; in case of EV-treated specimens, DS16570511 was added 10 minutes before the EVs. MCU-i11, MCU complex inhibitor: fibroblasts were treated with 10 µM MCU-i11 (Thermo Fisher Scientific) for 48 hours at 37°C in cell culture medium supplemented with exosome-depleted Fetal Bovine Serum; in case of EV-treated specimens, MCU-i11 was added 10 minutes before the EVs. Ruthenium Red (RR), Transient Receptor Potential (TRP) cation channels inhibitor: fibroblasts were treated with 10 µM Ruthenium Red (Sigma-Aldrich) for 20 minutes at 37°C in KRB/Ca 2+ saline; the protocol by Trollinger and colleagues was adapted [ 43 ]. 2.9. In vitro Statistical analysis Data are expressed as a means, with an indication of the standard error of the mean (SEM) obtained from at least three independent replicas of the experiment. The significant difference between conditions was determined by analysis of variance (ANOVA) and multiple comparisons by post hoc Bonferroni test with the GraphPad Prism software. 2.10. Cell cultures and transfection Established techniques were utilized to create stable Chinese hamster ovary (CHO) cell lines that expressed KOP receptors and stably expressed the Gα qi5 protein and fibroblast (Sigma) [ 44 ]. These cells were sustained in a culture medium consisting of DMEM and Ham F-12 (mixed at a 1:1 ratio) along with 5% fetal calf serum, penicillin (100 IU/mL), streptomycin (100 µg/ mL), and fungizone (2.5 µg/ mL). Additional supplements of geneticin (G418, 200 µg/ml) and hygromycin B (200 µg/ mL) were added to stock cultures. For transfection studies, CHO cells were seeded onto glass coverslips of varying dimensions, depending on the specific assay: 13 mm diameter for aequorin experiments and 24 mm diameter for Fura-2/AM measurements. Prior to transfection, cells were allowed to reach 50% confluence and then subjected to a standard Ca 2+ -phosphate procedure. All experiments were conducted for 36 hours of post-transfection. 2.11. Aequorin measurements Aequorin assessments were carried out to gauge mitochondrial calcium concentrations through established methodologies (Pinton P et al, Methods Cell Biol, 2007). Initially, cells were seeded onto 13-mm glass coverslips and allowed to reach 75% confluence. Then, 4 µg of mitochondrial-targeted aequorin was transfected into the cells. After 36 hours of transfection, cells were cultured for 2 hours at 37°C in Krebs–Ringer modified buffer (KRB) supplemented with 5 µM coelenterazine to activate the aequorin. Following this, measurements were taken using an automated luminometer (MicrobetaJET, PerkinElmer, CA, USA). KRB containing various concentrations of HA was injected, and luminescence was recorded for 60 seconds. To conclude the experiments and normalize the acquired values, a hypotonic solution containing 500 µM digitonin and 50 mM CaCl 2 was introduced to discharge the remaining aequorin. The results were presented as % of probe discharged ± standard error (SE). This computation is possible because aequorin undergoes an irreversible reaction upon binding to three high-affinity sites (EF-hand type), emitting a photon and deactivating the protein (discharged). To translate the aequorin luminescence data into [Ca 2+ ] values, a computer algorithm based on the Ca 2+ response curve of aequorins was utilized, as previously outlined [ 45 ]. All cell cultures were maintained at 37°C in a humidified atmosphere containing 5% carbon dioxide. 2.12. In vivo Study Design, population and assessment A prospective, single-center, non-comparative study was performed in accordance with the ethical standards of institutional and national legislation and in accordance with the Declaration of Helsinki on human experimentation. All the participants signed informed consent, and the present study was approved by the Ethics Committee of Marche Region (protocol n. 154/2021). TMJ OA was defined according to the American College of Rheumatology criteria for clinical hand OA [ 46 ] or as the presence of structural abnormalities. Between 2020 and 2022, patients were consecutively enrolled if they were diagnosed with TMJ OA that not requiring surgical treatment and provided written informed consent. Patients were consecutively enrolled if they met the following inclusion criteria: (i) age between 45 and 70 years, (ii) diagnosis of TMC osteoarthritis, Eaton-Littler stage II, confirmed radiographically, (iii) persistent pain and functional limitation, (iv) failure of at least 6 months of conservative treatment (including physical therapy and NSAIDs), and (v) ability to provide written informed consent. Exclusion criteria included: 1. Previous intra-articular injection of corticosteroids or hyaluronic acid within the last 6 months, 2. Prior surgery on the affected TMC joint, 3. Uncontrolled systemic diseases (e.g., poorly managed diabetes, active cancer), 4. Autoimmune or inflammatory rheumatic diseases (e.g., rheumatoid arthritis, psoriatic arthritis, lupus erythematous), 5. Neurological or orthopedic comorbidities affecting hand function (e.g., carpal tunnel syndrome, adjacent tendinopathies). Patient demographic characteristics are summarized as follows: The mean age was 59.3 ± 6.4 years; 17 patients were female and 8 male. The mean BMI was 26.1 ± 3.2 kg/m². The most frequent comorbidities included controlled hypertension (32%) and type 2 diabetes (12%). No patients presented with active infection, severe obesity (BMI > 35), or recent trauma to the affected hand. 2.13. Surgical Procedure to inject the microfragmented adipose tissue Each patient received an autologous intra-articular injection of aMAT, processed from their own lipoaspirate under sterile conditions using the Rigenera® system. Before injecting, the skin was sterilely dressed, and the injection of 2–3 mL of aMAT was performed into TMC joint with a superolateral approach, under fluoroscopic X-ray guidance and using a disposable 20G needle and a 3 mL Luer-Lock Syringe. After application of dressings, a plaster cast was applied for 14 days. Patients were advised not use nonsteroidal anti-inflammatory drugs following the procedure. Otherwise, mild activities and a gradual resumption of daily activity were allowed directly after splint removal. All patients followed the same standardized rehabilitation protocol. 2.14. Radiographic assessment Anterior–posterior, lateral and oblique radiographic views of the thumb aid in confirming the diagnosis [ 47 ]. Findings consistent with osteoarthritis include narrowing of the joint space (JSN) and osteophytes, in addition to subchondral sclerosis and cysts, which can be used to stage disease using the Eaton and Littler classification [ 48 ]. 2.15. Postoperative protocol At the end of the surgical procedure and prior to discharge, all patients were evaluated for wound condition and fitted with a custom-made thermoplastic splint immobilizing the first TMC joint. This rigid immobilization was maintained for 2 weeks. Afterwards, patients transitioned to a soft neoprene splint, which was worn during daytime activities for an additional 4 weeks. Rehabilitation began after the initial 2-week immobilization period, at the time of transition to the neoprene splint. The program included passive mobilization exercises followed by active-assisted range of motion and progressive grip strengthening. Rehabilitation was standardized and supervised by certified hand therapists, with two in-clinic sessions per week for 6 weeks, along with a prescribed home exercise regimen. Exercises included thumb circumduction, opposition drills, and resistance training using therapy putty and elastic bands. Patients were instructed to avoid nonsteroidal anti-inflammatory drugs (NSAIDs) for 12 weeks following the procedure to enable an accurate assessment of pain-related outcome measures. Adherence to the rehabilitation protocol was verified at each follow-up visit. Clinical follow-up appointments were scheduled at 6 weeks, 6 months, and 12 months post-intervention. At each visit, patients were systematically assessed for clinical status, functional outcomes, and any potential adverse events. 2.16. Outcome measures Outcomes were evaluated by two different members of the clinical team to minimize assessment bias: preoperative assessments and the surgical procedure were conducted by one surgeon, while postoperative evaluations were performed by a different clinician who was not involved in the treatment phase. Validated clinical tools and objective tests (e.g., MHQ, Kapandji score, dynamometry) were used to ensure reproducibility and minimize subjectivity. Patient-reported outcomes included: (i) The numeric rating score (NRS) for pain has been widely used to evaluate clinical pain intensity [ 49 ]. Joint pain intensity is assessed on a 100-mm score at baseline, after 6 and 12 months of follow up, where 0 indicates no pain and 100 indicates extreme pain; (ii) The Michigan Hand Outcomes Questionnaire (MHQ) is a validated patient-rated questionnaire, which means the patients evaluate their hand health state [ 50 ], where the major items include six categories (satisfaction, pain, work, daily living, aesthetics, function). The MHQ score ranged from 0 to 100, with higher scores indicating better hand function. Active flexion and extension of the TMC joint were measured with a goniometer and the total range of motion (ROM) was calculated, together with Kapandji opposition score. Measure of maximum grip strength was done in a standardized sitting position using a Jamar dynamoter for assessment of lateral, tip and palmar pinch test. Patient safety was systematically assessed at each follow-up visit (6 weeks, 6 months, and 12 months post-procedure). Evaluations included clinical examinations, patient-reported symptoms, and specific screening for potential adverse events such as infection, joint effusion, persistent or worsening pain, and neurological deficits. No adverse events were observed in any patient during the 12-month follow-up period. The statistical variables will be described using appropriate summary measures. The comparison between the NRS scale score with respect to the contralateral hand was performed using the Wilcoxon test for paired data, using the patient's last observation at 12 months as the response. Statistical significance was set at a probability level of 5%. For the evaluation of the cut-off capable of discriminating against the functional level of the intervention, Kapandji and MHQ scales were used. 2.17. Statistical analysis Continuous variables were reported as either means and standard deviation or median and interquartile ranges (IQRs) according to their distribution, as assessed by the Shapiro-Wilk normality test. Categorical variables were reported as absolute frequencies and percentages. Linear Mixed Models (LMM) were performed to estimate the covariates longitudinal effect on strength assessment with Tripod, Key, Tip test, and NRS. Mixed effects regression model uses all available data and can properly account for correlation between repeated measures. The covariates included in the model were group-status (i.e., treated and controlateral groups) and time-points as categorical variable and their interaction. To minimize the risk of type I error due to multiple comparisons, repeated-measures ANOVA models and LMM were followed by Bonferroni or Tukey post hoc correction where appropriate. For groups effect testing, the Tukey post-hoc test was performed. For NRS on activity, Thumb Opposition (i.e., Kapandji score), and MHQ scores, repeated measures ANOVA model was performed with p-value adjusted by Bonferroni approach. All p-values were interpreted in the context of these corrections. 3. Results 3.1. Adipose tissue-EVs characterization The adipose tissue derived extracellular vesicles were isolated from the adipose tissue and characterized as described in the “Method” section. The mean diameter measured by tunable resistive pulse sensing (TRPS) was 113 (SD=51) nm (Fig. 1a), and the mean protein concentration was 1115.37 ± 229.10 µg/mL (Fig. 1b). A semi-quantitative exosome antibody array was performed to profile the internal and surface proteins in aMAT (Fig. 1c). EVs were positive for programmed cell death 6 interacting protein (ALIX), tumour susceptibility gene 101 (TSG101), and annexin A5 (ANXA5). A weaker signal was detected for intercellular adhesion molecule 1 (ICAM), epithelial cell adhesion molecule (EpCAM), flotillin 1 (FLOT1), CD63, and CD81. MSC-EVs were negative for cis-Golgi matrix protein (GM130), the cellular protein contamination marker. The positivity for established exosomal markers CD63 and CD81 was confirmed by bead-based flow cytometry assay (Fig. 1d,f). MSC-EVs collected using anti-CD63 (Fig. 1D) or anti-CD81 (Fig. 1e) coated magnetic beads were stained with PE-conjugated anti-CD63 or anti-CD81, respectively. In Fig 1f, magnetic beads coated with anti-CD81 were stained with PE-conjugated anti-CD63 to detect double positivity to both markers. TEM images revealed nanovesicles with the cup-shaped morphology typical of exosomes (Fig. 1g). The internalization of MSC-EVs into target cells was demonstrated through fluorescence microscopy. Fluorescent red spots appeared inside fibroblasts after incubation with PKH26-stained MSC-EVs for 48h. In contrast, incubation with negative control did not show this phenomenon (Fig. 1h). 3.2. aMAT -EVs proteomic profiling Proteomic analysis was performed to evaluate the protein cargo of MSC-EVs. 1246 proteins belonging to Homo sapiens and 10097 predicted protein interactions were identified in the MSC-EV samples through the STRING software ( https://string-db.org/ ) (Fig. 2a). Pathway enrichment analysis of the proteomic results was performed to determine protein involvement in the gene ontology domains biological pathway (BP), molecular function (MF) and cellular component (CC) (BP ≥ 0.43, MF ≥ 0.49, CC ≥ 0.55) (Fig. S1a). BP analysis highlighted that the most noteworthy nodes are “Chronic inflammatory response” (GO:0002544), “Epithelial cell-cell adhesion” (GO:0090136), “Collagen fibril organization” (GO:0030199), “Extracellular matrix organization” (GO:0030198), “Regulation of wound healing” (GO:0061041). 8 clusters are obtained with the “Clusters” function and k-means = 8 in STRING The first cluster outnumbers the others and almost coincides in number of nodes (1130 out of 1246 proteins); it is to be noted that 100% of the BPs found via the enrichment analysis are part of the main cluster as showed in Fig. S1 (b, c, d). CC enrichment located most proteins in the collagen domain – for instance, “Collagen type XI trimer” (GO:0005592), “Fibrillar collagen trimer” (GO:0005583) and “Complex of collagen trimers” (GO:0098644). MF enrichment arranged 61 functions, with the most interesting ones being “Structural molecule activity conferring elasticity” (GO:0097493), “Collagen binding” (GO:0005518), “Extracellular matrix structural constituent” (GO:0005201) and “Antioxidant activity” (GO:0016209). 3.3. Characterization of Ca 2+ signalling triggered by Adipose tissue EVs We investigated whether aMAT-EVs elicit alterations in intracellular Ca 2+ fluxes on recipient fibroblasts. The concentration of Ca 2+ was measured in the cytosol of fibroblasts loaded with the ratiometric Ca 2+ indicator Fura-2 AM in a Ca 2+ -enriched KRB saline. Upon EV stimulation, an increase in Fura-2 AM ratio was recorded, indicating that EVs induce a transient elevation in the concentration of Ca 2+ in the cytoplasm ([Ca 2+ ] c ) (Fig. 2a and 2b). The positive and negative response rates of fibroblasts to EV treatment are 63.4% and 36.6% respectively (Fig. 2c). To determine whether calcium perturbation depended on intracellular reservoirs or extracellular influxes, we stimulated fibroblasts with MSC-EV in Ca 2+ -free KRB media enriched with the Ca 2+ chelator EGTA. Under these conditions, MSC-EV stimulation led to minimal or no variations in the Fura-2 AM ratio compared to fibroblasts maintained in KRB/Ca 2+ saline (Fig. 2d). The Area Under Curve (AUC) analysis of fibroblasts in KRB/EGTA saline showed a significant reduction compared to those in KRB/Ca 2+ saline (Fig. 2e). Various channels facilitate rapid Ca 2+ flux from the extracellular milieu. We focused on the Transient Receptor Potential (TRP) ion channels, a family that includes channels permeable to both monovalent ions (e.g., Na + , K + ) and divalent ions (e.g., Ca 2+ , Mg 2+ ). These channels can act as sensors for a variety of cellular and environmental signals [51]. Notably, certain TRP channel members are known to open in response to different extracellular matrix components [52]. Since our proteomic analysis identified extracellular matrix components within MSC-EV, we speculated that TRP channels might mediate the perturbation of Ca 2+ homeostasis. To test this hypothesis, we used the low-specificity inhibitor Ruthenium Red (RR) to block TRP channels. Fibroblasts were pretreated with 10 µM RR for 20 minutes before recording Ca 2+ signals [53]. Upon MSC-EV stimulation, fibroblasts exposed to RR showed no perturbation of [Ca 2+ ] c , unlike cells treated with the vehicle (Fig. 2f). The AUC analysis of fibroblast pretreated with RR showed a significant decrease (Fig. 2g). Perturbations in [Ca 2+ ] c are often linked to mitochondrial Ca 2+ uptake. To verify this hypothesis, fibroblasts were transfected with the ratiometric GFP-based indicator 2mt-GCaMP6s. Upon MSC-EV stimulation, an increase in the 2mt-GCaMP6s ratio was observed, indicating a transient accumulation of Ca 2+ in the mitochondrial matrix (Fig. 2h and 2i). 