Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes.

Cite this article: Bone Joint Res 2024;13(9):462–473.

Macrophages play a critical role in innate immunity by promoting or inhibiting tissue inflammation and repair. Classically, macrophages can differentiate into either pro-inflammatory (M1) or pro-reparative (M2) phenotypes in response to various stimuli. Therefore, this study aimed to address how extracellular vesicles (EVs) derived from polarized macrophages can affect the inflammatory response of tendon cells. For that purpose, human THP-1 cells were stimulated with lipopolysaccharide (LPS), and interleukins -4 and -13 (IL- 4, IL-13), to induce macrophages polarization into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, the EVs were isolated from the culture medium by ultracentrifugation. The impact of these nanovesicles on the inflammation and injury scenarios of human tendon-derived cells (hTDCs), which had previously been stimulated with interleukin- 1 beta (IL-1ß) to mimic an inflammatory scenario, was assessed. We were able to isolate three different nanovesicles populations, showing the typical shape, size and surface markers of EVs. By extensively analyzing the proteomic expression profiles of M1, M2, and M1/M2, distinct proteins that were upregulated in each type of macrophage-derived EVs were identified. Notably, most of the detected pro- inflammatory cytokines and chemokines had higher expression levels in M1-derived EVs and were mostly absent in M2-derived EVs. Hence, by acting as a biological cue, we observed that M2 macrophage-derived EVs increased the expression of the tendon-related marker tenomodulin (TNMD) and tended to reduce the presence of pro-inflammatory markers in hTDCs. Overall, these preliminary results show that EVs derived from polarized macrophages might be a potential tool to modulate the immune system responses becoming a valuable asset in the tendon repair and regeneration fields worthy to be further explored

Back pain is a leading cause of disability worldwide and it is primarily considered to be triggered by intervertebral disc (IVD) degeneration (IVDD). Current treatments may improve pain and mobility, but carry high costs and fail to address IVD repair or regeneration. As no effective therapeutic approach has been proposed to restore inflamed and degenerated IVDs, there is the urgent need to clarify the key pathomechanism of IVDD, the involvement of inflammation, particularly complement activation in matrix catabolism, and how to target them towards tissue repair/regeneration. Mesenchymal stem cell (MSC)-based therapies have become the focus of several regenerative IVD studies. Although patients in clinical trials reported less pain after cell therapy, the long-term success of cell engraftment is unclear due to the hostile IVD environment. The mechanism-of-action of MSCs is mostly dependent on the secreted soluble factors. Moreover, priming of MSC with interleukin (IL)-1β modulates the secretome content, improving its anti-inflammatory and regenerative effect on IVDD organ culture models. MSC-derived extracellular vesicles (EVs) have also been shown to modulate human IVD cells towards a healthy IVD phenotype in vitro. However, the mechanisms involved in the effect of secretome and EVs, particularly with regard to immunomodulation and matrix metabolism, are not fully understood. Our work investigates the effects of secretome and EVs secreted by IL-1β-primed MSCs to impair IVD matrix degradation and/or improve matrix formation in IVDD

