1. Investigación

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Osteoarthritis (OA) is a degenerative condition of the articular cartilage with chronic low-grade inflammation. Monocytes have a fundamental role in the progression of OA, given their implication in inflammatory responses and their capacity to differentiate into bone-resorbing osteoclasts (OCLs). This observational–experimental study attempted to better understand the molecular pathogenesis of OA through the examination of osteoclast progenitor (OCP) cells from both OA patients and healthy individuals (25 OA patients and healthy samples). The expression of osteoclastogenic and inflammatory genes was analyzed using RT-PCR. The OA monocytes expressed significantly higher levels of CD16, CD115, TLR2, Mincle, Dentin-1, and CCR2 mRNAs. Moreover, a flow cytometry analysis showed a significantly higher surface expression of the CD16 and CD115 receptors in OA vs. healthy monocytes, as well as a difference in the distribution of monocyte subsets. Additionally, the OA monocytes showed a greater osteoclast differentiation capacity and an enhanced response to an inflammatory stimulus. The results of this study demonstrate the existence of significant differences between the OCPs of OA patients and those of healthy subjects. These differences could contribute to a greater understanding of the molecular pathogenesis of OA and to the identification of new biomarkers and potential drug targets for OA.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes progressive joint destruction. Despite the advances in the treatment of this condition there remains a clinical need for safe therapies leading to clinical remission. Mesenchymal stem/stromal cells (MSCs) play immunomodulatory and regenerative roles which can be partly mediated by their secretome. In recent years, the important contribution of extracellular vesicles (EVs) to MSC actions has received an increasing interest as a new therapeutic approach. We provide an extensive overview of the immunomodulatory properties of MSC EVs and their effects on articular cells such as fibroblast-like synoviocytes that play a central role in joint destruction. This review discusses the anti-arthritic effects of MSC EVs in vitro and in animal models of RA as well as their potential mechanisms. Recent preclinical data suggest that transfer of non-coding RNAs by MSC EVs regulates key signaling pathways involved in the pathogenesis of RA. We also examine a number of EV modifications for improving their anti-arthritic efficacy and carrier ability for drug delivery.

The use of mesenchymal stem cells constitutes a promising therapeutic approach, as it has shown beneficial effects in different pathologies. Numerous in vitro, pre-clinical, and, to a lesser extent, clinical trials have been published for osteoarthritis. Osteoarthritis is a type of arthritis that affects diarthritic joints in which the most common and studied effect is cartilage degradation. Nowadays, it is known that osteoarthritis is a disease with a very powerful inflammatory component that affects the subchondral bone and the rest of the tissues that make up the joint. This inflammatory component may induce the differentiation of osteoclasts, the bone-resorbing cells. Subchondral bone degradation has been suggested as a key process in the pathogenesis of osteoarthritis. However, very few published studies directly focus on the activity of mesenchymal stem cells on osteoclasts, contrary to what happens with other cell types of the joint, such as chondrocytes, synoviocytes, and osteoblasts. In this review, we try to gather the published bibliography in relation to the effects of mesenchymal stem cells on osteoclastogenesis. Although we find promising results, we point out the need for further studies that can support mesenchymal stem cells as a therapeutic tool for osteoclasts and their consequences on the osteoarthritic joint.

Objective: The aim of this paper was to study the outcome of patients who underwent cranioplasty with cryopreserved autologous bone after decompressive craniectomy. Methods: Data from 74 patients were retrospectively analyzed. They were divided into groups according to the storage time and the age at cranioplasty. To assess its predictive potential for complication, factors were related to successive stages (preoperative, craniectomy, tissue processing, cranioplasty, and postoperative). Cooling and warming rates applied on bone flap were calculated. The ability to inhibit microbial growth was determined exposing bone fragments to a panel of microorganisms. The concentration of antibiotics eluted from the bone was also determined. A bone explant culture method was used to detect living cells in the thawed cranial bone. Results: Hydrocephalus was significantly more frequent in pediatric patients (26.7%) than in adults (5.1%). The overall rate of bone flap resorption was 21.6% (43.7% of them requiring reoperation). Surgical site infection after cranioplasty was detected in 6.8% of patients. There was no correlation between infection as postoperative complication and previous microbiological positive culture during processing. The etiology of craniectomy did not influence the risk of bone flap contamination. Vancomycin was the only antibiotic detected in the supernatant where the bone was incubated. Outgrowth from bone explants was observed in 36.8% of thawed skulls. An early start of bone flap processing at the tissue bank had a positive effect on cell viability. Conclusion: The outcome after autologous cranioplasty is a multifactorial process, which is modulated by patient-, surgery-, and bone-related factors.