3.4. Characterization of mitochondrial physiology after aMAT-EV treatment Mitochondrial Ca 2+ perturbations are usually linked to changes in the mitochondrial physiology, including stimulation of tricarboxylic cycle and mitochondrial respiration [54]. We utilized mitochondrial membrane potential (ΔΨm) as a readout of mitochondrial respiration after Adipose tissue-EV stimulation. Fibroblasts were treated with MSC-EVs for 48 hours and then incubated with the potentiometric dye TMRM. To ensure that aspecific accumulation of TMRM was not accounted in the measurement, cells were stimulated with the mitochondrial uncoupler FCCP, to allow the release of only mitochondrial TMRM. The difference in signal before and after FCCP administration, accounted as readout of ΔΨm, resulted significantly more elevated in EV-treated fibroblasts (Fig. 3a and 3b). The elevation of ΔΨm suggests an elevation of mitochondrial respiration. To confirm this hypothesis, oxygen consumption rate (OCR) was measured in fibroblasts treated with MSC-EVs or vehicles. No effect was observed on basal respiration or respiration related to ATP production (Fig. 3d and 3e), nonetheless the maximal respiration and spare respiratory capacity resulted significantly elevated in cells exposed to EV compared to cells (Fig. 3f, g). To understand if the alteration of mitochondrial bioenergetics was dependent on mitochondrial Ca 2+ uptake, fibroblasts were pretreated with two different inhibitors of the mitochondrial calcium uniporter (MCU, DS16570511 and MCU-i11) before exposure to MSC-EV, then ΔΨm was measured. We observed that both inhibitors prevented the elevation of ΔΨm after 48 hours of exposure to MSC-EV (Fig. 3h, i). 3.5. Characterization of Ca 2+ signalling and the mitochondrial physiology in inflamed cells Extracellular vesicles (EVs) have frequently been associated with the modulation of responses to pro-inflammatory cytokines. Additionally, various perturbations in mitochondrial physiology have been reported as pivotal in the inflammatory response and the propagation of an inflammatory state [55,56]. To mimic an inflammatory condition, fibroblasts were treated with 10 ng/mL TNFα for 24 hours, after which [Ca 2+ ] c levels were measured. EV stimulation elicited a transient increase in [Ca 2+ ] c in vehicle-treated fibroblasts. Interestingly, the Ca 2+ mobilization induced by MSC-EVs was significantly stronger in fibroblasts pretreated with TNFα (Fig. 4a, c). Exposure to TNFα induced a reduction in ΔΨm in fibroblasts, as confirmed by multiple reports in other cell models [57–59]. Remarkably, administration of MSC-EVs to TNFα-treated fibroblasts was able to restore ΔΨm to levels comparable to those of untreated fibroblasts (Fig. 4b). 3.6. Aequorin measurements The aim of this technology is to evaluate whether SF exosomes can reduce pain by affecting nociceptors. It is evident that the selective binding of agonists, such as nociceptin, to their respective receptors leads to a significant increase in mitochondrial calcium levels (Fig. 4d). This effect is notably absent in the negative control group, which lacks agonistic stimulation. Fig. 4 Adipose tissue-EV effect of TNFα-treated fibroblasts. (a) Representative traces of TNFα inflamed and non-inflamed fibroblasts stimulated with EVs in KRB/Ca2+ saline show an increase in Fura-2 AM ratio, indicating a transient increase in the cytosolic Ca2+ levels; (b)ΔΨm of inflamed cells is reduced compared to normal cells. ΔΨm of EV-treated fibroblasts is enhanced compared to the untreated condition. EV treatment on inflamed fibroblasts leads to a recovery in the ΔΨm; (c) Fura-2 AM ratio quantification as AUC is increased in inflamed fibroblasts; (d) percentage of Calcium Variation. Stable cell lineages of CHO cells bearing KOP receptors underwent transfection with a mitochondria-targeted aequorin sensor, following which they were subjected to various concentrations of hyaluronic acid (HA), as delineated in the illustration. The resultant mitochondrial calcium responses are delineat-ed as a percentage of the mitochondrial calcium response, relative to receptor stimulation with a canonical agonist. Delving further into the experimental investigation, the same cell models were exposed to a substance designated as aMAT-EV and hyaluronic acid, applied in a wide range of concentrations from 0.005 to 5 mg/ mL. Interestingly, low molecular weight hyaluronic acid did not induce any noticeable increase in luminescence. In contrast, high molecular weight hyaluronic acid elicited a significant luminescent response in CHO cells expressing KOP receptors (Fig. 4d). The maximal effect of hyaluronic acid was approximately half that of the robust response induced by dynorphin A, suggesting partial agonism at KOP receptors. Notably, aMAT EV also demonstrated the ability to activate receptors, as clearly observed in the experiments. 3.7. Clinical results Twenty-five participants were enrolled in the study. We enrolled 25 patients with symptomatically and radiographically diagnosed TMC OA joints (Grade II Eaton classification). All patients had unilateral involvement, while patients with bilateral involvement were excluded from the study. The mean age was 56.2 years (range 45–70 years, SD 8.71). 60% of the participants were male and 40% were male. Eighteen were dominant right hand. Patients were monitored according to our follow-up schedule. No patient reported worsening of symptoms. Fig. 5 Radiographic images, procedural steps, hand functionality, and outcome graphs. (a) and (b) show preoperative radiographs of Stage II CMJ osteoarthritis. Panel (c) to (i) depict the procedural workflow, including preparation and injection. (j) and (k) show post-operative follow up (1 year). Panels (l) to (o) present data trends for pinch strength and pain levels over time, comparing affected and contralateral groups. The affected group demonstrates improvements in pinch strength (tripod, key, and tip) and a reduction in pain (NRS), while the contralateral group remains stable across all metrics. Statistical analysis performed using Linear Mixed Models with Tukey’s post hoc test for between-group comparisons. Significance was set at p < 0.05. There were no postoperative complications, no infections, and no significant adverse reactions or side effects. All patients were assessed at regular intervals (Table 1). Results at 12 months postoperatively were used to assess the efficacy of the procedure (Fig. 5). Table 1 Overview of the Study with Preoperative and Postoperative Evaluations Assessments Preoperative assessment - Pain- Range of motion test- Function- Strength test- Opposition testDate for surgery programmed NRS at rest and after activityMHQKey, Tripod and Tip pinchKapandji Date of surgery Application of a thermoplastic splint 2 weeks post treatment Application of a soft splint 6 weeks post treatment - Soft splint removedThe exercise routine can now be progressively intensified in order to fully utilize the thumb. 6 months post treatment assessment - Pain- Range of motion test- Function- Strength test- Opposition test NRS at rest and after activityMHQKey, Tripod and Tip pinchKapandji 12 months post treatment assessment - Pain- Range of motion test- Function- Strength test- Opposition test NRS at rest and after activityMHQKey, Tripod and Tip pinchKapandji 3.7.1. Strength and NRS activity Strength assessed with Tripod, Key and Tip pinch test. Tripod pinch test assessed a significant improvement from 6.152 Kg (SD 0.81) to 8.588 (SD 1.59) after 6 months (p<0.001) and to 9.688 (SD 1.92) after 12 months (p<0.001) (Fig. 6a). Compared to the contralateral healthy hand, tripod pinch test showed no significant differences 6 months (p=0.20) and 12 months (p=0.5) after treatment (Fig. 6b). Key pinch test assessed a significant improvement from 7.02 Kg (SD 1.38) to 8.66 (SD 2) after 6 months (p<0.001) and to 9.28 (SD 1.61) after 12 months (p<0.001) (Fig. 6c). Compared to the contralateral healthy hand, key pinch test showed no significant differences between 6 months (p=0.87) and 12 months (p=0.22) after treatment (Fig. 6d). Tip pinch test assessed a significant improvement from 4.68 Kg (SD 0.63) to 6.06 (SD 0.68) after 6 months (p<0.001) and to 6.99 (SD 0.81) after 12 months (p<0.001) (Fig. 6e). Compared to the contralateral healthy hand, tip pinch test showed no significant differences 6 months (p=0.77) and 12 months (p=0.002) after treatment (Fig. 6f). The comparative evaluation of strength compared to the contralateral hand is strongly limited and influenced by the patient's dominance, as the dominant hand tends to be naturally stronger and more coordinated than the non-dominant hand. An improvement in pain both at rest and after activity was seen. NRS at rest preoperatively was 55.16±16.02 improving to 34.73±15.57 after 6 months (p<0.0001) and 14.73±13.46 after 12 months (p<0.0001). NRS on activity preoperatively was 80.78±11.4 improving to 63.09±12.35 after 6 months (p<0.0001) and 23.47±10.89 after 12 months (p<0.0001) (Fig. 6g, h and Fig. 7c). 3.7.2. Thumb Opposition (Kapandji score) and MHQ Kapandji score improved significantly from 6.56/10 (SD 0.89) preoperatively to 7.12/10 (SD 0.58) at 6 months post treatment and 8.44/10 (SD 0.49) at 12 months post treatment (Fig. 7a). Fig. 7 Box plot analyses showing temporal changes in KAPANDJI scores (a) and MHQ scores (b) over a 12-month peri-od. Panel (a) demonstrates significant improvement in KAPANDJI scores (ANOVA, F(1.51, 36.33) = 138.38, p < 0.0001, η²g = 0.57) with Bonferroni-adjusted pairwise comparisons (****p < 0.0001) between time points. Panel (b) shows significant enhancement in MHQ scores (ANOVA, F(1.35, 32.3) = 268.4, p < 0.0001, η²g = 0.73) with similar statistical significance between time points. Both metrics demonstrate progressive improvement from base-line (T0) through 6 months (T6) to 12 months (T12). Panel (c) presents a box plot analysis showing significant pain reduction over time (ANOVA, F(2,48) = 993.7, p < 0.0001, η²g = 0.81) with Bonferroni-adjusted pairwise comparisons (****p < 0.0001) between all time points. The data demonstrate a progressive decrease in pain scores from baseline (T0) through 6 months (T6) to 12 months (T12) post-intervention. Compared to baseline (84.76±4.63), MHQ scores were significantly higher at six months (90.86±2.94) (p<0.0001) and twelve months (98.14±1.97) (p<0.0001), indicating improvement (Fig. 7b). 3.7.3. Postoperative X-rays All patients who had an X-ray 12 months postoperatively were still classified at the same Dell stage and therefore did not show any obvious worsening. 4. Discussion Until now, research on MSC-derived extracellular vesicles has primarily focused on their molecular content and regenerative potential [ 60 , 61 ]. However, the cellular mechanisms they trigger upon interaction with recipient cells remain largely unexplored. In this study, aMAT-EV were isolated and characterized according to MISEV guidelines. Size analysis using tunable resistive pulse sensing and TEM imaging confirmed their nanometric dimensions and typical cup-shaped morphology. Key exosomal markers (ALIX, TSG101, ANXA5, CD81, and CD63) were detected using immunoblotting and flow cytometry. Proteomic analysis revealed proteins linked to extracellular matrix organization, cell adhesion, collagen fibril formation, wound healing, and inflammation regulation—hallmarks of regenerative activity [ 41 , 62 ]. Although chondrocytes are commonly studied in the context of osteoarthritis, we selected fibroblasts for in vitro investigation based on their active involvement in periarticular remodeling, matrix turnover, and inflammatory signaling within the osteoarthritic joint. Fibroblasts represent a reproducible and well-characterized model and are key contributors to tissue homeostasis and degeneration in the context of thumb carpometacarpal osteoarthritis, where multiple periarticular structures are affected. GO enrichment analysis of EV proteomics indicated the presence of proteins involved in mitochondrial function, extracellular matrix organization, and calcium-dependent signaling cascades. Calcium signaling was selected as a focal pathway based on its central role in mitochondrial regulation, cellular metabolism, and the activation of stress and inflammatory responses. The observed modulation of intracellular Ca²⁺ dynamics in fibroblasts supports the hypothesis that aMAT-derived EVs may exert their therapeutic effects by restoring cellular homeostasis and dampening pro-inflammatory signaling in target stromal cells. To explore their effects on recipient cells, aMAT-EV was shown to be internalized by human dermal fibroblasts within 24 hours, as evidenced by PKH26 staining. This uptake underscores their potential for therapeutic delivery and intercellular communication. The study also revealed that aMAT-EV trigger intracellular calcium signaling in fibroblasts, a critical pathway for cellular responses. Using Fura-2 AM, a significant rise in cytosolic calcium levels was observed upon EV stimulation. Further experiments confirmed that this calcium influx originates from the extracellular space, mediated by transient receptor potential (TRP) channels. Ruthenium Red, a TRP channel inhibitor, effectively blocked the calcium response, highlighting the role of mechanical activation in this process. Mitochondrial calcium uptake, crucial for cellular bioenergetics, was also evaluated. aMAT-EV induced transient calcium accumulation in the mitochondrial matrix, enhancing the mitochondrial membrane potential (ΔΨm) and oxygen consumption during maximal respiration. However, inhibiting the mitochondrial calcium uniporter (MCU) did not affect ΔΨm, suggesting an alternate pathway for calcium entry. Moreover, the therapeutic potential of aMAT-EV was validated in inflamed fibroblasts treated with TNF-α. EV treatment restored ΔΨm to levels comparable to non-inflamed cells, indicating their ability to mitigate inflammation-induced mitochondrial dysfunction. This regenerative effect, likely mediated by their anti-inflammatory properties, highlights aMAT-EV as promising candidates for novel therapies targeting inflammation and oxidative stress-related disorders [ 63 , 64 ]. The mitochondrial improvements observed in EV-treated fibroblasts may have functional relevance in the context of trapeziometacarpal osteoarthritis (TMC OA). Fibroblasts in osteoarthritic tissues are known to undergo mitochondrial dysfunction, increased oxidative stress, and adopt a pro-inflammatory secretory phenotype—collectively contributing to matrix degradation and persistent inflammation. By restoring mitochondrial membrane potential and improving bioenergetic capacity, extracellular vesicles may help reprogram fibroblasts toward a more quiescent and homeostatic state. This process could mitigate senescence-associated secretory activity and the production of catabolic mediators. Prior studies have shown that cellular senescence and mitochondrial dysfunction in fibroblasts are closely linked to OA progression and joint pain, particularly through the accumulation of reactive oxygen species and inflammatory cytokines [ 65 , 66 ]. Our findings support the hypothesis that the EV-induced modulation of mitochondrial function may represent a key mechanism in alleviating stromal inflammation and tissue degeneration in TMC OA This investigation adds further evidence to the concept that mitochondrial modulation plays a pivotal role in the therapeutic potential of EVs. Enhanced ΔΨm and improved mitochondrial respiration upon aMAT-EV stimulation are consistent with increased cellular bioenergetics, which supports tissue regeneration and repair. Furthermore, the ability of aMAT-EVs to restore mitochondrial function in inflamed fibroblasts suggests their dual action in both repairing metabolic dysfunction and mitigating inflammation. These properties are critical for chronic conditions such as OA, where mitochondrial dysfunction and inflammatory pathways converge to exacerbate joint degeneration. Lastly in vitro study was conducted to assess the impact of aMAT-EV on opioid receptors (OPr) within an in vitro model. To analyze this, we used CHO cell lines expressing the Gα qi5 protein, which couples any G-protein-coupled receptor (GPCR) to an increase in intracellular Ca²⁺ concentration ([Ca²⁺]c). The observed Ca²⁺ responses confirmed that aMAT-EVs selectively activates the KOP receptor as a partial agonist, suggesting that the therapeutic effect may be partially mediated by modulation of nociceptive pathways, resulting in reduced pain perception in patients. In vitro results support the efficacy of aMAT-EVs in reducing nociceptive pain through selective KOP receptor activation. Aequorin-based assays were employed, showing a transient increase in mitochondrial Ca²⁺ concentration ([Ca²⁺]m), which is at least one order of magnitude higher than cytosolic levels, upon receptor activation. These findings imply that aMAT-EVs may play a role in pain reduction via specific nociceptive pathways [ 67 , 68 ]. The implications of these findings extend beyond nociceptive modulation. The dual action of aMAT-EV, combining mitochondrial restoration with KOP receptor activation, introduces a novel paradigm in pain management. Traditional pharmacological interventions for OA and related pain disorders primarily target symptom relief without addressing underlying cellular dysfunctions. The EV-based approach, by contrast, offers a multifaceted mechanism that not only alleviates pain but also promotes cellular homeostasis and repair, which could delay disease progression. Recent studies have demonstrated that mitochondrial function and calcium signaling play a pivotal role in nociceptive modulation. Mitochondrial Ca²⁺ uptake, ROS production, and cross-talk with the endoplasmic reticulum have been shown to influence neuronal excitability and pain sensitivity in both inflammatory and neuropathic contexts [ 69 – 71 ]. These findings provide a mechanistic rationale supporting the analgesic potential of aMAT-derived EVs, which modulate these same pathways in vitro . Clinically, patients treated with aMAT demonstrated significant improvement in pain and functional parameters. The Michigan Hand Outcomes Questionnaire (MHQ) showed a notable improvement (p < 