Worldwide, tendon disorders are one of the main causes of disability that decrease the quality of life of individuals and represent a substantial economic burden on society. Currently, the main therapies used for tendon injuries are not able to restore tendon functionality, and due to tendons' hypovascular and hypocellular nature, they present a reduced healing capacity, which also limits the success of the available therapies. In order to discover new therapies, extracellular vesicles (EVs), key players in cell-cell communication, have been widely explored for tissue engineering and regenerative medicine applications. Thus, the aim of this study is to assess the role of EVs derived from platelets in stem cell tenogenic commitment using a bioengineered tendon in vitro model for potential use as tendon therapeutic agents. Biomimetic platelet-derived EVs were produced by freeze-thaw cycles of platelets and isolation at different centrifugation speed. To recreate the architecture of tendons, a 3D system consisting of electrospun anisotropic nanofiber scaffolds coated with collagen encapsulating human adipose stem cells (hASCs) and different types of platelet-derived EVs, were produced. Then, the influence of the tendon-mimetic constructs and the distinct EVs populations in the hASCs tenogenic differentiation were assessed over culture time. We observed that the hASCs on the nanofibrous tendon scaffolds, show high cytoskeleton anisotropic organization that is characteristic of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted hASCs tenogenic commitment, supported by the increased gene expression of tendon-related markers (SCX and TNMD). Additionally, EVs enhanced the deposition of tendon like extracellular matrix (ECM), as evidenced by the increased gene expression of ECM-related markers such as COL1, COL3, DCN, TNC, and MMP-3, which are fundamental for ECM synthesis and degradation balance. Moreover, EVs induced lower collagen matrix contraction on hASCs, which has been related with lower myofibroblast differentiation. Overall, the results revealed that EVs are capable of modulating stem cells' behavior boosting their tenogenic commitment, through the increased expression of healthy tendon cell markers, potentiating ECM deposition and decreasing cell contractility. Therefore, platelet EVs are a promising biochemical tool, worthy to be further explored, as paracrine signaling that might potentiate tendon repair and regeneration

Despite promising results in attempting intervertebral disc regeneration, intradiscal cell transplantation is affected by several drawbacks, including poor viability in the harsh disc environment, low cost-effectiveness, and immunogenic/tumorigenic concerns. Recently, the development of cell-free approaches is gaining increasing interest in the field, with a particular regard towards extracellular vesicles (EVs). Nucleus pulposus cell (NPC) progenitors characterized by Tie2 expression have shown a higher chondrogenic differentiation potential compared to MSCs. The aim of this study was to investigate the putative regenerative effects of EVs isolated from Tie2-overexpressing NPC progenitors on degenerative NPCs. NPCs were isolated from young donors and underwent an optimized culture protocol to maximize Tie2 expression (NPCs. Tie2+. ) or a standard protocol (NPCs. STD. ). Following EV characterization, NPC isolated from patients affected by intervertebral disc degeneration (IDD) were treated with either NPCs. Tie2+. -EVs or NPCs. STD. -EVs. Cell proliferation and viability were assessed with the CCK-8 assay. Cell apoptosis and necrosis were evaluated with the Annexin V/PI assay. Cell senescence was investigated with b-galactosidase staining. EV uptake was assessed with PKH26 staining of EVs under confocal microscopy. Treatment with EVs isolated from young NPC donors significantly increased degenerative NPC viability, especially in samples treated with NPCs. Tie2+. -EVs. Likewise, NPCs. Tie2+. -EVs significantly reduced cell senescence and did not show to exert necrotic nor apoptotic effects on recipient cells. Furthermore, EV uptake was successfully observed in all treated cells. NPCs. Tie2+. -EVs demonstrated to significantly enhance degenerative NPC viability, senescence and apoptosis. The use of committed progenitors naturally residing the in the nucleus pulposus may optimize EV regenerative properties and constitute the basis for a new therapy for IDD

Mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) have great promise in the field of orthopaedic nanomedicine due to their regenerative, as well as immunomodulatory and anti-inflammatory properties. Researchers are interested in harnessing these biologically sourced nanovesicles as powerful therapeutic tools with intrinsic bioactivity to help treat various orthopaedic diseases and defects. Recently, a new class of EV mimetics has emerged known as nanoghosts (NGs). These vesicles are derived from the plasma membrane of ghost cells, thus inheriting the surface functionalities and characteristics of the parent cell while at the same time allowing for a more standardized and reproducible production and significantly greater yield when compared to EVs. This study aims to investigate and compare the osteoinductive potential of MSC-EVs and MSC-NGs in vitro as novel tools in the field of bone tissue engineering and nanomedicine. To carry out this investigation, MSC-EVs were isolated from serum-free MSC conditioned media through differential ultracentrifugation. The remaining cells were treated with hypotonic buffer to produce MSC-ghosts that were then homogenized and serially extruded through 400 and 200 nm polycarbonate membranes to form the MSC-NGs. The concentration, size distribution, zeta potential, and protein content of the isolated nanoparticles were assessed. Afterwards, MSCs were treated with either MSC-EVs or MSC-NGs under osteogenic conditions, and their differentiation was assessed through secreted ALP assay, qPCR, and Alizarin Red mineralization staining. Isolation of MSC-EVs and MSC-NGs was successful, with relatively similar mean diameter size and colloidal stability. No effect on MSC viability and metabolic activity was observed with either treatment. Both MSC-EV and MSC-NG groups had enhanced osteogenic outcomes compared to the control; however, a trend was observed that suggests MSC-NGs as better osteoinductive mediators compared to MSC-EVs. Acknowledgements: The authors would like to acknowledge Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, CHRP, and McGill's Faculty of Dental Medicine and Oral Health Sciences for their financial support