Background: Osteoarthritis (OA) is a joint disease characterized by cartilage degradation, low-grade synovitis and subchondral bone alterations. In the damaged joint, there is a progressive increase of oxidative stress leading to disruption of chondrocyte homeostasis. The modulation of oxidative stress could control the expression of inflammatory and catabolic mediators involved in OA. We have previously demonstrated that extracellular vesicles (EVs) present in the secretome of human mesenchymal stem cells from adipose tissue (AD-MSCs) exert antiinflammatory and anti-catabolic effects in OA chondrocytes. In the current work, we have investigated whether AD-MSC EVs could regulate oxidative stress in OA chondrocytes as well as the possible contribution of peroxiredoxin 6 (Prdx6). Methods: Microvesicles (MV) and exosomes (EX) were isolated from AD-MSC conditioned medium by differential centrifugation with size filtration. The size and concentration of EVs were determined by resistive pulse sensing. OA chondrocytes were isolated from knee articular cartilage of advanced OA patients. 4-Hydroxynonenal adducts, IL-6 and MMP-13 were determined by enzyme-linked immunosorbent assay. Expression of Prdx6 and autophagic markers was assessed by immunofluorescence and Western blotting. Prdx6 was downregulated in AD-MSCs by transfection with a specific siRNA. Results: MV and to a lesser extent EX significantly reduced the production of oxidative stress in OA chondrocytes stimulated with IL-1β. Treatment with MV resulted in a dramatic upregulation of Prdx6. MV also enhanced the expression of autophagy marker LC3B. We downregulated Prdx6 in AD-MSCs by using a specific siRNA and then MV were isolated. These Prdx6-silenced MV failed to modify oxidative stress and the expression of autophagy markers. We also assessed the possible contribution of Prdx6 to the effects of MV on IL-6 and MMP-13 production. The reduction in the levels of both mediators induced by MV was partly reverted after Prdx6 silencing. Conclusion: Our results indicate that EVs from AD-MSCs regulate the production of oxidative stress in OA chondrocytes during inflammation. Prdx6 may mediate the antioxidant and protective effects of MV. The translational potential of this article: This study gives insight into the protective properties of EVs from AD-MSCs in OA chondrocytes. Our findings support the development of novel therapies based on EVs to prevent or treat cartilage degradation.

Adipose tissue represents an abundant source of mesenchymal stem cells (MSC) for therapeutic purposes. Previous studies have demonstrated the anti-inflammatory potential of adipose tissue-derived MSC (ASC). Extracellular vesicles (EV) present in the conditioned medium (CM) have been shown to mediate the cytoprotective effects of human ASC secretome. Nevertheless, the role of EV in the anti-inflammatory effects of mouse-derived ASC is not known. The current study has investigated the influence of mouse-derived ASC CM and its fractions on the response of mouse-derived peritoneal macrophages against lipopolysaccharide (LPS). CM and its soluble fraction reduced the release of pro-inflammatory cytokines, adenosine triphosphate and nitric oxide in stimulated cells. They also enhanced the migration of neutrophils or monocytes, in the absence or presence of LPS, respectively, which is likely related to the presence of chemokines, and reduced the phagocytic response. The anti-inflammatory effect of CM may be dependent on the regulation of toll-like receptor 4 expression and nuclear factor- B activation. Our results demonstrate the antiinflammatory effects of mouse-derived ASC secretome in mouse-derived peritoneal macrophages stimulated with LPS and show that they are not mediated by EV.

In order to study the relationship of the patient’s anxiety level from Corah’s Dental Anxiety Scale (DAS) vs different physiological parameters: pre and post-operative blood pressure, and pre and post-operative heart rates, and subsequently, relate the results to the patient’s post-operative anti-inflammatory analgesic need, 185 patients requiring a simple dental extraction were recruited. They filled out the DAS in the waiting room prior to their procedure and once in the examination room, their preoperative blood pressure and heart rate was measured. Once the dental extraction had been completed, their blood pressure and heart rate were measured again. Before leaving the clinic, the patient was given an analgesic form in which they had to indicate whether or not they had required analgesia after the procedure. Diastolic blood pressure (DBP) showed statistically significant differences between pre-operative and post-operative (P = 0.001). DAS was related with pre-operative diastolic blood pressure (pre-DBP) (P = 0.001) and post-operative diastolic blood pressure (post-DBP) as well as pre-operative heart rate (pre-HR) (P = 0.027) and post-operative heart rate (post-HR) (P = 0.013). Patients with high levels of DAS tend to take more Ibuprofen 400 mg (P = 0.038). The different levels of anxiety will determine what type of anti-inflammatory analgesia the patient will take, if necessary.

Mesenchymal stem/stromal cells (MSCs) represent a promising therapy for musculoskeletal diseases. There is compelling evidence indicating that MSC e ects are mainly mediated by paracrine mechanisms and in particular by the secretion of extracellular vesicles (EVs). Many studies have thus suggested that EVs may be an alternative to cell therapy with MSCs in tissue repair. In this review, we summarize the current understanding of MSC EVs actions in preclinical studies of (1) immune regulation and rheumatoid arthritis, (2) bone repair and bone diseases, (3) cartilage repair and osteoarthritis, (4) intervertebral disk degeneration and (5) skeletal muscle and tendon repair. We also discuss the mechanisms underlying these actions and the perspectives of MSC EVs-based strategies for future treatments of musculoskeletal disorders.