0.0001), particularly in pain relief. NRS scores decreased significantly both at rest and during activity after treatment. At rest, the NRS score dropped from 55.16 to 34.73 after six months and to 14.73 after 12 months (p < 0.0001 for both periods), while activity-related pain scores decreased from 80.78 to 63.09 after six months and to 23.47 after 12 months. These improvements indicate substantial pain relief and functional recovery, with a more pronounced impact on activity-related pain. Objective measures, including pinch strength and Kapandji opposition score, showed significant improvements from baseline to 6 and 12 months. All three pinch strength tests (Tripod, Key, and Tip) improved, suggesting that aMAT was effective in enhancing hand strength. Comparison with the healthy contralateral hand showed no significant differences in Tripod and Key pinch tests at both follow-up periods, while a significant improvement was observed in the Tip pinch test after 12 months. The Kapandji score also showed a statistically significant increase over time, reflecting enhanced thumb opposition and overall hand functionality. Although no radiographic structural improvements were observed over the 12-month follow-up period, it is important to consider that trapeziometacarpal osteoarthritis is typically characterized by a progressive course. Therefore, the absence of radiographic deterioration within this timeframe can be regarded as a positive outcome, especially from the perspective of hand surgeons managing this condition. Moreover, the imaging modality employed—standard plain radiographs—may not be sensitive enough to detect subtle tissue-level changes induced by extracellular vesicles. Future studies incorporating advanced imaging techniques such as magnetic resonance imaging (MRI) may provide more detailed insights into the microstructural and microenvironmental effects of aMAT-derived EVs. Interestingly, these functional improvements align with the observed cellular and molecular changes. The enhanced mitochondrial respiration and bioenergetic state in vitro could underpin the clinical improvements in grip strength and thumb opposition. Such correlations highlight the translational relevance of the findings, suggesting that aMAT-EVs could provide a mechanistic link between cellular metabolism and clinical recovery. No significant radiological changes were observed after one year, although pain reduction was evident, aligning with findings from other studies [ 72 ]. However, radiography may be limited in assessing early cartilage and synovial microenvironment changes. As shown by Mayoly A et al. [ 73 ], pain reduction may correlate more closely with reduced edema in the cartilage and synovium. Future studies employing advanced imaging modalities could provide a clearer view of structural changes associated with pain relief. Additionally, exploring advanced imaging techniques such as MRI or positron emission tomography (PET) could shed light on EV-induced microenvironmental changes, such as reductions in synovial inflammation or cartilage repair. Such approaches could validate the observed therapeutic benefits at the molecular and structural levels, further reinforcing the clinical utility of aMAT-EVs. The study highlights the potential of microfragmented adipose tissue in managing TMC joint osteoarthritis, aligning with recent advances in regenerative medicine. However, consistent methodologies for adipose tissue processing and controlled, randomized trials will be essential to confirm the efficacy of this treatment in broader patient populations. 5. Study Limitations and Future Directions While the results of this study are promising, several limitations must be acknowledged. The relatively small sample size (n = 25) and single-center design may constrain statistical power and limit generalizability. Nevertheless, the consistent clinical improvements observed across all validated metrics indicate that aMAT-EVs hold substantial therapeutic promise. This study was not designed as a randomized controlled trial (RCT), but rather as an exploratory, single-arm clinical investigation aimed at evaluating the safety and early clinical efficacy of aMAT-EVs. Given the ethical constraints associated with the use of placebo or sham injections in intra-articular procedures, a control group was not included. While the findings are encouraging, future RCTs with appropriate comparators will be required to confirm and generalize these results. Additionally, structural changes were assessed exclusively by conventional X-rays, which have limited sensitivity for detecting early tissue-level improvements such as cartilage repair or synovial inflammation. The inclusion of advanced imaging techniques such as MRI or ultrasound in future trials will help to more accurately capture microstructural responses and their correlation with clinical outcomes. Finally, while the in vitro findings provide mechanistic insights into the action of aMAT-EVs, the translation of these molecular and cellular effects to clinical outcomes remains partially speculative. However, even if this study provides converging evidence from in vitro and in vivo data supporting the therapeutic relevance of aMAT-derived EVs, the precise molecular mechanisms underlying the observed effects remain incompletely delineated. The modulation of mitochondrial dynamics and calcium signaling in fibroblasts aligns with known pathways involved in cellular stress and inflammation; however, direct causal links between these molecular events and clinical improvements were not established. Further mechanistic studies are needed to dissect the contribution of individual EV cargo molecules—such as specific proteins, miRNAs, or lipids—and their downstream targets in recipient cells. Approaches such as pathway inhibition, gene knockdown, or selective EV subpopulation profiling could help elucidate the key effectors mediating analgesic and anti-inflammatory actions. Further investigations are needed to delineate the precise pathways through which aMAT-EVs exert their regenerative and anti-nociceptive effects, particularly in inflamed and degenerated tissues. 6. Conclusion This study demonstrates the potential of extracellular vesicles (EVs) derived from microfragmented adipose tissue (aMAT-EVs) as a novel, minimally invasive therapeutic strategy for managing trapeziometacarpal (TMC) osteoarthritis. While several recent clinical studies have reported the therapeutic effects of autologous microfragmented adipose tissue (aMAT) in large-joint osteoarthritis (e.g., knee or hip OA) [ 74 – 76 ], our study provides novel insights in three key aspects. First, it targets trapeziometacarpal osteoarthritis (TMC OA), a distinct and under-investigated form of OA with unique anatomical and functional characteristics. Second, it combines clinical outcomes with a mechanistic investigation focused on aMAT-derived extracellular vesicles (EVs), which have not been explored in previous trials. Third, we demonstrate the impact of these EVs on mitochondrial function and calcium signaling in fibroblasts, supporting a potential cellular mechanism underlying clinical pain relief. This integrative approach strengthens the translational relevance of the findings and highlights the therapeutic potential of EVs as a future standalone option. Through comprehensive in vitro and in vivo analyses, we have shown that aMAT-EVs possess a unique capacity to modulate inflammation, enhance mitochondrial bioenergetics, and influence nociceptive pathways via selective activation of KOP receptors. Clinically, these effects translate into significant reductions in pain and improvements in hand functionality and strength, as evidenced by decreased NRS scores, increased Kapandji opposition scores, and enhanced pinch strength across multiple measures. Importantly, the absence of radiological progression over 12 months suggests that this approach may offer both symptomatic relief and a protective effect against joint deterioration. The findings provide a promising foundation for the development of EV-based therapies, which could address the unmet clinical needs of patients with OA and related musculoskeletal disorders. By targeting both the underlying cellular dysfunctions and the symptomatic burden, aMAT-EVs represent an innovative therapeutic modality with the potential to redefine current treatment paradigms. While the results of this study are promising, several limitations must be acknowledged. First, the sample size of the clinical cohort was relatively small, and the study was conducted at a single center. This may limit the generalizability of the findings to broader and more diverse patient populations. Future multicenter, randomized controlled trials with larger cohorts are necessary to validate these results and establish robust efficacy and safety profiles. Second, while significant functional and symptomatic improvements were observed, radiological assessments showed no discernible changes in joint structure over the 12-month period. Advanced imaging modalities such as MRI or PET scans were not employed, which might have provided deeper insights into microenvironmental changes, including cartilage repair or synovial inflammation. Incorporating such techniques in future studies could offer a more comprehensive evaluation of the therapeutic effects of aMAT-EVs. Finally, while the in vitro findings provide mechanistic insights into the action of aMAT-EVs, the translation of these molecular and cellular effects to clinical outcomes remains partially speculative. Further investigations are needed to delineate the precise pathways through which aMAT-EVs exert their regenerative and anti-nociceptive effects, particularly in inflamed and degenerated tissues. Future research should focus on optimizing EV isolation and characterization methods to enhance reproducibility and scalability for clinical use. Additionally, the long-term effects of aMAT-EV therapy on joint health, as well as its potential applications in other chronic pain and degenerative conditions, warrant exploration. The integration of advanced omics technologies and imaging tools will be critical for unraveling the full therapeutic potential of aMAT-EVs and refining their application in personalized medicine. Abbreviations EVs Extracellular vesicles MSDs Musculoskeletal diseases RA rheumatoid arthritis OA osteoarthritis LBP low-back pain NP neck pain TMC trapeziometacarpal PRP platelet-rich plasma MSCs mesenchymal stem cells AD-MSCs adipose-derived MSCs aMAT microfragmented adipose tissue aMAT-EVs aMAT contained Extracellular Vesicles NTA Nanoparticle tracking analysis TEM Transmission electron microscopy MCU mitochondrial calcium uniporter MHQ Michigan Hand Outcomes Questionnaire IQRs interquartile ranges OCR oxygen consumption rate Declarations Ethic approvals and consent to participate All the procedures were approved by Ethics Committee of Marche Region (protocol n. 154/2021).. Informed Patient Consent Statement: Informed consent was obtained from all individual participants included in the study, following the guidelines of the Human Research Approval Committee protocol number 2/2019. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request Competing interests The authors declare that they have no competing interests Funding This research did not receive any specific grant from founding agencies in the public, commercial, or non-profit sectors. Authors' contributions F.D.F : Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing—original draft, Writing—review and editing, Visualization. L.F : Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing—original draft, Writing—review and editing, Visualization. I.Z .: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing—original draft. L.S .: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing—review and editing, Visualization. A.M.M . Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing—original draft, Writing—review and editing, Visualization: M.P.C .: Formal analysis, Investigation, Data curation, Writing—review and editing. M.F .: Data curation and statistical analysis. L.S .: Conceptualization, Methodology, Formal analysis, Investigation. E.T .: Conceptualization, Methodology, Formal analysis, Investigation. I.P.C .: Validation, Formal analysis, Investigation, Data curation, Writing—review and editing. E.M.S .: Validation, Formal analysis, Investigation, Data curation, Writing—review and editing. M.B .: Validation, Formal analysis, Investigation, Data curation, Writing—review and editing. A.R : Validation, Data curation, Writing—review and editing. UDA : Validation, Data curation, Writing—review and editing. N.D.C. : Validation, Data curation, Writing—review and editing L.L : Formal analysis, Investigation, Data curation, Writing—review and editing. 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Adipose-derived stem cell exosomes act as delivery vehicles of microRNAs in a dog model of chronic hepatitis. Nanotheranostics. 2024;8:298–311. Benderdour M, Martel-Pelletier J, Pelletier J-P, Kapoor M, Zunzunegui M-V, Fahmi H. Cellular Aging, Senescence and Autophagy Processes in Osteoarthritis. CAS. 2015;8:147–57. Jeon OH, David N, Campisi J, Elisseeff JH. Senescent cells and osteoarthritis: a painful connection. J Clin Invest. 2018;128:1229–37. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003;4:517–29. Zavan B, Ferroni L, Giorgi C, Calò G, Brun P, Cortivo R, et al. Hyaluronic acid induces activation of the κ-opioid receptor. PLoS ONE. 2013;8:e55510. Yousuf MS, Maguire AD, Simmen T, Kerr BJ. Endoplasmic reticulum–mitochondria interplay in chronic pain: The calcium connection. Mol Pain. 2020;16:1744806920946889. Silva Santos Ribeiro P, Willemen HLDM, Eijkelkamp N. Mitochondria and sensory processing in inflammatory and neuropathic pain. Front Pain Res. 2022;3:1013577. Kim HY, Lee KY, Lu Y, Wang J, Cui L, Kim SJ, et al. Mitochondrial Ca 2+ Uptake Is Essential for Synaptic Plasticity in Pain. J Neurosci. 2011;31:12982–91. Erne HC, Cerny MK, Ehrl D, Bauer AT, Schmauss V, Moog P, et al. Autologous Fat Injection versus Lundborg Resection Arthroplasty for the Treatment of Trapeziometacarpal Joint Osteoarthritis. Plast Reconstr Surg. 2018;141:119–24. Mayoly A, Witters M, Jouve E, Bec C, Iniesta A, Kachouh N, et al. Intra Articular Injection of Autologous Microfat and Platelets-Rich Plasma in the Treatment of Wrist Osteoarthritis: A Pilot Study. JCM. 2022;11:5786. Richter DL, Harrison JL, Faber L, Schrader S, Zhu Y, Pierce C et al. Microfragmented Adipose Tissue Injection Reduced Pain Compared With a Saline Control Among Patients With Symptomatic Osteoarthritis of the Knee During 1-Year Follow-Up: A Randomized Controlled Trial. Arthroscopy: The Journal of Arthroscopic & Related Surgery. 2025;41:248–60. Zaffagnini S, Andriolo L, Boffa A, Poggi A, Cenacchi A, Busacca M, et al. Microfragmented Adipose Tissue Versus Platelet-Rich Plasma for the Treatment of Knee Osteoarthritis: A Prospective Randomized Controlled Trial at 2-Year Follow-up. Am J Sports Med. 2022;50:2881–92. Ulivi M, Meroni V, Viganò M, Colombini A, Lombardo MDM, Rossi N, et al. Micro-fragmented adipose tissue (mFAT) associated with arthroscopic debridement provides functional improvement in knee osteoarthritis: a randomized controlled trial. Knee Surg Sports Traumatol Arthrosc. 2023;31:3079–90. 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Ferrara","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Zavan","suffix":""}],"badges":[],"createdAt":"2025-05-22 12:51:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6725135/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6725135/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-025-06930-4","type":"published","date":"2025-08-18T16:12:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85384210,"identity":"cd126de8-5d0d-4772-bc12-8b7d288fcff7","added_by":"auto","created_at":"2025-06-25 09:38:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":17883237,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of MSC-EVs. (a) Representative trace of particle diameter (nm) and concentration (particle/mL) of MSC-derived EVs (MSC-EVs) measured through Tunable Resistive Pulse Sensing (TRPS). (b) Mean protein con-centration (µg/mL) of MSC-EVs evaluated with BCA assay. (c) Exosomal markers detection with Exo-Check Exo-some Antibody Array. (D-F) Flow cytometry detected EV markers: CD63 (d), CD81 (e), double positivity CD63 and CD81 (f). (g) Representative images of cup-shaped EVs visualized via Transmission Electron Microscopy (TEM). (h) Representative image of fibroblasts treated with PKH26-stained EVs (left panel) or vehicle (right pan-el). Scale bar 10 µm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6725135/v1/9efcf045a41787d4800df9a7.png"},{"id":85384113,"identity":"bd59888e-5016-4156-86aa-d3e25b582658","added_by":"auto","created_at":"2025-06-25 09:38:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5138192,"visible":true,"origin":"","legend":"\u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e signalling is elicited by MSC-EVs. (a) Representative traces of fibroblasts stimulated with EVs in KRB/Ca\u003csup\u003e2+\u003c/sup\u003e saline show an increase in Fura-2 AM ratio intensity, indicating a transient increase in the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e levels. (b) Representative Fura-2 AM ratio images of the transient increase of cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e levels 10 seconds before, and 130 seconds and 250 seconds after EV treatment. (c) Positive response rate (in cyan) of fibroblasts to EV treatment was 63,4%, negative response (in red) to EVs was 36,6%. (d) Representative traces of fibroblasts stimulated with EVs in KRB/Ca\u003csup\u003e2+\u003c/sup\u003e saline (in blue) and KRB/EGTA saline (in orange) show a different effect on the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e levels, with no increase in fluorescence intensity registered in presence of EGTA 100 µM. (e) Quantitation of the amount of mobilized Ca\u003csup\u003e2+\u003c/sup\u003e expressed as area under the curve (AUC) calculated on Fura-2 ratio traces in fibroblasts stimulated with MSC-EV and recorded in KRB/Ca\u003csup\u003e2+\u003c/sup\u003e saline Ca\u003csup\u003e2+\u003c/sup\u003e free KRB saline\u0026nbsp; + 100 µM EGTA. (f) Representative traces of normal fibroblasts and fibroblasts pre-treated with Ruthenium Red (RR) show no increase in Fura-2 AM ratio in presence of RR 10 µM. (g) Fura-2 AM fluorescence ratio quantification as AUC of fibroblasts pretreated with RR or vehicle. (h) Representative trace of mt-GCaMP6-transfected fibroblasts stimulated with EVs show an increase in fluorescence ratio, indicating a transient increase in the mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels. (i) Representative images of mt-GCaMP6 ratio of the transient increase of mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels 10 seconds before, and 230 seconds and 800 seconds after EV treatment.