Osteoarthritis (OA) is a degenerative disease that lacks regenerative treatment options. Current research focuses on mesenchymal stem cells (MSCs) and Platelet-Rich Plasma (PRP) as regenerative therapies, but extracellular vesicles (EVs) have shown to be more advantageous. This study compares the regenerative potential of human umbilical cord MSC-derived EVs (cEVs) and platelet-derived EVs (pEVs) in ex vivo and in vivo OA models. In the ex vivo study, OA conditions were induced in human cartilage explants, which were then treated either with pEVs or cEVs. Results showed a higher content of DNA and collagen in the pEVs group compared to control and cEVs groups, suggesting that pEVs could be a potential alternative to cEVs. In the in vivo study, an OA model was established in the knee joints of rats through MIA (monoiodoacetate) injection and then treated either with pEVs or cEVs. Results showed that pEVs-treated knee joints had better subchondral bone integrity and greater OA reversion, particularly in female rats, indicating that pEVs are a viable regeneration treatment for OA and outperform cEVs in terms of efficacy. Overall, the study demonstrates the potential of EVs as a regenerative treatment for OA, with pEVs showing promising results in both ex vivo and in vivo models. The use of pEVs in clinical practice could provide a faster path to translation due to the established use of platelet concentrates in therapeutics. However, further studies are needed to fully evaluate the potential of pEVs for OA treatment and to elucidate the mechanisms behind their regenerative effects. Acknowledgments: The authors thank Dr Fernando Hierro (UIB) for their technical contribution with TEM, Mª Trinidad García (UIB) for the access to radioactivity facilities, Aina Arbós (IUNICS) for her contribution in the histology staining, María Tortosa (IdISBa) for her assistance with the animal care and ADEMA School of Dentistry for the access to the cone beam computed tomography (CBCT). Funding: This research was funded by Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (MS16/00124; CP16/00124), PROGRAMA JUNIOR del proyecto TALENT PLUS, construyendo SALUD, generando VALOR (JUNIOR01/18), financed by the sustainable tourism tax of the Balearic Islands; the Direcció General d'Investigació and Conselleria d'Investigació, Govern Balear (FPI/2046/2017); the Mecanisme de Recuperació i Resiliència, intended to execute research projects of «Noves polítiques públiques per a un mercat de treball dinàmic, resilient i inclusiu», collected in Pla de Recuperació, Transformació i Resiliència, financed by European Union-Next Generation EU and driven by SOIB and Conselleria de Fons Europeus, Universitat i Cultura i la Conselleria de Model Econòmic, Turisme i Treball (NG0421) and the grant SYN20/03 from IdISBa