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6725135/v1/fe19b8e30997f2971c42d5ac.png"},{"id":85384153,"identity":"60900579-006a-485c-b2c4-a6e82c65f1c7","added_by":"auto","created_at":"2025-06-25 09:38:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1835492,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;aMAT-EVs affect the mitochondrial physiology of fibroblasts. (a) Representative traces of the mitochondrial membrane potential (ΔΨm) reporter, TMRM, in fibroblasts untreated (in red) or exposed to EVs for 48 hours (in blue), showing the slope of the kinetics following 1 µM FCCP stimulus. (b) TMRM fluorescence intensity quantification of fibroblasts treated with EVs for 48 hours in KRB/Ca\u003csup\u003e2+\u003c/sup\u003e saline is significantly increased compared to the fluorescence intensity of untreated fibroblasts. (c-g) Representative traces of oxygen consumption rates in fibroblasts untreated (blue line) or exposed to MSC-EV for 48 hours (red line). OCR was analyzed in real time using the XF96 extracellular flux analyzer to quantify basal respiration rates (d), ATP-linked mitochondrial respiration (e), maximal respiration capacity (f) and spare respiratory capacity (g). While basal respiration and ATP production are not significantly different compared to untreated cells (d and e respectively), maximal respiration (f) and spare respiratory capacity (g) are increased in EV-treated fibroblasts. (h) ΔΨm of fibroblasts pre-treated with the mitochondrial calcium uniporter (MCU) complex inhibitor DS16570511 and stimulated with EVs for 48 hours shows no significant difference compared to only EV-treated fibroblasts. (i) ΔΨm of fibroblasts pre-treated with the MCU complex inhibitor MCU-i11 and stimulated with EVs shows no significant difference compared to only EV-treated fibroblasts.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6725135/v1/25d060de1e8345a299208309.png"},{"id":85384112,"identity":"e2b03706-4acf-4fab-908b-a743799ece36","added_by":"auto","created_at":"2025-06-25 09:38:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":999974,"visible":true,"origin":"","legend":"\u003cp\u003eAdipose tissue-EV effect of TNFα-treated fibroblasts. (a) Representative traces of TNFα inflamed and non-inflamed fibroblasts stimulated with EVs in KRB/Ca2+ saline show an increase in Fura-2 AM ratio, indicating a transient increase in the cytosolic Ca2+ levels; (b)ΔΨm of inflamed cells is reduced compared to normal cells. ΔΨm of EV-treated fibroblasts is enhanced compared to the untreated condition. EV treatment on inflamed fibroblasts leads to a recovery in the ΔΨm; (c) Fura-2 AM ratio quantification as AUC is increased in inflamed fibroblasts; (d) percentage of Calcium Variation. Stable cell lineages of CHO cells bearing KOP receptors underwent transfection with a mitochondria-targeted aequorin sensor, following which they were subjected to various concentrations of hyaluronic acid (HA), as delineated in the illustration. The resultant mitochondrial calcium responses are delineat-ed as a percentage of the mitochondrial calcium response, relative to receptor stimulation with a canonical agonist.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6725135/v1/19e2e181d3e511edfe02310f.png"},{"id":85384139,"identity":"623d083d-c7d1-43d9-9ae1-382762b443d0","added_by":"auto","created_at":"2025-06-25 09:38:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31599624,"visible":true,"origin":"","legend":"\u003cp\u003eRadiographic images, procedural steps, hand functionality, and outcome graphs. (a) and (b) show preoperative radiographs of Stage II CMJ osteoarthritis. Panel (c) to (i) depict the procedural workflow, including preparation and injection. (j) and (k) show post-operative follow up (1 year). Panels (l) to (o) present data trends for pinch strength and pain levels over time, comparing affected and contralateral groups. The affected group demonstrates improvements in pinch strength (tripod, key, and tip) and a reduction in pain (NRS), while the contralateral group remains stable across all metrics. Statistical analysis performed using Linear Mixed Models with Tukey’s post hoc test for between-group comparisons. Significance was set at p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6725135/v1/cb9cef62cffa3e9323f58936.png"},{"id":85384156,"identity":"c41b19bc-62ea-4c27-892c-d6c09cd65616","added_by":"auto","created_at":"2025-06-25 09:38:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3060986,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical analysis of pinch strength measurements (Tripod, Key, and Tip pinch) comparing affected and contralateral groups over time (T0, T6, T12). Statistical comparisons assessed by repeated measures ANOVA with Bonferroni correction. Effect sizes are reported as generalized eta-squared (η²g). Panels (a), (c), and (e) display adjusted means with confidence intervals for the affected and contralateral groups, while Panels (b), (d), and (f) illustrate temporal progression of the affected group. The data demonstrates significant improvements in pinch strength for the affected group across all pinch types, with consistent stability in the contralateral group. Error bars represent confidence intervals, and black dots indicate point estimates. Statistical analysis of NRS (Numeric Rating Scale) pain scores. (g) shows adjusted means with confidence intervals comparing affected and contralateral groups at different time points (T0, T6, T12). (h) displays the temporal progression of NRS scores for both affected and contralateral groups.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6725135/v1/175493fea96465811bc95134.png"},{"id":85384132,"identity":"bfdf9fda-4c6a-4b00-88fb-e056a133be42","added_by":"auto","created_at":"2025-06-25 09:38:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":897886,"visible":true,"origin":"","legend":"\u003cp\u003eBox plot analyses showing temporal changes in KAPANDJI scores (a) and MHQ scores (b) over a 12-month peri-od. Panel (a) demonstrates significant improvement in KAPANDJI scores (ANOVA, F(1.51, 36.33) = 138.38, p \u0026lt; 0.0001, η²g = 0.57) with Bonferroni-adjusted pairwise comparisons (****p \u0026lt; 0.0001) between time points. Panel (b) shows significant enhancement in MHQ scores (ANOVA, F(1.35, 32.3) = 268.4, p \u0026lt; 0.0001, η²g = 0.73) with similar statistical significance between time points. Both metrics demonstrate progressive improvement from base-line (T0) through 6 months (T6) to 12 months (T12). Panel (c) presents a box plot analysis showing significant pain reduction over time (ANOVA, F(2,48) = 993.7, p \u0026lt; 0.0001, η²g = 0.81) with Bonferroni-adjusted pairwise comparisons (****p \u0026lt; 0.0001) between all time points. The data demonstrate a progressive decrease in pain scores from baseline (T0) through 6 months (T6) to 12 months (T12) post-intervention.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6725135/v1/37adab289f4b928aeab23174.png"},{"id":89847150,"identity":"8f6cbfed-00c6-487f-b99f-e6ab03bac593","added_by":"auto","created_at":"2025-08-25 16:41:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":58298351,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6725135/v1/20845148-1eca-4715-af7e-bcfe9c049a41.pdf"},{"id":85384194,"identity":"95775f88-3371-44a4-ba7a-514314e8cff1","added_by":"auto","created_at":"2025-06-25 09:38:38","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":1664545,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6725135/v1/b754bdb645166b1cb247759a.docx"}],"financialInterests":"","formattedTitle":"Mechanism of Action Behind the Pain-Relief Effects of Extracellular Vesicles in Microfragmented Adipose Tissue: An In vitro and In vivo Study","fulltext":[{"header":"1. Background","content":"\u003cp\u003eExtracellular vesicles (EVs) have emerged as promising biological agents, capable of delivering active biomolecules to target cells and modulating a range of biological processes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] These nano-sized particles, secreted by cells, facilitate intercellular communication and influence tissue repair, inflammation, and immune responses [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Interest in EVs has grown recently due to their therapeutic potential in regenerative medicine and pain management, underscoring the importance of elucidating their precise mechanisms of action [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Given their ability to deliver proteins, lipids, and genetic material to target cells, EVs represent a promising approach for treating chronic conditions, particularly those involving tissue degeneration and pain, such as musculoskeletal diseases [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Musculoskeletal diseases (MSDs) are a significant health concern globally, affecting millions and imposing a high socioeconomic burden. Key MSDs include rheumatoid arthritis (RA), osteoarthritis (OA), low-back pain (LBP), neck pain (NP), and gout [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], with OA standing out as the most common degenerative joint disease among adults, significantly impacting daily functioning and quality of life. According to the Centers for Disease Control and Prevention, arthritis affects one in four adults, with prevalence surging by over 113% from 1990 to 2020 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. OA frequently affects the hand, knee, and hip joints, leading to substantial healthcare costs [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Notably, hand OA has a high clinical impact due to the extensive loss of functionality in small, complex joints, which contributes to pain and significant disability [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. OA is often diagnosed at an advanced stage, when joint degeneration is irreversible, necessitating surgical intervention [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], especially in joints like the thumb's trapeziometacarpal (TMC) joint [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTrapeziometacarpal osteoarthritis (TMC OA) is a multifactorial degenerative disease that affects not only the articular cartilage, but also the synovial membrane, joint capsule, and periarticular soft tissues. Within this microenvironment, fibroblasts play a central role by producing extracellular matrix components, modulating local inflammation, and influencing tissue remodeling [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Emerging evidence has implicated synovial fibroblasts in the pathogenesis of OA through their secretion of pro-inflammatory cytokines, matrix-degrading enzymes, and their ability to respond to mechanical and biochemical stress [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Given their functional relevance and accessibility for \u003cem\u003ein vitro\u003c/em\u003e modeling, fibroblasts represent a suitable cellular system to investigate the mechanistic effects of EV-based therapies in OA.\u003c/p\u003e \u003cp\u003eIn earlier stages of joint degeneration, conservative treatments such as splinting and the use of analgesics or NSAIDs are typically prescribed [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Corticosteroid and hyaluronic acid injections have also been applied, though their efficacy remains inconclusive, with guidelines recommending these treatments mainly for mildly symptomatic cases [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. When conservative measures fail, surgical options such as joint arthroplasty or ligament repair become necessary, particularly in cases like TMC joint OA hands [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEmerging therapies, including the use of platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs), offer promising alternatives. MSCs from sources like bone marrow, umbilical cord, synovium, and adipose tissue have demonstrated potential in reducing inflammation and alleviating symptoms through the secretion of paracrine factors [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong these sources, adipose tissue offers distinct advantages that make it particularly suitable for clinical use. Compared to bone marrow or umbilical cord-derived MSCs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], adipose-derived MSCs (AD-MSCs) are more abundant, easier to harvest via minimally invasive procedures, and exhibit a strong paracrine secretory profile. Moreover, the use of autologous adipose tissue reduces immunogenicity risks and complies more readily with current regulatory frameworks, especially when processed via minimal manipulation methods such as microfragmentation. These characteristics support the rationale for selecting microfragmented adipose tissue (aMAT) as the tissue source in this study and highlight its translational potential in regenerative approaches for osteoarthritis and pain.\u003c/p\u003e \u003cp\u003eIn particular, micro-fragmented adipose tissue has gained attention for its regenerative potential in joint pathologies [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This tissue is commonly prepared using non-enzymatic methods, resulting in minimally manipulated products that comply with regulatory standards [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eaMAT contains a heterogeneous population of cells, including MSCs, pericytes, preadipocytes, and immune cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Among them, AD-MSCs are particularly abundant and play a crucial role in tissue repair due to their potent paracrine activity. AD-MSCs are known to secrete EVs enriched in cytokines, growth factors, and microRNAs that can modulate inflammation, promote matrix remodeling, and influence pain signaling [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These properties make them a promising candidate for regenerative approaches in osteoarthritis and pain management.\u003c/p\u003e \u003cp\u003eDespite advancements in MSC-based therapies, significant gaps remain in understanding the mechanisms through which EVs modulate nociceptive pathways. EVs derived from aMAT-EVs may bridge this gap by providing a minimally invasive therapeutic option. Their ability to influence inflammation, extracellular matrix organization, and cellular bioenergetics highlights their potential not only for joint preservation but also for effective pain management. Given the limitations of conventional therapies, exploring EV-mediated mechanisms in pain modulation could revolutionize treatment paradigms for OA and other chronic conditions.\u003c/p\u003e \u003cp\u003eConsidering such consideration in this study, we focus on characterizing EVs derived from treated adipose grafts, specifically analyzing their potential to alleviate osteoarticular pain. Building on our expertise in EV isolation and characterization, we aim to identify the mechanisms by which these vesicles interact with nociceptive receptors to modulate pain. Our approach includes morphological, proteomic, and functional analyses of these vesicles, with the goal of elucidating pathways involved in pain perception and assessing their efficacy in clinical pain.\u003c/p\u003e \u003cp\u003eThrough both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e on human studies, this investigation aims to advance our understanding of EV-mediated pain modulation, potentially paving the way for innovative, minimally invasive treatments for OA and related chronic pain conditions.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Surgical procedure to obtain adipose micro-grafts tissue (aMAT)\u003c/h2\u003e \u003cp\u003ePatients were positioned supine, and local anesthesia was administered. A small incision was made to introduce a blunt cannula attached to a Luer-lock 60-cc syringe. Klein sterile solution, containing saline and lidocaine, was injected into the subcutaneous fat layer of either the abdominal or thigh region. Subsequently, approximately 30 mL of adipose tissue was extracted.\u003c/p\u003e \u003cp\u003eThe collected lipoaspirate was processed under sterile conditions in a closed system using Rigenera\u0026reg; technology (HBW, Turin, Italy). This non-enzymatic approach is specifically designed to disaggregate human tissues such as adipose tissue, dental pulp, cartilage, dermis, and bone for reapplication. The process has been documented in multiple studies [\u003cspan additionalcitationids=\"CR36 CR37 CR38\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This disposable device progressively reduces the size of adipose tissue clusters from their initial dimensions of 1\u0026ndash;3.5 mm to finer fragments measuring 0.2\u0026ndash;0.8 mm, resulting in an adipose micro-graft. The surgical workflow consists of two stages: liposuction followed by tissue disaggregation to create micro-grafts. In this procedure, 4 mL of lipoaspirate was mechanically fragmented using Rigeneracons\u0026reg; (Human Brain Wave S.r.L., Turin, Italy) while mixing with 4 mL of saline solution. This process yielded approximately 6\u0026ndash;7 mL of micro-fragmented adipose tissue, ready for direct application.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Isolation and Characterization of aMAT contained Extracellular Vesicles (aMAT-EVs)\u003c/h2\u003e \u003cp\u003eaMAT was resuspended in 10 mL of PBS and applied to Amicon Ultra-15 100 kDa centrifugal devices (Millipore, MA, USA), which had been pre-sterilized with 70% ethyl alcohol (Sigma-Aldrich, Saint Louis, MA, USA). The devices were centrifuged at 2000xg for 20 minutes at 4\u0026deg;C. The resulting filtrate was then washed with sterile PBS (Euroclone, Milan, Italy) via another centrifugation step at 2000xg for 20 minutes at +\u0026thinsp;4\u0026deg;C. The isolated aMAT-EVs were recovered from the filtering unit, quantified using the Pierce\u0026trade; BCA protein assay kit (Thermo Fisher Scientific), and promptly stored in small aliquots at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Nanoparticle tracking analysis (NTA)\u003c/h2\u003e \u003cp\u003eNanoparticle tracking analysis (NTA) employs laser light scattering and Brownian motion to assess the size and concentration of EVs. Using the NanoSight NS300 instrument (Malvern, UK) equipped with a 488 nm laser, particle size and distribution were measured. Samples were diluted in filtered PBS to a final volume of one mL. Optimal measurement concentrations were determined by pre-testing the ideal particle per frame value (20\u0026ndash;100 particles/frame). Each measurement involved capturing five 1-minute videos at a temperature of 25\u0026deg;C and a syringe pump speed of 30. The data are presented as averaged finite track length adjustment (FTLA) concentration/size.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Transmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy (TEM) was utilized for imaging. The sEVs were first fixed in a 2% glutaraldehyde solution in phosphate buffer (1:1 ratio), following a previously outlined protocol [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Subsequently, the fixed sEVs were deposited, rinsed, and stained with heavy metal compounds on a gridded slide using standard procedures. Imaging was performed using a TEM Zeiss EM 910 instrument (Zeiss, Oberkochen, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. aMAT-EVs markers analysis\u003c/h2\u003e \u003cp\u003eSuperficial Extracellular Vescicles markers were detected with Exosome-human CD81 Flow Detection Reagent (Thermo Fisher Scientific) as described elsewhere [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. PE-conjugated anti-human CD81 monoclonal antibody or PE-conjugated anti-human CD63 monoclonal antibody (Thermo Fisher Scientific) were used to label exosome captured by beads. Negative control was performed by staining EV resuspension medium (without EVs). Data collection and analysis were performed with Attune\u0026trade; NxT Acoustic Focusing Cytometer (Life Technologies, Carlsbad, California, USA) and Attune NxT Software version 2.5 data, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Proteomic analyses\u003c/h2\u003e \u003cp\u003eProteomic sample preparation and analysis of extracellular vesicles followed established protocols. Protein digestion and clean-up were conducted as described earlier [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The proteomic profiling was performed using an Ultimate 3000 nanoLC system coupled to an Orbitrap Lumos tribrid mass spectrometer, both from Thermo Fisher Scientific. Peptides were trapped using a PepMap trap-cartridge and separated on a C18 reversed-phase column with a 90-minute linear gradient. The mass spectrometry (MS) analysis employed a data-dependent acquisition (DDA) approach, with a mass range of 400\u0026ndash;1500 m/z, using HCD fragmentation at 27 normalized collision energy. The resolution was set to 120,000 for MS1 and 15,000 for MS/MS scans. Peptides with single or unassigned charges were excluded, and a quadrupole isolation width of 1.6 Da was used. Data analysis was performed in Proteome Discoverer v2.5, applying search parameters including trypsin as the enzyme, a maximum of one missed cleavage, and mass tolerances of 10 ppm for precursors and 0.6 Da for fragments. Proteins were identified based on at least one unique peptide, with a false discovery rate (FDR) below 0.1. All analyses were carried out in triplicate. Pathway enrichment of the proteomic data was conducted using the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein identifiers were entered, and the analysis was focused on Homo sapiens, with k-means clustering (k\u0026thinsp;=\u0026thinsp;8) applied to organize functional protein networks. Enriched Gene Ontology (GO) terms for biological processes, cellular components, and molecular functions were prioritized. Visualization and data integration were done using the STRING database and SR plot web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn/srplot\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn/srplot\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Fura-2 AM calcium measurements\u003c/h2\u003e \u003cp\u003eThe intracellular calcium indicator Fura-2 AM was used to ratiometrically evaluate the change of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels upon EV stimulation. Fibroblasts were grown on 24 mm \u0026Oslash; cover glasses and then incubated for 30 min at 37\u0026deg;C in Krebs-Ringer modified buffer (KRB: 125mM NaCl, 5mM KCl, 1mM Na\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1mM MgSO\u003csub\u003e4\u003c/sub\u003e, 5.5mM glucose, and 20mM HEPES, pH 7.4, at 37\u0026deg;C) supplemented with 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e or 100 \u0026micro;M EGTA, supplemented with 2.5 mM Fura-2 AM (Thermo Fisher Scientific), 0.02% Pluronic F-68 (Sigma-Aldrich), and 0.1 mM sulfinpyrazone (Sigma-Aldrich). Cells were washed and the saline was replaced accordingly. Next, the cells were placed in an open Leyden chamber on a 37\u0026deg;C thermostated stage and exposed to 340/380 wavelength light using the Olympus xcellence (Olympus, Shinjuku, Tokyo, Japan) multiple wavelength high-resolution fluorescence microscopy system equipped with a Hamamatsu ORCA ER CCD camera (Hamamatsu Photonics) and a 40x UPLXAPO40XO oil immersion objective (n.a. 1.4) to determine the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e response.\u003c/p\u003e \u003cp\u003eTo test mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e levels, fibroblasts were grown on 24 mm \u0026Oslash; coverslips and transfected with a last generation mitochondrial mt-GCaMP6-encoding plasmid. Calcium Imaging was performed on Olympus excellence multiple wavelength high-resolution fluorescence microscopy system. Cells were alternatively illuminated at 474 and 410 nm and fluorescence was collected through a 515/30‐nm band‐pass filter. Exposure time was set to 200 ms at 474 nm and to 400 ms at 410 nm, to account for the low quantum yield at the latter wavelength. Images were analyzed with ImageJ software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMitochondrial membrane potential (ΔΨm) measurements\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFibroblasts were grown on 24 mm \u0026Oslash; cover glasses and treated for 48 hours with 1 mg/mL MSC-EVs. To measure mitochondrial membrane potential (ΔΨm), fibroblasts were incubated for 30 min at 37\u0026deg;C in KRB/Ca\u003csup\u003e2+\u003c/sup\u003e saline supplemented with 2 nM Tetramethylrhodamine methyl ester (TMRM; Thermo Fisher Scientific) and 10 \u0026micro;M Verapamil (Thermo Fisher Scientific) at 37\u0026deg;C for 30 minutes. After 60 seconds of baseline recording, fibroblasts were stimulated with 1 \u0026micro;M carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP; Thermo Fisher Scientific) to dissipate ΔΨm. Measurements were performed by using the Olympus xcellence workstation equipped with an Olympus 40x UPLXAPO40XO oil immersion objective (n.a. 1.4) and a temperature-controlled stage set at 37\u0026deg;C. Images were analysed with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Cell treatments with inhibitors\u003c/h2\u003e \u003cp\u003eDS16570511, mitochondrial calcium uniporter (MCU) complex inhibitor: fibroblasts were treated with 3 \u0026micro;M DS16570511 (Thermo Fisher Scientific) for 48 hours at 37\u0026deg;C in cell culture medium supplemented with exosome-depleted Fetal Bovine Serum; in case of EV-treated specimens, DS16570511 was added 10 minutes before the EVs.\u003c/p\u003e \u003cp\u003eMCU-i11, MCU complex inhibitor: fibroblasts were treated with 10 \u0026micro;M MCU-i11 (Thermo Fisher Scientific) for 48 hours at 37\u0026deg;C in cell culture medium supplemented with exosome-depleted Fetal Bovine Serum; in case of EV-treated specimens, MCU-i11 was added 10 minutes before the EVs.\u003c/p\u003e \u003cp\u003eRuthenium Red (RR), Transient Receptor Potential (TRP) cation channels inhibitor: fibroblasts were treated with 10 \u0026micro;M Ruthenium Red (Sigma-Aldrich) for 20 minutes at 37\u0026deg;C in KRB/Ca\u003csup\u003e2+\u003c/sup\u003e saline; the protocol by Trollinger and colleagues was adapted [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. \u003cem\u003eIn vitro\u003c/em\u003e Statistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as a means, with an indication of the standard error of the mean (SEM) obtained from at least three independent replicas of the experiment. The significant difference between conditions was determined by analysis of variance (ANOVA) and multiple comparisons by post hoc Bonferroni test with the GraphPad Prism software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Cell cultures and transfection\u003c/h2\u003e \u003cp\u003eEstablished techniques were utilized to create stable Chinese hamster ovary (CHO) cell lines that expressed KOP receptors and stably expressed the Gα\u003csub\u003eqi5\u003c/sub\u003e protein and fibroblast (Sigma) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. These cells were sustained in a culture medium consisting of DMEM and Ham F-12 (mixed at a 1:1 ratio) along with 5% fetal calf serum, penicillin (100 IU/mL), streptomycin (100 \u0026micro;g/ mL), and fungizone (2.5 \u0026micro;g/ mL). Additional supplements of geneticin (G418, 200 \u0026micro;g/ml) and hygromycin B (200 \u0026micro;g/ mL) were added to stock cultures.\u003c/p\u003e \u003cp\u003eFor transfection studies, CHO cells were seeded onto glass coverslips of varying dimensions, depending on the specific assay: 13 mm diameter for aequorin experiments and 24 mm diameter for Fura-2/AM measurements. Prior to transfection, cells were allowed to reach 50% confluence and then subjected to a standard Ca\u003csup\u003e2+\u003c/sup\u003e-phosphate procedure. All experiments were conducted for 36 hours of post-transfection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Aequorin measurements\u003c/h2\u003e \u003cp\u003eAequorin assessments were carried out to gauge mitochondrial calcium concentrations through established methodologies (Pinton P et al, Methods Cell Biol, 2007). Initially, cells were seeded onto 13-mm glass coverslips and allowed to reach 75% confluence. Then, 4 \u0026micro;g of mitochondrial-targeted aequorin was transfected into the cells. After 36 hours of transfection, cells were cultured for 2 hours at 37\u0026deg;C in Krebs\u0026ndash;Ringer modified buffer (KRB) supplemented with 5 \u0026micro;M coelenterazine to activate the aequorin. Following this, measurements were taken using an automated luminometer (MicrobetaJET, PerkinElmer, CA, USA). KRB containing various concentrations of HA was injected, and luminescence was recorded for 60 seconds. To conclude the experiments and normalize the acquired values, a hypotonic solution containing 500 \u0026micro;M digitonin and 50 mM CaCl\u003csub\u003e2\u003c/sub\u003e was introduced to discharge the remaining aequorin. The results were presented as % of probe discharged\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). This computation is possible because aequorin undergoes an irreversible reaction upon binding to three high-affinity sites (EF-hand type), emitting a photon and deactivating the protein (discharged). To translate the aequorin luminescence data into [Ca\u003csup\u003e2+\u003c/sup\u003e] values, a computer algorithm based on the Ca\u003csup\u003e2+\u003c/sup\u003e response curve of aequorins was utilized, as previously outlined [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. All cell cultures were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% carbon dioxide.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. \u003cem\u003eIn vivo\u003c/em\u003e Study Design, population and assessment\u003c/h2\u003e \u003cp\u003e A prospective, single-center, non-comparative study was performed in accordance with the ethical standards of institutional and national legislation and in accordance with the Declaration of Helsinki on human experimentation. All the participants signed informed consent, and the present study was approved by the Ethics Committee of Marche Region (protocol n. 154/2021). TMJ OA was defined according to the American College of Rheumatology criteria for clinical hand OA [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] or as the presence of structural abnormalities. Between 2020 and 2022, patients were consecutively enrolled if they were diagnosed with TMJ OA that not requiring surgical treatment and provided written informed consent.\u003c/p\u003e \u003cp\u003ePatients were consecutively enrolled if they met the following inclusion criteria: (i) age between 45 and 70 years, (ii) diagnosis of TMC osteoarthritis, Eaton-Littler stage II, confirmed radiographically, (iii) persistent pain and functional limitation, (iv) failure of at least 6 months of conservative treatment (including physical therapy and NSAIDs), and (v) ability to provide written informed consent.\u003c/p\u003e \u003cp\u003eExclusion criteria included: 1. Previous intra-articular injection of corticosteroids or hyaluronic acid within the last 6 months, 2. Prior surgery on the affected TMC joint, 3. Uncontrolled systemic diseases (e.g., poorly managed diabetes, active cancer), 4. Autoimmune or inflammatory rheumatic diseases (e.g., rheumatoid arthritis, psoriatic arthritis, lupus erythematous), 5. Neurological or orthopedic comorbidities affecting hand function (e.g., carpal tunnel syndrome, adjacent tendinopathies). Patient demographic characteristics are summarized as follows: The mean age was 59.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4 years; 17 patients were female and 8 male. The mean BMI was 26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 kg/m\u0026sup2;. The most frequent comorbidities included controlled hypertension (32%) and type 2 diabetes (12%). No patients presented with active infection, severe obesity (BMI\u0026thinsp;\u0026gt;\u0026thinsp;35), or recent trauma to the affected hand.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Surgical Procedure to inject the microfragmented adipose tissue\u003c/h2\u003e \u003cp\u003eEach patient received an autologous intra-articular injection of aMAT, processed from their own lipoaspirate under sterile conditions using the Rigenera\u0026reg; system. Before injecting, the skin was sterilely dressed, and the injection of 2\u0026ndash;3 mL of aMAT was performed into TMC joint with a superolateral approach, under fluoroscopic X-ray guidance and using a disposable 20G needle and a 3 mL Luer-Lock Syringe. After application of dressings, a plaster cast was applied for 14 days. Patients were advised not use nonsteroidal anti-inflammatory drugs following the procedure. Otherwise, mild activities and a gradual resumption of daily activity were allowed directly after splint removal. All patients followed the same standardized rehabilitation protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Radiographic assessment\u003c/h2\u003e \u003cp\u003eAnterior\u0026ndash;posterior, lateral and oblique radiographic views of the thumb aid in confirming the diagnosis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Findings consistent with osteoarthritis include narrowing of the joint space (JSN) and osteophytes, in addition to subchondral sclerosis and cysts, which can be used to stage disease using the Eaton and Littler classification [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Postoperative protocol\u003c/h2\u003e \u003cp\u003eAt the end of the surgical procedure and prior to discharge, all patients were evaluated for wound condition and fitted with a custom-made thermoplastic splint immobilizing the first TMC joint. This rigid immobilization was maintained for 2 weeks. Afterwards, patients transitioned to a soft neoprene splint, which was worn during daytime activities for an additional 4 weeks. Rehabilitation began after the initial 2-week immobilization period, at the time of transition to the neoprene splint. The program included passive mobilization exercises followed by active-assisted range of motion and progressive grip strengthening. Rehabilitation was standardized and supervised by certified hand therapists, with two in-clinic sessions per week for 6 weeks, along with a prescribed home exercise regimen. Exercises included thumb circumduction, opposition drills, and resistance training using therapy putty and elastic bands. Patients were instructed to avoid nonsteroidal anti-inflammatory drugs (NSAIDs) for 12 weeks following the procedure to enable an accurate assessment of pain-related outcome measures. Adherence to the rehabilitation protocol was verified at each follow-up visit. Clinical follow-up appointments were scheduled at 6 weeks, 6 months, and 12 months post-intervention. At each visit, patients were systematically assessed for clinical status, functional outcomes, and any potential adverse events.