Macrophages (Mφ) are immune cells that play a crucial role in both innate and adaptive immunity as they are involved in a wide range of physiological and pathological processes. Depending on the microenvironment and signals present, Mφ can polarize into either M1 or M2 phenotypes, with M1 macrophages exhibiting pro-inflammatory and cytotoxic effects, while M2 macrophages having immunosuppressive and tissue repair properties. Macrophages have been shown to play key roles in the development and progression or inhibition of various diseases, including cancer. For example, macrophages can stimulate tumor progression by promoting immunosuppression, angiogenesis, invasion, and metastasis. This work aimed to investigate the effect of extracellular vesicles (EVs)-derived from polarized macrophages on an osteosarcoma cell line. Monocytes were extracted from buffy coats and cultured in RPMI medium with platelet lysate or M-CSF. After 6 days of seeding, Mφ were differentiated into M1 and M2 with INF-γ/LPS and IL-4/IL-13, respectively. The medium with M1 or M2 derived EVs was collected and EVs were isolated by differential centrifugation and size exclusion chromatography and its morphology and size were characterized with SEM and NTA, respectively. The presence of typical EVs markers (CD9, CD63) was assessed by Western Blot. Finally, EVs from M1 or M2-polarized Mφ were added onto osteosarcoma cell cultures and their effect on cell viability and cell cycle, proliferation, and gene expression was assessed. The EVs showed the typical shape, size and surface markers of EVs. Overall, we observed that osteosarcoma cells responded differentially to EVs isolated from the M1 and M2-polarized Mφ. In summary, the use of Mφ-derived EVs for the treatment of osteosarcoma and other cancers deserves further study as it could benefit from interesting traits of EVs such as low immunogenicity, nontoxicity, and ability to pass through tissue barriers. Acknowledgements: Carlos III Health Institute and the European Social Fund for contract CP21/00136 and project PI22/01686

Growing evidence has suggested that paracrine mechanisms of Mesenchymal stem cell (MSC) may be involved in the underlying mechanism of MSC after transplantation, and extracellular vesicles (EVs) are an important component of this paracrine role. The aim of this study was to investigate the in vitro osteogenic effects of EVs derived from undifferentiated mesenchymal stem cells and from chemically induced to differentiate into osteogenic cells for 7 days. Further, the osteoinductive potential of EVs for bone regeneration in rat calvarial defects was assessed. We could isolate and characterize EVs from naïve and osteogenic-induced MSCs. Proteomic analysis revealed that EVs contained distinct protein profiles, with Osteo-EVs having more differentially expressed proteins with osteogenic properties. EVs were found to enhance the proliferation and migration of cultured MSC. In addition, the study found that Osteo-EVs/MEM combination scaffolds could enhance greater bone formation after 4 weeks as compared to native MEM loaded with serum-free media. The study suggests that EVs derived from chemically osteogenic-induced MSCs for 7 days can significantly enhance both the osteogenic differentiation activity of cultured hMSCs and the osteoinductivity of MEM scaffolds. The results indicate that Osteo-MSC-secreted nanocarriers-EVs combined with MEM scaffolds can be used for repairing bone defects

Nanovesicle-based therapy is increasingly being pursued as a safe, cell-free strategy to combat various immunological, musculoskeletal and neurodegenerative diseases. Small secreted extracellular vesicles (sEVs) obtained from multipotent mesenchymal stromal cells (MSCs) are of particular interest for therapeutic use since they convey anti-inflammatory, anti-scarring and neuroprotective activities to the recipient cells. Cell-derived vesicles (CDVs) produced by a proprietary extrusion process are surrounded by a lipid bilayer membrane with correct membrane topology, display biological activities similar to MSC-derived EVs and may find specific application for organ-targeted drug delivery systems. Translation of nanovesicle-based therapeutics into clinical application requires quantitative and reproducible analysis of bioactivity and stability, and the potential for GMP-compliant manufacturing. Manufacturing and regulatory considerations as well as preclinical models to support clinical translation will be discussed