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16. Outcome measures\u003c/h2\u003e \u003cp\u003eOutcomes were evaluated by two different members of the clinical team to minimize assessment bias: preoperative assessments and the surgical procedure were conducted by one surgeon, while postoperative evaluations were performed by a different clinician who was not involved in the treatment phase. Validated clinical tools and objective tests (e.g., MHQ, Kapandji score, dynamometry) were used to ensure reproducibility and minimize subjectivity. Patient-reported outcomes included: (i) The numeric rating score (NRS) for pain has been widely used to evaluate clinical pain intensity [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Joint pain intensity is assessed on a 100-mm score at baseline, after 6 and 12 months of follow up, where 0 indicates no pain and 100 indicates extreme pain; (ii) The Michigan Hand Outcomes Questionnaire (MHQ) is a validated patient-rated questionnaire, which means the patients evaluate their hand health state [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], where the major items include six categories (satisfaction, pain, work, daily living, aesthetics, function). The MHQ score ranged from 0 to 100, with higher scores indicating better hand function. Active flexion and extension of the TMC joint were measured with a goniometer and the total range of motion (ROM) was calculated, together with Kapandji opposition score. Measure of maximum grip strength was done in a standardized sitting position using a Jamar dynamoter for assessment of lateral, tip and palmar pinch test.\u003c/p\u003e \u003cp\u003ePatient safety was systematically assessed at each follow-up visit (6 weeks, 6 months, and 12 months post-procedure). Evaluations included clinical examinations, patient-reported symptoms, and specific screening for potential adverse events such as infection, joint effusion, persistent or worsening pain, and neurological deficits. No adverse events were observed in any patient during the 12-month follow-up period.\u003c/p\u003e \u003cp\u003eThe statistical variables will be described using appropriate summary measures. The comparison between the NRS scale score with respect to the contralateral hand was performed using the Wilcoxon test for paired data, using the patient's last observation at 12 months as the response. Statistical significance was set at a probability level of 5%. For the evaluation of the cut-off capable of discriminating against the functional level of the intervention, Kapandji and MHQ scales were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17. Statistical analysis\u003c/h2\u003e \u003cp\u003eContinuous variables were reported as either means and standard deviation or median and interquartile ranges (IQRs) according to their distribution, as assessed by the Shapiro-Wilk normality test. Categorical variables were reported as absolute frequencies and percentages. Linear Mixed Models (LMM) were performed to estimate the covariates longitudinal effect on strength assessment with Tripod, Key, Tip test, and NRS. Mixed effects regression model uses all available data and can properly account for correlation between repeated measures. The covariates included in the model were group-status (i.e., treated and controlateral groups) and time-points as categorical variable and their interaction. To minimize the risk of type I error due to multiple comparisons, repeated-measures ANOVA models and LMM were followed by Bonferroni or Tukey post hoc correction where appropriate. For groups effect testing, the Tukey post-hoc test was performed. For NRS on activity, Thumb Opposition (i.e., Kapandji score), and MHQ scores, repeated measures ANOVA model was performed with p-value adjusted by Bonferroni approach. All p-values were interpreted in the context of these corrections.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Adipose tissue-EVs characterization \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe adipose tissue derived extracellular vesicles were isolated from the adipose tissue and characterized as described in the \u0026ldquo;Method\u0026rdquo; section. The mean diameter measured by tunable resistive pulse sensing (TRPS) was 113 (SD=51) nm (Fig. 1a), and the mean protein concentration was 1115.37 \u0026plusmn; 229.10 \u0026micro;g/mL (Fig. 1b). A semi-quantitative exosome antibody array was performed to profile the internal and surface proteins in aMAT (Fig. 1c).\u003c/p\u003e\n\u003cp\u003eEVs were positive for programmed cell death 6 interacting protein (ALIX), tumour susceptibility gene 101 (TSG101), and annexin A5 (ANXA5). A weaker signal was detected for intercellular adhesion molecule 1 (ICAM), epithelial cell adhesion molecule (EpCAM), flotillin 1 (FLOT1), CD63, and CD81. MSC-EVs were negative for cis-Golgi matrix protein (GM130), the cellular protein contamination marker. The positivity for established exosomal markers CD63 and CD81 was confirmed by bead-based flow cytometry assay (Fig. 1d,f). MSC-EVs collected using anti-CD63 (Fig. 1D) or anti-CD81 (Fig. 1e) coated magnetic beads were stained with PE-conjugated anti-CD63 or anti-CD81, respectively. In Fig 1f, magnetic beads coated with anti-CD81 were stained with PE-conjugated anti-CD63 to detect double positivity to both markers. TEM images revealed nanovesicles with the cup-shaped morphology typical of exosomes (Fig. 1g). The internalization of MSC-EVs into target cells was demonstrated through fluorescence microscopy. Fluorescent red spots appeared inside fibroblasts after incubation with PKH26-stained MSC-EVs for 48h. In contrast, incubation with negative control did not show this phenomenon (Fig. 1h).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. aMAT -EVs proteomic profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteomic analysis was performed to evaluate the protein cargo of MSC-EVs. 1246 proteins belonging to \u003cem\u003eHomo sapiens\u003c/em\u003e and 10097 predicted protein interactions were identified in the MSC-EV samples through the STRING software (\u003ca href=\"https://string-db.org/\"\u003ehttps://string-db.org/\u003c/a\u003e) (Fig. 2a). Pathway enrichment analysis of the proteomic results was performed to determine protein involvement in the gene ontology domains biological pathway (BP), molecular function (MF) and cellular component (CC) (BP \u0026ge; 0.43, MF \u0026ge; 0.49, CC \u0026ge; 0.55) (Fig. S1a).\u003c/p\u003e\n\u003cp\u003eBP analysis highlighted that the most noteworthy nodes are \u0026ldquo;Chronic inflammatory response\u0026rdquo; (GO:0002544), \u0026ldquo;Epithelial cell-cell adhesion\u0026rdquo; (GO:0090136), \u0026ldquo;Collagen fibril organization\u0026rdquo; (GO:0030199), \u0026ldquo;Extracellular matrix organization\u0026rdquo; (GO:0030198), \u0026ldquo;Regulation of wound healing\u0026rdquo; (GO:0061041). 8 clusters are obtained with the \u0026ldquo;Clusters\u0026rdquo; function and k-means = 8 in STRING The first cluster outnumbers the others and almost coincides in number of nodes (1130 out of 1246 proteins); it is to be noted that 100% of the BPs found via the enrichment analysis are part of the main cluster as showed in Fig. S1 (b, c, d).\u003c/p\u003e\n\u003cp\u003eCC enrichment located most proteins in the collagen domain \u0026ndash; for instance, \u0026ldquo;Collagen type XI trimer\u0026rdquo; (GO:0005592), \u0026ldquo;Fibrillar collagen trimer\u0026rdquo; (GO:0005583) and \u0026ldquo;Complex of collagen trimers\u0026rdquo; (GO:0098644).\u003c/p\u003e\n\u003cp\u003eMF enrichment arranged 61 functions, with the most interesting ones being \u0026ldquo;Structural molecule activity conferring elasticity\u0026rdquo; (GO:0097493), \u0026ldquo;Collagen binding\u0026rdquo; (GO:0005518), \u0026ldquo;Extracellular matrix structural constituent\u0026rdquo; (GO:0005201) and \u0026ldquo;Antioxidant activity\u0026rdquo; (GO:0016209).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Characterization of Ca\u003csup\u003e2+\u003c/sup\u003e signalling triggered by Adipose tissue EVs \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated whether aMAT-EVs elicit alterations in intracellular Ca\u003csup\u003e2+\u003c/sup\u003e fluxes on recipient fibroblasts. The concentration of Ca\u003csup\u003e2+\u003c/sup\u003e was measured in the cytosol of fibroblasts loaded with the ratiometric Ca\u003csup\u003e2+\u003c/sup\u003e indicator Fura-2 AM in a Ca\u003csup\u003e2+\u003c/sup\u003e-enriched KRB saline. Upon EV stimulation, an increase in Fura-2 AM ratio was recorded, indicating that EVs induce a transient elevation in the concentration of Ca\u003csup\u003e2+\u003c/sup\u003e in the cytoplasm ([Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ec\u003c/sub\u003e) (Fig. 2a and 2b). The positive and negative response rates of fibroblasts to EV treatment are 63.4% and 36.6% respectively (Fig. 2c).\u003c/p\u003e\n\u003cp\u003eTo determine whether calcium perturbation depended on intracellular reservoirs or extracellular influxes, we stimulated fibroblasts with MSC-EV in Ca\u003csup\u003e2+\u003c/sup\u003e-free KRB media enriched with the Ca\u003csup\u003e2+\u003c/sup\u003e chelator EGTA. Under these conditions, MSC-EV stimulation led to minimal or no variations in the Fura-2 AM ratio compared to fibroblasts maintained in KRB/Ca\u003csup\u003e2+\u003c/sup\u003e saline (Fig. 2d). The Area Under Curve (AUC) analysis of fibroblasts in KRB/EGTA saline showed a significant reduction compared to those in KRB/Ca\u003csup\u003e2+\u003c/sup\u003e saline (Fig. 2e).\u003c/p\u003e\n\u003cp\u003eVarious channels facilitate rapid Ca\u003csup\u003e2+\u003c/sup\u003e flux from the extracellular milieu. We focused on the Transient Receptor Potential (TRP) ion channels, a family that includes channels permeable to both monovalent ions (e.g., Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e) and divalent ions (e.g., Ca\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e). These channels can act as sensors for a variety of cellular and environmental signals [51]. Notably, certain TRP channel members are known to open in response to different extracellular matrix components [52]. Since our proteomic analysis identified extracellular matrix components within MSC-EV, we speculated that TRP channels might mediate the perturbation of Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis.\u003c/p\u003e\n\u003cp\u003eTo test this hypothesis, we used the low-specificity inhibitor Ruthenium Red (RR) to block TRP channels. Fibroblasts were pretreated with 10 \u0026micro;M RR for 20 minutes before recording Ca\u003csup\u003e2+\u003c/sup\u003e signals [53]. Upon MSC-EV stimulation, fibroblasts exposed to RR showed no perturbation of [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ec\u003c/sub\u003e, unlike cells treated with the vehicle (Fig. 2f). The AUC analysis of fibroblast pretreated with RR showed a significant decrease (Fig. 2g).\u003c/p\u003e\n\u003cp\u003ePerturbations in [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ec\u003c/sub\u003e are often linked to mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake. To verify this hypothesis, fibroblasts were transfected with the ratiometric GFP-based indicator 2mt-GCaMP6s. Upon MSC-EV stimulation, an increase in the 2mt-GCaMP6s ratio was observed, indicating a transient accumulation of Ca\u003csup\u003e2+\u003c/sup\u003e in the mitochondrial matrix (Fig. 2h and 2i).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Characterization of mitochondrial physiology after aMAT-EV treatment \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e perturbations are usually linked to changes in the mitochondrial physiology, including stimulation of tricarboxylic cycle and mitochondrial respiration [54]. We utilized mitochondrial membrane potential (\u0026Delta;\u0026Psi;m) as a readout of mitochondrial respiration after Adipose tissue-EV stimulation. Fibroblasts were treated with MSC-EVs for 48 hours and then incubated with the potentiometric dye TMRM. To ensure that aspecific accumulation of TMRM was not accounted in the measurement, cells were stimulated with the mitochondrial uncoupler FCCP, to allow the release of only mitochondrial TMRM. The difference in signal before and after FCCP administration, accounted as readout of \u0026Delta;\u0026Psi;m, resulted significantly more elevated in EV-treated fibroblasts (Fig. 3a and 3b).\u003c/p\u003e\n\u003cp\u003eThe elevation of \u0026Delta;\u0026Psi;m suggests an elevation of mitochondrial respiration. To confirm this hypothesis, oxygen consumption rate (OCR) was measured in fibroblasts treated with MSC-EVs or vehicles. No effect was observed on basal respiration or respiration related to ATP production (Fig. 3d and 3e), nonetheless the maximal respiration and spare respiratory capacity resulted significantly elevated in cells exposed to EV compared to cells (Fig. 3f, g).\u003c/p\u003e\n\u003cp\u003eTo understand if the alteration of mitochondrial bioenergetics was dependent on mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uptake, fibroblasts were pretreated with two different inhibitors of the mitochondrial calcium uniporter (MCU, DS16570511 and MCU-i11) before exposure to MSC-EV, then \u0026Delta;\u0026Psi;m was measured. We observed that both inhibitors prevented the elevation of \u0026Delta;\u0026Psi;m after 48 hours of exposure to MSC-EV (Fig. 3h, i).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Characterization of Ca\u003csup\u003e2+\u003c/sup\u003e signalling and the mitochondrial physiology in inflamed cells \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtracellular vesicles (EVs) have frequently been associated with the modulation of responses to pro-inflammatory cytokines. Additionally, various perturbations in mitochondrial physiology have been reported as pivotal in the inflammatory response and the propagation of an inflammatory state [55,56]. To mimic an inflammatory condition, fibroblasts were treated with 10 ng/mL TNF\u0026alpha; for 24 hours, after which [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ec\u003c/sub\u003e levels were measured. EV stimulation elicited a transient increase in [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ec\u003c/sub\u003e in vehicle-treated fibroblasts. Interestingly, the Ca\u003csup\u003e2+\u003c/sup\u003e mobilization induced by MSC-EVs was significantly stronger in fibroblasts pretreated with TNF\u0026alpha; (Fig. 4a, c). Exposure to TNF\u0026alpha; induced a reduction in \u0026Delta;\u0026Psi;m in fibroblasts, as confirmed by multiple reports in other cell models [57\u0026ndash;59]. Remarkably, administration of MSC-EVs to TNF\u0026alpha;-treated fibroblasts was able to restore \u0026Delta;\u0026Psi;m to levels comparable to those of untreated fibroblasts (Fig. 4b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. Aequorin measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe aim of this technology is to evaluate whether SF exosomes can reduce pain by affecting nociceptors. It is evident that the selective binding of agonists, such as nociceptin, to their respective receptors leads to a significant increase in mitochondrial calcium levels (Fig. 4d). This effect is notably absent in the negative control group, which lacks agonistic stimulation.\u003c/p\u003e\n\u003cp\u003eFig. 4 Adipose tissue-EV effect of TNF\u0026alpha;-treated fibroblasts. (a) Representative traces of TNF\u0026alpha; inflamed and non-inflamed fibroblasts stimulated with EVs in KRB/Ca2+ saline show an increase in Fura-2 AM ratio, indicating a transient increase in the cytosolic Ca2+ levels; (b)\u0026Delta;\u0026Psi;m of inflamed cells is reduced compared to normal cells. \u0026Delta;\u0026Psi;m of EV-treated fibroblasts is enhanced compared to the untreated condition. EV treatment on inflamed fibroblasts leads to a recovery in the \u0026Delta;\u0026Psi;m; (c) Fura-2 AM ratio quantification as AUC is increased in inflamed fibroblasts; (d) percentage of Calcium Variation. Stable cell lineages of CHO cells bearing KOP receptors underwent transfection with a mitochondria-targeted aequorin sensor, following which they were subjected to various concentrations of hyaluronic acid (HA), as delineated in the illustration. The resultant mitochondrial calcium responses are delineat-ed as a percentage of the mitochondrial calcium response, relative to receptor stimulation with a canonical agonist.\u003c/p\u003e\n\u003cp\u003eDelving further into the experimental investigation, the same cell models were exposed to a substance designated as aMAT-EV and hyaluronic acid, applied in a wide range of concentrations from 0.005 to 5 mg/ mL. Interestingly, low molecular weight hyaluronic acid did not induce any noticeable increase in luminescence. In contrast, high molecular weight hyaluronic acid elicited a significant luminescent response in CHO cells expressing KOP receptors (Fig. 4d). The maximal effect of hyaluronic acid was approximately half that of the robust response induced by dynorphin A, suggesting partial agonism at KOP receptors. Notably, aMAT EV also demonstrated the ability to activate receptors, as clearly observed in the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7. Clinical results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-five participants were enrolled in the study. We enrolled 25 patients with symptomatically and radiographically diagnosed TMC OA joints (Grade II Eaton classification). All patients had unilateral involvement, while patients with bilateral involvement were excluded from the study. The mean age was 56.2 years (range 45\u0026ndash;70 years, SD 8.71). 60% of the participants were male and 40% were male. Eighteen were dominant right hand. Patients were monitored according to our follow-up schedule. No patient reported worsening of symptoms.\u003c/p\u003e\n\u003cp\u003eFig. 5 Radiographic images, procedural steps, hand functionality, and outcome graphs. (a) and (b) show preoperative radiographs of Stage II CMJ osteoarthritis. Panel (c) to (i) depict the procedural workflow, including preparation and injection. (j) and (k) show post-operative follow up (1 year). Panels (l) to (o) present data trends for pinch strength and pain levels over time, comparing affected and contralateral groups. The affected group demonstrates improvements in pinch strength (tripod, key, and tip) and a reduction in pain (NRS), while the contralateral group remains stable across all metrics. Statistical analysis performed using Linear Mixed Models with Tukey\u0026rsquo;s post hoc test for between-group comparisons. Significance was set at p \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003eThere were no postoperative complications, no infections, and no significant adverse reactions or side effects. All patients were assessed at regular intervals (Table 1). Results at 12 months postoperatively were used to assess the efficacy of the procedure (Fig. 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable \u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e Overview of the Study with Preoperative and Postoperative Evaluations\u003c/p\u003e\n\u003ctable\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"236\"\u003e\u003cstrong\u003eAssessments\u003c/strong\u003e\u003c/td\u003e\n\u003ctd width=\"236\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"236\"\u003e\u003cem\u003ePreoperative assessment \u003c/em\u003e- Pain- Range of motion test- Function- Strength test- Opposition testDate for surgery programmed\u003c/td\u003e\n\u003ctd width=\"236\"\u003eNRS at rest and after activityMHQKey, Tripod and Tip pinchKapandji\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"472\"\u003e\u003cem\u003eDate of surgery\u003c/em\u003eApplication of a thermoplastic splint\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"472\"\u003e\u003cem\u003e2 weeks post treatment\u003c/em\u003eApplication of a soft splint\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"472\"\u003e\u003cem\u003e6 weeks post treatment\u003c/em\u003e- Soft splint removedThe exercise routine can now be progressively intensified in order to fully utilize the thumb.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"236\"\u003e\u003cem\u003e6 months post treatment assessment \u003c/em\u003e- Pain- Range of motion test- Function- Strength test- Opposition test\u003c/td\u003e\n\u003ctd width=\"236\"\u003eNRS at rest and after activityMHQKey, Tripod and Tip pinchKapandji\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"236\"\u003e\u003cem\u003e12 months post treatment assessment \u003c/em\u003e- Pain- Range of motion test- Function- Strength test- Opposition test\u003c/td\u003e\n\u003ctd width=\"236\"\u003eNRS at rest and after activityMHQKey, Tripod and Tip pinchKapandji\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.7.1. Strength and NRS activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrength assessed with Tripod, Key and Tip pinch test. Tripod pinch test assessed a significant improvement from 6.152 Kg (SD 0.81) to 8.588 (SD 1.59) after 6 months (p\u0026lt;0.001) and to 9.688 (SD 1.92) after 12 months (p\u0026lt;0.001) (Fig. 6a). Compared to the contralateral healthy hand, tripod pinch test showed no significant differences 6 months (p=0.20) and 12 months (p=0.5) after treatment (Fig. 6b). Key pinch test assessed a significant improvement from 7.02 Kg (SD 1.38) to 8.66 (SD 2) after 6 months (p\u0026lt;0.001) and to 9.28 (SD 1.61) after 12 months (p\u0026lt;0.001) (Fig. 6c). Compared to the contralateral healthy hand, key pinch test showed no significant differences between 6 months (p=0.87) and 12 months (p=0.22) after treatment (Fig. 6d). Tip pinch test assessed a significant improvement from 4.68 Kg (SD 0.63) to 6.06 (SD 0.68) after 6 months (p\u0026lt;0.001) and to 6.99 (SD 0.81) after 12 months (p\u0026lt;0.001) (Fig. 6e). Compared to the contralateral healthy hand, tip pinch test showed no significant differences 6 months (p=0.77) and 12 months (p=0.002) after treatment (Fig. 6f).\u003c/p\u003e\n\u003cp\u003eThe comparative evaluation of strength compared to the contralateral hand is strongly limited and influenced by the patient's dominance, as the dominant hand tends to be naturally stronger and more coordinated than the non-dominant hand.\u003c/p\u003e\n\u003cp\u003eAn improvement in pain both at rest and after activity was seen. NRS at rest preoperatively was 55.16\u0026plusmn;16.02 improving to 34.73\u0026plusmn;15.57 after 6 months (p\u0026lt;0.0001) and 14.73\u0026plusmn;13.46 after 12 months (p\u0026lt;0.0001). NRS on activity preoperatively was 80.78\u0026plusmn;11.4 improving to 63.09\u0026plusmn;12.35 after 6 months (p\u0026lt;0.0001) and 23.47\u0026plusmn;10.89 after 12 months (p\u0026lt;0.0001) (Fig. 6g, h and Fig. 7c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7.2. Thumb Opposition (Kapandji score) and MHQ\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKapandji score improved significantly from 6.56/10 (SD 0.89) preoperatively to 7.12/10 (SD 0.58) at 6 months post treatment and 8.44/10 (SD 0.49) at 12 months post treatment (Fig. 7a).\u003c/p\u003e\n\u003cp\u003eFig. 7 Box plot analyses showing temporal changes in KAPANDJI scores (a) and MHQ scores (b) over a 12-month peri-od. Panel (a) demonstrates significant improvement in KAPANDJI scores (ANOVA, F(1.51, 36.33) = 138.38, p \u0026lt; 0.0001, \u0026eta;\u0026sup2;g = 0.57) with Bonferroni-adjusted pairwise comparisons (****p \u0026lt; 0.0001) between time points. Panel (b) shows significant enhancement in MHQ scores (ANOVA, F(1.35, 32.3) = 268.4, p \u0026lt; 0.0001, \u0026eta;\u0026sup2;g = 0.73) with similar statistical significance between time points. Both metrics demonstrate progressive improvement from base-line (T0) through 6 months (T6) to 12 months (T12). Panel (c) presents a box plot analysis showing significant pain reduction over time (ANOVA, F(2,48) = 993.7, p \u0026lt; 0.0001, \u0026eta;\u0026sup2;g = 0.81) with Bonferroni-adjusted pairwise comparisons (****p \u0026lt; 0.0001) between all time points. The data demonstrate a progressive decrease in pain scores from baseline (T0) through 6 months (T6) to 12 months (T12) post-intervention.\u003c/p\u003e\n\u003cp\u003eCompared to baseline (84.76\u0026plusmn;4.63), MHQ scores were significantly higher at six months (90.86\u0026plusmn;2.94) (p\u0026lt;0.0001) and twelve months (98.14\u0026plusmn;1.97) (p\u0026lt;0.0001), indicating improvement (Fig. 7b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7.3. Postoperative X-rays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients who had an X-ray 12 months postoperatively were still classified at the same Dell stage and therefore did not show any obvious worsening.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eUntil now, research on MSC-derived extracellular vesicles has primarily focused on their molecular content and regenerative potential [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. However, the cellular mechanisms they trigger upon interaction with recipient cells remain largely unexplored. In this study, aMAT-EV were isolated and characterized according to MISEV guidelines. Size analysis using tunable resistive pulse sensing and TEM imaging confirmed their nanometric dimensions and typical cup-shaped morphology. Key exosomal markers (ALIX, TSG101, ANXA5, CD81, and CD63) were detected using immunoblotting and flow cytometry. Proteomic analysis revealed proteins linked to extracellular matrix organization, cell adhesion, collagen fibril formation, wound healing, and inflammation regulation\u0026mdash;hallmarks of regenerative activity [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Although chondrocytes are commonly studied in the context of osteoarthritis, we selected fibroblasts for in vitro investigation based on their active involvement in periarticular remodeling, matrix turnover, and inflammatory signaling within the osteoarthritic joint. Fibroblasts represent a reproducible and well-characterized model and are key contributors to tissue homeostasis and degeneration in the context of thumb carpometacarpal osteoarthritis, where multiple periarticular structures are affected. GO enrichment analysis of EV proteomics indicated the presence of proteins involved in mitochondrial function, extracellular matrix organization, and calcium-dependent signaling cascades. Calcium signaling was selected as a focal pathway based on its central role in mitochondrial regulation, cellular metabolism, and the activation of stress and inflammatory responses. The observed modulation of intracellular Ca\u0026sup2;⁺ dynamics in fibroblasts supports the hypothesis that aMAT-derived EVs may exert their therapeutic effects by restoring cellular homeostasis and dampening pro-inflammatory signaling in target stromal cells.\u003c/p\u003e \u003cp\u003eTo explore their effects on recipient cells, aMAT-EV was shown to be internalized by human dermal fibroblasts within 24 hours, as evidenced by PKH26 staining. This uptake underscores their potential for therapeutic delivery and intercellular communication. The study also revealed that aMAT-EV trigger intracellular calcium signaling in fibroblasts, a critical pathway for cellular responses. Using Fura-2 AM, a significant rise in cytosolic calcium levels was observed upon EV stimulation. Further experiments confirmed that this calcium influx originates from the extracellular space, mediated by transient receptor potential (TRP) channels. Ruthenium Red, a TRP channel inhibitor, effectively blocked the calcium response, highlighting the role of mechanical activation in this process. Mitochondrial calcium uptake, crucial for cellular bioenergetics, was also evaluated. aMAT-EV induced transient calcium accumulation in the mitochondrial matrix, enhancing the mitochondrial membrane potential (ΔΨm) and oxygen consumption during maximal respiration. However, inhibiting the mitochondrial calcium uniporter (MCU) did not affect ΔΨm, suggesting an alternate pathway for calcium entry. Moreover, the therapeutic potential of aMAT-EV was validated in inflamed fibroblasts treated with TNF-α. EV treatment restored ΔΨm to levels comparable to non-inflamed cells, indicating their ability to mitigate inflammation-induced mitochondrial dysfunction. This regenerative effect, likely mediated by their anti-inflammatory properties, highlights aMAT-EV as promising candidates for novel therapies targeting inflammation and oxidative stress-related disorders [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The mitochondrial improvements observed in EV-treated fibroblasts may have functional relevance in the context of trapeziometacarpal osteoarthritis (TMC OA). Fibroblasts in osteoarthritic tissues are known to undergo mitochondrial dysfunction, increased oxidative stress, and adopt a pro-inflammatory secretory phenotype\u0026mdash;collectively contributing to matrix degradation and persistent inflammation. By restoring mitochondrial membrane potential and improving bioenergetic capacity, extracellular vesicles may help reprogram fibroblasts toward a more quiescent and homeostatic state. This process could mitigate senescence-associated secretory activity and the production of catabolic mediators. Prior studies have shown that cellular senescence and mitochondrial dysfunction in fibroblasts are closely linked to OA progression and joint pain, particularly through the accumulation of reactive oxygen species and inflammatory cytokines [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Our findings support the hypothesis that the EV-induced modulation of mitochondrial function may represent a key mechanism in alleviating stromal inflammation and tissue degeneration in TMC OA\u003c/p\u003e \u003cp\u003eThis investigation adds further evidence to the concept that mitochondrial modulation plays a pivotal role in the therapeutic potential of EVs. Enhanced ΔΨm and improved mitochondrial respiration upon aMAT-EV stimulation are consistent with increased cellular bioenergetics, which supports tissue regeneration and repair. Furthermore, the ability of aMAT-EVs to restore mitochondrial function in inflamed fibroblasts suggests their dual action in both repairing metabolic dysfunction and mitigating inflammation. These properties are critical for chronic conditions such as OA, where mitochondrial dysfunction and inflammatory pathways converge to exacerbate joint degeneration.\u003c/p\u003e \u003cp\u003eLastly \u003cem\u003ein vitro\u003c/em\u003e study was conducted to assess the impact of aMAT-EV on opioid receptors (OPr) within an \u003cem\u003ein vitro\u003c/em\u003e model. To analyze this, we used CHO cell lines expressing the Gα\u003csub\u003eqi5\u003c/sub\u003e protein, which couples any G-protein-coupled receptor (GPCR) to an increase in intracellular Ca\u0026sup2;⁺ concentration ([Ca\u0026sup2;⁺]c). The observed Ca\u0026sup2;⁺ responses confirmed that aMAT-EVs selectively activates the KOP receptor as a partial agonist, suggesting that the therapeutic effect may be partially mediated by modulation of nociceptive pathways, resulting in reduced pain perception in patients. \u003cem\u003eIn vitro\u003c/em\u003e results support the efficacy of aMAT-EVs in reducing nociceptive pain through selective KOP receptor activation. Aequorin-based assays were employed, showing a transient increase in mitochondrial Ca\u0026sup2;⁺ concentration ([Ca\u0026sup2;⁺]m), which is at least one order of magnitude higher than cytosolic levels, upon receptor activation. These findings imply that aMAT-EVs may play a role in pain reduction via specific nociceptive pathways [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe implications of these findings extend beyond nociceptive modulation. The dual action of aMAT-EV, combining mitochondrial restoration with KOP receptor activation, introduces a novel paradigm in pain management. Traditional pharmacological interventions for OA and related pain disorders primarily target symptom relief without addressing underlying cellular dysfunctions. The EV-based approach, by contrast, offers a multifaceted mechanism that not only alleviates pain but also promotes cellular homeostasis and repair, which could delay disease progression. Recent studies have demonstrated that mitochondrial function and calcium signaling play a pivotal role in nociceptive modulation. Mitochondrial Ca\u0026sup2;⁺ uptake, ROS production, and cross-talk with the endoplasmic reticulum have been shown to influence neuronal excitability and pain sensitivity in both inflammatory and neuropathic contexts [\u003cspan additionalcitationids=\"CR70\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. These findings provide a mechanistic rationale supporting the analgesic potential of aMAT-derived EVs, which modulate these same pathways \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eClinically, patients treated with aMAT demonstrated significant improvement in pain and functional parameters. The Michigan Hand Outcomes Questionnaire (MHQ) showed a notable improvement (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), particularly in pain relief. NRS scores decreased significantly both at rest and during activity after treatment. At rest, the NRS score dropped from 55.16 to 34.73 after six months and to 14.73 after 12 months (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for both periods), while activity-related pain scores decreased from 80.78 to 63.09 after six months and to 23.47 after 12 months. These improvements indicate substantial pain relief and functional recovery, with a more pronounced impact on activity-related pain.\u003c/p\u003e \u003cp\u003eObjective measures, including pinch strength and Kapandji opposition score, showed significant improvements from baseline to 6 and 12 months. All three pinch strength tests (Tripod, Key, and Tip) improved, suggesting that aMAT was effective in enhancing hand strength. Comparison with the healthy contralateral hand showed no significant differences in Tripod and Key pinch tests at both follow-up periods, while a significant improvement was observed in the Tip pinch test after 12 months. The Kapandji score also showed a statistically significant increase over time, reflecting enhanced thumb opposition and overall hand functionality. Although no radiographic structural improvements were observed over the 12-month follow-up period, it is important to consider that trapeziometacarpal osteoarthritis is typically characterized by a progressive course. Therefore, the absence of radiographic deterioration within this timeframe can be regarded as a positive outcome, especially from the perspective of hand surgeons managing this condition. Moreover, the imaging modality employed\u0026mdash;standard plain radiographs\u0026mdash;may not be sensitive enough to detect subtle tissue-level changes induced by extracellular vesicles. Future studies incorporating advanced imaging techniques such as magnetic resonance imaging (MRI) may provide more detailed insights into the microstructural and microenvironmental effects of aMAT-derived EVs.\u003c/p\u003e \u003cp\u003eInterestingly, these functional improvements align with the observed cellular and molecular changes. The enhanced mitochondrial respiration and bioenergetic state \u003cem\u003ein vitro\u003c/em\u003e could underpin the clinical improvements in grip strength and thumb opposition. Such correlations highlight the translational relevance of the findings, suggesting that aMAT-EVs could provide a mechanistic link between cellular metabolism and clinical recovery.