Approximately 30% of general practice consultations for musculoskeletal pain are related to tendon disorders, causing substantial personal suffering and enormous related healthcare costs. Treatments are often prone to long rehabilitation times, incomplete functional recovery, and secondary complications following surgical repair. Overall, due to their hypocellular and hypovascular nature, the regenerative capacity of tendons is very poor and intrinsically a disorganized scar tissue with inferior biomechanical properties forms after injury. Therefore, advanced therapeutic modalities need to be developed to enable functional tissue regeneration within a degenerative environment, moving beyond pure mechanical repair and overcoming the natural biological limits of tendon healing. Our recent studies have focused on developing biologically augmented treatment strategies for tendon injuries, aiming at restoring a physiological microenvironment and boosting endogenous tissue repair. Along these lines, we have demonstrated that the local application of mesenchymal stromal cell-derived small extracellular vesicles (sEVs) has the potential to improve rotator cuff tendon repair by modulating local inflammation and reduce fibrotic scarring. In another approach, we investigated if the local delivery of the tendon ECM protein SPARC, which we previously demonstrated to be essential for tendon maturation and tissue homeostasis, has the potential to enhance tendon healing. Finally, I will present results demonstrating the utility of nanoparticle-delivered, chemically modified mRNAs (cmRNA) to improve tendon repair

Within the field of disc degeneration-related low back pain, the spine community has been increasingly acknowledging the regenerative potential of extracellular vesicles (EVs). EVs are small lipid bilayer-delimited particles naturally released by cells, involved in intercellular signaling. They do so by interacting with recipient cells and releasing their biological cargo (e.g., mRNA, miRNA, DNA, protein, lipid). EVs derived from mesenchymal stromal cells and, more recently, also EVs from notochordal cells, the cells residing within the core of the juvenile human disc, are being actively studied. In general, they have been proposed to mitigate inflammation/catabolic processes, reduce apoptosis, stimulate proliferation and even improve the matrix producing capacity of the treated cells. Within this context, appropriate characterization of EVs is essential to increase the level of evidence that the reported effects are indeed EV-associated. To analyze the purity and biochemical composition of EV preparations the International Society for Extracellular Vesicles (ISEV) has prepared guidelines recommending the analysis of multiple (EV) markers, as well as proteins co-isolated/recovered with EVs. Alongside, to prove that the effects are EV-associated and not due to co-isolated factors from the tissue or cells used to derive the EVs, appropriate technical controls need to be taken along (during cell/tissue culture). As such the question arises: “what is the evidence so far?”. While from a fundamental perspective EVs are very appealing, the use of natural EVs in clinical applications is challenging. It comes with drawbacks, including biologic variability, yield, cumbersome isolation, and challenging upscaling and storage to achieve industrial levels. To date there is no FDA-approved EV-based therapy for disc-related lower back pain. Nonetheless, EV-based therapeutic approaches have unique advantages over the use of (pluripotent) stem cell-based therapies, such as a high biologic, but low immunogenic and tumorigenic potential. Acknowledgements: This talk is based on experiences from part of the project NC-CHOICE [no. 19251] of the research talent programme VICI financed by the Dutch Research Council (NWO) and the iPSpine project that receives funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 825925

Mesenchymal stem cells (MSCs) have been studied for the treatment of Osteoarthritis (OA), a potential mechanism of MSC therapies has been attributed to paracrine activity, in which extracellular vesicles (EVs) may play a major role. It is suggested that MSCs from younger donor compete with adult MSC in their EV production capabilities. Therefore, MSCs generated from induced pluripotent mesenchymal stem cells (iMSC) appear to provide a promising source. In this study, MSCs and iMSC during long term-expansion using a serum free clinical grade condition, were characterized for surface expression pattern, proliferation and differentiation capacity, and senescence rate. Culture media were collected continuously during cell expansion, and EVs were isolated. Nanoparticle tracking analysis (NTA), transmission electron microscopy, western blots, and flow cytometry were used to identify EVs. We evaluated the biological effects of MSC and iMSC-derived EVs on human chondrocytes treated with IL-1α, to mimic the OA environment. In both cell types, from early to late passages, the amount of EVs detected by NTA increased significantly, EVs collected during cells expansion, retained tetraspanins (CD9, CD63 and CD81) expression. The anti-inflammatory activity of MSC-EVs was evaluated in vitro using OA chondrocytes, the expression of IL-6, IL-8 and COX-2 was significantly reduced after the treatment with hMSC-derived EVs isolated at early passage. The miRNA content of EVs was also investigated, we identify miRNA that are involved in specific biological function. At the same time, we defined the best culture conditions to maintain iMSC and define the best time window in which to isolate EVs with highest biological activity. In conclusion, a clinical grade serum-free medium was found to be suitable for the isolation and expansion of MSCs and iMSC with increased EVs production for therapeutic applications. Acknowledgments: This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874671