\u003c/p\u003e \u003cp\u003eNo significant radiological changes were observed after one year, although pain reduction was evident, aligning with findings from other studies [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. However, radiography may be limited in assessing early cartilage and synovial microenvironment changes. As shown by Mayoly A et al. [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], pain reduction may correlate more closely with reduced edema in the cartilage and synovium. Future studies employing advanced imaging modalities could provide a clearer view of structural changes associated with pain relief.\u003c/p\u003e \u003cp\u003eAdditionally, exploring advanced imaging techniques such as MRI or positron emission tomography (PET) could shed light on EV-induced microenvironmental changes, such as reductions in synovial inflammation or cartilage repair. Such approaches could validate the observed therapeutic benefits at the molecular and structural levels, further reinforcing the clinical utility of aMAT-EVs.\u003c/p\u003e \u003cp\u003eThe study highlights the potential of microfragmented adipose tissue in managing TMC joint osteoarthritis, aligning with recent advances in regenerative medicine. However, consistent methodologies for adipose tissue processing and controlled, randomized trials will be essential to confirm the efficacy of this treatment in broader patient populations.\u003c/p\u003e"},{"header":"5. Study Limitations and Future Directions","content":"\u003cp\u003eWhile the results of this study are promising, several limitations must be acknowledged. The relatively small sample size (n\u0026thinsp;=\u0026thinsp;25) and single-center design may constrain statistical power and limit generalizability. Nevertheless, the consistent clinical improvements observed across all validated metrics indicate that aMAT-EVs hold substantial therapeutic promise. This study was not designed as a randomized controlled trial (RCT), but rather as an exploratory, single-arm clinical investigation aimed at evaluating the safety and early clinical efficacy of aMAT-EVs. Given the ethical constraints associated with the use of placebo or sham injections in intra-articular procedures, a control group was not included. While the findings are encouraging, future RCTs with appropriate comparators will be required to confirm and generalize these results.\u003c/p\u003e \u003cp\u003eAdditionally, structural changes were assessed exclusively by conventional X-rays, which have limited sensitivity for detecting early tissue-level improvements such as cartilage repair or synovial inflammation. The inclusion of advanced imaging techniques such as MRI or ultrasound in future trials will help to more accurately capture microstructural responses and their correlation with clinical outcomes. Finally, while the \u003cem\u003ein vitro\u003c/em\u003e findings provide mechanistic insights into the action of aMAT-EVs, the translation of these molecular and cellular effects to clinical outcomes remains partially speculative. However, even if this study provides converging evidence from in vitro and in vivo data supporting the therapeutic relevance of aMAT-derived EVs, the precise molecular mechanisms underlying the observed effects remain incompletely delineated. The modulation of mitochondrial dynamics and calcium signaling in fibroblasts aligns with known pathways involved in cellular stress and inflammation; however, direct causal links between these molecular events and clinical improvements were not established. Further mechanistic studies are needed to dissect the contribution of individual EV cargo molecules\u0026mdash;such as specific proteins, miRNAs, or lipids\u0026mdash;and their downstream targets in recipient cells. Approaches such as pathway inhibition, gene knockdown, or selective EV subpopulation profiling could help elucidate the key effectors mediating analgesic and anti-inflammatory actions. Further investigations are needed to delineate the precise pathways through which aMAT-EVs exert their regenerative and anti-nociceptive effects, particularly in inflamed and degenerated tissues.\u003c/p\u003e"},{"header":"6. Conclusion","content":"\u003cp\u003eThis study demonstrates the potential of extracellular vesicles (EVs) derived from microfragmented adipose tissue (aMAT-EVs) as a novel, minimally invasive therapeutic strategy for managing trapeziometacarpal (TMC) osteoarthritis. While several recent clinical studies have reported the therapeutic effects of autologous microfragmented adipose tissue (aMAT) in large-joint osteoarthritis (e.g., knee or hip OA) [\u003cspan additionalcitationids=\"CR75\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e], our study provides novel insights in three key aspects. First, it targets trapeziometacarpal osteoarthritis (TMC OA), a distinct and under-investigated form of OA with unique anatomical and functional characteristics. Second, it combines clinical outcomes with a mechanistic investigation focused on aMAT-derived extracellular vesicles (EVs), which have not been explored in previous trials. Third, we demonstrate the impact of these EVs on mitochondrial function and calcium signaling in fibroblasts, supporting a potential cellular mechanism underlying clinical pain relief. This integrative approach strengthens the translational relevance of the findings and highlights the therapeutic potential of EVs as a future standalone option. Through comprehensive \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e analyses, we have shown that aMAT-EVs possess a unique capacity to modulate inflammation, enhance mitochondrial bioenergetics, and influence nociceptive pathways via selective activation of KOP receptors. Clinically, these effects translate into significant reductions in pain and improvements in hand functionality and strength, as evidenced by decreased NRS scores, increased Kapandji opposition scores, and enhanced pinch strength across multiple measures. Importantly, the absence of radiological progression over 12 months suggests that this approach may offer both symptomatic relief and a protective effect against joint deterioration.\u003c/p\u003e \u003cp\u003eThe findings provide a promising foundation for the development of EV-based therapies, which could address the unmet clinical needs of patients with OA and related musculoskeletal disorders. By targeting both the underlying cellular dysfunctions and the symptomatic burden, aMAT-EVs represent an innovative therapeutic modality with the potential to redefine current treatment paradigms.\u003c/p\u003e \u003cp\u003eWhile the results of this study are promising, several limitations must be acknowledged. First, the sample size of the clinical cohort was relatively small, and the study was conducted at a single center. This may limit the generalizability of the findings to broader and more diverse patient populations. Future multicenter, randomized controlled trials with larger cohorts are necessary to validate these results and establish robust efficacy and safety profiles.\u003c/p\u003e \u003cp\u003eSecond, while significant functional and symptomatic improvements were observed, radiological assessments showed no discernible changes in joint structure over the 12-month period. Advanced imaging modalities such as MRI or PET scans were not employed, which might have provided deeper insights into microenvironmental changes, including cartilage repair or synovial inflammation. Incorporating such techniques in future studies could offer a more comprehensive evaluation of the therapeutic effects of aMAT-EVs.\u003c/p\u003e \u003cp\u003eFinally, while the \u003cem\u003ein vitro\u003c/em\u003e findings provide mechanistic insights into the action of aMAT-EVs, the translation of these molecular and cellular effects to clinical outcomes remains partially speculative. Further investigations are needed to delineate the precise pathways through which aMAT-EVs exert their regenerative and anti-nociceptive effects, particularly in inflamed and degenerated tissues.\u003c/p\u003e \u003cp\u003eFuture research should focus on optimizing EV isolation and characterization methods to enhance reproducibility and scalability for clinical use. Additionally, the long-term effects of aMAT-EV therapy on joint health, as well as its potential applications in other chronic pain and degenerative conditions, warrant exploration. The integration of advanced omics technologies and imaging tools will be critical for unraveling the full therapeutic potential of aMAT-EVs and refining their application in personalized medicine.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eEVs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003eExtracellular vesicles\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eMSDs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003eMusculoskeletal diseases\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eRA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003erheumatoid arthritis\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eOA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003eosteoarthritis\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eLBP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003elow-back pain\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eNP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003eneck pain\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eTMC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003etrapeziometacarpal\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003ePRP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003eplatelet-rich plasma\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eMSCs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003emesenchymal stem cells\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eAD-MSCs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003eadipose-derived MSCs\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eaMAT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003emicrofragmented adipose tissue\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eaMAT-EVs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003eaMAT contained Extracellular Vesicles\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eNTA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003eNanoparticle tracking analysis\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eTEM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003eTransmission electron microscopy\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eMCU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003emitochondrial calcium uniporter\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eMHQ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003eMichigan Hand Outcomes Questionnaire\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eIQRs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003einterquartile ranges\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eOCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"283\"\u003e\n\u003cp\u003eoxygen consumption rate\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthic approvals and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the procedures were approved by Ethics Committee of Marche Region (protocol n. 154/2021).. Informed Patient Consent Statement: Informed consent was obtained from all individual participants included in the study, following the guidelines of the Human Research Approval Committee protocol number 2/2019.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from founding agencies in the public, commercial, or non-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.D.F\u003c/strong\u003e: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing\u0026mdash;original draft, Writing\u0026mdash;review and editing, Visualization. \u003cstrong\u003eL.F\u003c/strong\u003e: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing\u0026mdash;original draft, Writing\u0026mdash;review and editing, Visualization. \u003cstrong\u003eI.Z\u003c/strong\u003e.: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing\u0026mdash;original draft. \u003cstrong\u003eL.S\u003c/strong\u003e.: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing\u0026mdash;review and editing, Visualization. \u003cstrong\u003eA.M.M\u003c/strong\u003e. Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing\u0026mdash;original draft, Writing\u0026mdash;review and editing, Visualization: \u003cstrong\u003eM.P.C\u003c/strong\u003e.: Formal analysis, Investigation, Data curation, Writing\u0026mdash;review and editing. \u003cstrong\u003eM.F\u003c/strong\u003e.: Data curation and statistical analysis. \u003cstrong\u003eL.S\u003c/strong\u003e.: Conceptualization, Methodology, Formal analysis, Investigation. \u003cstrong\u003eE.T\u003c/strong\u003e.: Conceptualization, Methodology, Formal analysis, Investigation. \u003cstrong\u003eI.P.C\u003c/strong\u003e.: Validation, Formal analysis, Investigation, Data curation, Writing\u0026mdash;review and editing. \u003cstrong\u003eE.M.S\u003c/strong\u003e.: Validation, Formal analysis, Investigation, Data curation, Writing\u0026mdash;review and editing. \u003cstrong\u003eM.B\u003c/strong\u003e.: Validation, Formal analysis, Investigation, Data curation, Writing\u0026mdash;review and editing. \u003cstrong\u003eA.R\u003c/strong\u003e: Validation, Data curation, Writing\u0026mdash;review and editing. \u003cstrong\u003eUDA\u003c/strong\u003e: Validation, Data curation, Writing\u0026mdash;review and editing. \u003cstrong\u003eN.D.C.\u003c/strong\u003e: Validation, Data curation, Writing\u0026mdash;review and editing \u003cstrong\u003eL.L\u003c/strong\u003e: Formal analysis, Investigation, Data curation, Writing\u0026mdash;review and editing. \u003cstrong\u003eM.R\u003c/strong\u003e.: Validation, Supervision, Project administration. \u003cstrong\u003eA.M.\u003c/strong\u003e: Writing\u0026mdash;review and editing, Supervision. \u003cstrong\u003eP.P\u003c/strong\u003e.: Writing\u0026mdash;review and editing, Supervision. \u003cstrong\u003eB.Z\u003c/strong\u003e: Writing\u0026mdash;review and editing, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCamussi G. 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Knee Surg Sports Traumatol Arthrosc. 2023;31:3079\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Adipose stem cells, non-enzymatic method, Rigenera protocol, trapeziometacarpal arthritis, microfragmented adipose tissue; extracellular vesicles","lastPublishedDoi":"10.21203/rs.3.rs-6725135/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6725135/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTrapeziometacarpal (TMC) osteoarthritis is a prevalent and debilitating condition that impairs hand functionality and reduces quality of life. Current treatments\u0026mdash;including conservative measures such as splinting and anti-inflammatory medications, as well as surgical interventions\u0026mdash;often exhibit limited efficacy or involve invasive procedures. Novel therapeutic approaches are necessary to address the pain and functional limitations experienced by affected patients.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study investigates the potential of extracellular vesicles (EVs) derived from microfragmented adipose tissue (aMAT) as a minimally invasive treatment for TMC osteoarthritis. EVs were characterized using morphological, proteomic, and functional analyses, revealing their ability to modulate cellular processes through proteins associated with extracellular matrix organization, wound healing, and inflammation regulation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFunctional studies demonstrated that EVs modulate calcium signaling and mitochondrial activity, enhancing cellular bioenergetics and mitigating inflammation-induced dysfunction.\u003c/p\u003e\u003ch2\u003eTrial registration\u003c/h2\u003e \u003cp\u003e This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and adhered to all relevant national and institutional ethical guidelines for research involving human participants. Approval for the study was obtained from the Ethics Committee of Marche Region, protocol n. 154/2021. All participants provided written informed consent before enrollment in the study. They were informed about the study\u0026rsquo;s purpose, procedures, potential risks, and their right to withdraw at any time without consequences. Informed Patient Consent Statement: Informed consent was obtained from all individual participants included in the study, following the guidelines of the Human Research Approval Committee protocol number 2/2019.\u003c/p\u003e","manuscriptTitle":"Mechanism of Action Behind the Pain-Relief Effects of Extracellular Vesicles in Microfragmented Adipose Tissue: An In vitro and In vivo Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 09:38:11","doi":"10.21203/rs.3.rs-6725135/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-06-18T06:30:09+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-18T04:18:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-23T13:54:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2025-05-22T08:51:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a4e1d3a8-0aac-401c-9708-ddae85479e79","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-25T16:31:11+00:00","versionOfRecord":{"articleIdentity":"rs-6725135","link":"https://doi.org/10.1186/s12967-025-06930-4","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2025-08-18 16:12:59","publishedOnDateReadable":"August 18th, 2025"},"versionCreatedAt":"2025-06-25 09:38:11","video":"","vorDoi":"10.1186/s12967-025-06930-4","vorDoiUrl":"https://doi.org/10.1186/s12967-025-06930-4","workflowStages":[]},"version":"v1","identity":"rs-6725135","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6725135","identity":"rs-6725135","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}