Aims. Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants. Methods. pEVs and cEVs were isolated by size exclusion chromatography (SEC) and physically characterized by nanoparticle tracking analysis (NTA), protein content, and purity. OA conditions were induced in human cartilage explants (10 ng/ml oncostatin M and 2 ng/ml tumour necrosis factor alpha (TNFα)) and treated with 1 × 10. 9. particles of pEVs or cEVs for 14 days. Then, DNA, glycosaminoglycans (GAG), and collagen content were quantified, and a histological study was performed. EV uptake was monitored using PKH26 labelled EVs. Results. Significantly higher content of DNA and collagen was observed for the pEV-treated group compared to control and cEV groups. No differences were found in GAG quantification nor in EVs uptake within any treated group. Conclusion. In conclusion, pEVs showed better performance than cEVs in our in vitro OA model. Although further studies are needed, pEVs are shown as a potential alternative to cEVs for cell-free regenerative medicine. Cite this article: Bone Joint Res 2023;12(10):667–676

Recent studies suggested that both the soluble protein of the mesenchymal stromal cell (MSC) secretome, as well as the secreted extracellular vesicles (EVs) promote bone regeneration. However, there is limited knowledge of the changes in MSC secretome vesicular fraction during aging. We therefore aimed to characterize the release profiles and cargo of EVs from MSCs of different chronological ages. Conditioned medium (CM) was collected from 13 bone marrow MSC strains (20-89 years) and from one MSC strain derived from human induced pluripotent stem cells (iPSCs). The EV-containing fraction was enriched with ultracentrifugation. The number of particles in the CM was evaluated by nanoparticle tracking analysis (NTA), and the number of EVs was evaluated by flow cytometry (FC) after staining with cell-mask-green and anti-CD81 antibody. EV cargo analysis was conducted using next-generation sequencing (NGS). Our data confirmed the release of EVs from all MSC strains used in the study. There were no correlations between the number of particles and the number of EVs released in the CM, and between the number of EVs released and the strain age. Nevertheless, some of the lowest concentrations of EVs were found in the CM of strains over 70 years of age, which exhibited a low/absent chondrogenic and osteogenic differentiation potential. In contrast, iPSC-MSCs, which exhibited a high growth and three-lineage differentiation potential, released a similar amount of EVs as the best performing bone marrow MSC strain. NGS analysis identified several microRNAs that were significantly enriched in EVs of young MSC strains exhibiting low senescence, and those that were enriched in EVs of strains exhibiting high differentiation potentials. Gender had no influence on microRNA profiles in EVs or releasing MSCs. Taken together, our data provides new insights into the properties of MSC vesicular secretome and its therapeutic potential during aging

Joint tissues release extracellular vesicles (EVs) that potentially sustain joint homeostasis and contribute to osteoarthritis (OA) pathogenesis. EVs are putative novel therapeutics for OA, and transport biologically active molecules (including small non-coding RNAs (SNCRNAs)) between cells. This study identified altering SNCRNA cargo in EVs in OA which may act as early diagnostic markers and treatment targets. OA was surgically induced in four skeletally mature Standardbred horses using an osteochondral fragment model in the left middle carpal joint. The right joint underwent sham surgery. Synovial fluid (SF) and plasma were obtained weekly throughout the 70-day study. EVs were isolated using size exclusion chromatography and characterised using nanoparticle tracking (Nanosight), and exosome fluorescence detection and tetraspanin phenotyping (Exoview). RNA was extracted from EVs derived from SF (sham and OA joints) and plasma collected at days 10, 35, 42, 49, 56, 63, and subjected to small RNA sequencing on a NovaSeq SP100 flow cell (Illumina). Nanosight-derived EV characteristics of size and concentration were not significantly different following disease induction. The diameter of the temporal population of plasma and SF-derived exosomes changed significantly for CD9 and CD81 following OA induction with significant temporal, and disease-related changes in CD63 and CD81 protein expressin in plasma and SF. In SF and plasma-derived EVs snoRNAs, snRNAs, tRNAs, lncRNA, y-RNA, piRNAs and scRNA were found. Following pairwise analysis of all-time points we identified 27 miRs DE in plasma and 45 DE miRs in SF. Seven were DE in plasma and SF; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a. In plasma and SF 35 and 21 snoRNAs were DE with four DE in plasma and SF; U3, snord15, snord46, snord58. This work has identified alterations to OA EV sncRNAs in plasma and SF providing a greater understanding of the role of EVs in early OA

Cite this article: Bone Joint Res 2023;12(1):5–8.

In the context of regenerative medicine for the treatment of musculoskeletal pathologies mesenchymal stromal cells (MSCs) have shown good results thanks to secretion of therapeutic factors, both free and conveyed within the extracellular vesicles (EV), which in their totality constitute the “secretome”. The portfolio and biological activity of these molecules can be modulated by both in vitro and in vivo conditions, thus making the analysis of these activities very complex. A deep knowledge of the targets regulated by the secretome has become a matter of fundamental importance and a homogeneous and complete molecular characterization is still lacking in the field of applications for the musculoskeletal system. Therefore, the aim of this work was to characterize the secretome obtained from adipose-derived MSCs (ASCs), and its modulation after pre-conditioning of the ASCs. Pre-conditioning was done by culturing cells in the presence of i) high levels of IFNγ, as proposed for the production of clinical grade secretome with enhanced regenerative potential, ii) low levels of inflammatory stimuli, mimicking conditions found in the osteoarthritis (OA) synovial fluid. Furthermore, EVs ability to migrate within cartilage, chondrocyte and synoviocytes obtained from OA patients was evaluated. The data showed that more than 50 cytokines / chemokines and more than 200 EV-microRNAs are detectable at various intensity levels in ASCs secretomes. The majority of the most abundantly present molecules are involved in the remodelling of the extracellular matrix and in the homeostasis and chemotaxis of inflammatory cells including macrophages, which in OA are often characterized by an M1 inflammatory polarization, promoting their transition to an M2 anti-inflammatory phenotype. Inflammatory priming with IFNγ and synovial fluid-like conditions were able to further increase the ability of the secretome to interact with inflammatory cells and modulate their migration. Finally, the penetration of the EVs in the cartilage explants resulted a rapid process, which begins a few minutes after administration of the EVs that are able to reach a depth of 30-40 μm in 5 hours. The same capacity for interaction was also verified in chondrocytes and synoviocytes isolated from the cartilage and synovial membrane of OA patients. Thanks to the soluble factors and EV-microRNAs, the ASCs secretome has shown a strong propensity to modulate the inflammatory and degenerative processes that characterize OA. The inflammatory pre-conditioning through high concentrations of inflammatory molecules or in conditions similar to the synovial fluid of OA patients was able to increase this capacity by increasing their chemotactic power. The microscopy data also support the hypothesis of the ability of MSC-EVs to influence the chondrocytes residing in the ECM of the cartilage and the synovial cells of the synovial membrane through active interaction and the release of their therapeutic content

Intervertebral disc degeneration (IDD) affects more than 80% of the population all over the world. Current strategies for the treatment of IDD are based on conservative or surgical procedures with the aim of relieving pain. Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy in recent decades, but studies showed that the particularly hostile microenvironment in the intervertebral disc (IVD) can compromise cells survival rate. The use of exosomes, extracellular vesicles released by various cell types, possess considerable economic advantages including low immunogenicity and toxicity. Exosomes allow intercellular communication by conveying functional proteins, RNA, miRNA and lipids between cells. The purpose of this study is to assess the therapeutic effects of exosomes derived from Wharton Jelly mesenchymal stromal cells (WJ-MSC) on human nucleuspulposus cells (hNPC) in an in vitro 3D culture model. Exosomes (exos) were isolated by tangential flow filtration of WJ-MSC conditioned media and characterized by: quantification with BCA test; morphological observation with TEM, surface marker expression by WB and size evaluation by NTA. Confocal microscopy has been used to identify exosomes marked with PKH26 and monitor fusion and/or incorporation in hNPC. hNPC were isolated from waste surgical material from patients undergoing discectomy (n = 5), expanded, encapsulated in alginate beads and treated with: culture medium (control group); WJ-MSC exos (WJ-exos) at different concentrations (10 μg/ml, 50 μg/ml and 100 μg/ml). They were then analysed for: cell proliferation (Trypan Blu); viability (Live/Dead Assay); quantification of nitrites (Griess) and glycosaminoglycans, GAG (DMBB). The hNPC in alginate beads treated for 7 days were included in paraffin and histologically analysed to determine the presence of extracellular matrix (ECM) components. Finally, the expression levels of catabolic and anabolic genes were evaluated through real-time polymerase chain reaction (qPCR). All concentrations of WJ-exos under exam were capable to induce a significant increase in cell proliferation after 10 and 14 days of treatment (p < 0.01 and p < 0.001, respectively). Live/Dead assay showed a decrease in cell death at 50 μg/ml of WJ-exos (p < 0.05). While cellular oxidative stress indicator, nitrite production, was reduced in a dose-dependent way and statistically significant only with 100 μg/ml of WJ-exos (p < 0.05). WJ-exos at 10 and 100 μg/ml induced a significant increase in GAG content (p < 0.05; p < 0.01, respectively) confirmed by Alcian Blu staining. Exos derived from WJ-MSC modulated gene expression levels by increasing expression of ACAN and SOX-9 genes and reducing significantly of IL-6, MMP-1, MMP-13 and ADAMTS-5 levels (p < 0.05; p < 0.01) compared to the control group. Our results supported the potential use of exosomes from WJ-MSC for the treatment of IDD. Exosomes improved hNPC growth, attenuated ECM degradation and reduced oxidative stress and inflammation. This study offers a new scenario in IVD regeneration, promoting the potential use of extracellular vesicles as an alternative strategy to cell therapy

Aim. Periprosthetic joint infections (PJI) are severe complications after total joint arthroplasty (TJA). Up to now, a gold standard in the diagnostics of PJI is missing. Small extracellular vesicles (sEVs) are secreted by all types of cells and play a key role in immune response in presence of infection (1). In this prospective study, the diagnostic accuracy of sEVs in the synovial fluid to detect PJI of knee, hip and shoulder joints was investigated. We hypothesized increased surface markers of sEVs in PJI compared to aseptic complications (e.g. implant loosening, stress shielding related pain). Method. Synovial fluid from 48 patients with painful arthroplasty was examined. The distinction between aseptic and infectious cases was made on the basis of the 2018 Definition of Periprosthetic Hip and Knee Infection (2). 35 (72,9%) probands assigned to aseptic and 13 patients (27,1%) to PJI group. Immuno-fluorescence flow cytometry served to document the concentrations of CD9, CD63, CD66b, CD82 and HLA-DR on sEVs. Results. The concentration of CD9 surface marker on sEVs in synovial fluid was significantly lower (p=0.002) in PJI group than in aseptic group. In contrast, the levels of CD82 on sEVs in synovial fluid was significantly higher (p<0.0001) in the PJI group than in aseptic group. The concentrations of CD63, CD66b and HLA-DR on sEVs in synovial fluid did not differ significantly between the two cohorts (CD63: p=0.372; CD66b: p=0.634; HLA-DR: p=0.558). Conclusions. Overall, the significance of sEVs in the diagnostics of PJI is not well enough understood and the subject of current research and scientific discussion. Our data suggest, that CD82 and CD9 on sEVs in synovial fluid are promising biomarkers to differentiate between PJI and aseptic complications