Introduction and Objective. Exosomal miRNA have been shown to regulate many myogenic and osteogenic pathways involved in injury repair and healing. It is also known that rehabilitation and exercise can improve muscle mass and bone growth. The mechanisms by which this occurs in vivo are well studied, but the impact exosomes and their associated miRNA cargo have is unclear. With this knowledge and question in mind, we hypothesized that C2C12 myoblasts subjected to in vitro mechanical stimulus (“exercise”) would exhibit improved exosome production and differentially expressed miRNA cargo when compared to their static (“unexercised”) counterparts. Materials and Methods. C2C12 myoblasts were cultured using the FlexCell FX-5000TT bioreactor. Two exercise regimens were programmed: 1) low intensity regimen (LIR) (0–15% strain at 0.5 Hz for 24 hours) 2) high intensity interval regimen (HIIR) (12–22% strain at 1 Hz for 10 minutes followed by 50 minutes of rest repeated for 24 hours). Unexercised (static) cells were cultured in parallel. Exosomes were isolated using the Invitrogen Total Exosome Isolation Reagent. The Pierce BCA Protein Assay, System Bioscience's ExoELISA-ULTRA CD81 Kit and, SBI's ExoFlow-ONE EV labeling kit were used to confirm and quantify exosome number and protein concentration. The SBI Exo-NGS service was used to perform miRNA sequencing on isolated exosomes. Results. All exercise regimens resulted in increased exosome concentrations as determined by CD81 exosome ELISA and flow-cytometry based exosome quantification. The LIR interestingly produced significantly more exosomes than static and HIIR. Within the exosomes from mechanically stimulated cells, 35 miRNAs were found to be differentially expressed when compared to exosomes from unexercised cells. Interestingly, this significance was only found within exosomes from the HIIR group. Specifically, upon investigation 8 of these miRNAs were found to be involved in myogenic and osteogenic proliferation and differentiation. These results correlate with our previous findings that exosomes from exercised cells improve the proliferation and myogenic differentiation of C2C12 myoblasts. Conclusions. Our results indicate that exercise can be optimized to improve the production and regenerative capacity of exosomes. These results also indicate that exosomes may be intimately involved in systemic health and repair during rehabilitation and exercise. To examine these results in vivo, mouse studies using a crush injury model and exosomes from mechanically stimulated cells are currently planned

MicroRNAs (miRNAs) are a class of small non-coding RNAs that have emerged as potential predictive, prognostic, and therapeutic biomarkers, relevant to many pathophysiological conditions including limb immobilization, osteoarthritis, sarcopenia, and cachexia. Impaired musculoskeletal homeostasis leads to distinct muscle atrophies. Understanding miRNA involvement in the molecular mechanisms underpinning conditions such as muscle wasting may be critical to developing new strategies to improve patient management. MicroRNAs are powerful post-transcriptional regulators of gene expression in muscle and, importantly, are also detectable in the circulation. MicroRNAs are established modulators of muscle satellite stem cell activation, proliferation, and differentiation, however, there have been limited human studies that investigate miRNAs in muscle wasting. This narrative review summarizes the current knowledge as to the role of miRNAs in the skeletal muscle differentiation and atrophy, synthesizing the findings of published data. Cite this article: Bone Joint Res 2020;9(11):798–807

Aims. Current diagnostic tools are not always able to effectively identify periprosthetic joint infections (PJIs). Recent studies suggest that circulating microRNAs (miRNAs) undergo changes under pathological conditions such as infection. The aim of this study was to analyze miRNA expression in hip arthroplasty PJI patients. Methods. This was a prospective pilot study, including 24 patients divided into three groups, with eight patients each undergoing revision of their hip arthroplasty due to aseptic reasons, and low- and high-grade PJI, respectively. The number of intraoperative samples and the incidence of positive cultures were recorded for each patient. Additionally, venous blood samples and periarticular tissue samples were collected from each patient to determine miRNA expressions between the groups. MiRNA screening was performed by small RNA-sequencing using the miRNA next generation sequencing (NGS) discovery (miND) pipeline. Results. Overall, several miRNAs in plasma and tissue were identified to be progressively deregulated according to ongoing PJI. When comparing the plasma samples, patients with a high-grade infection showed significantly higher expression levels for hsa-miR-21-3p, hsa-miR-1290, and hsa-miR-4488, and lower expression levels for hsa-miR-130a-3p and hsa-miR-451a compared to the aseptic group. Furthermore, the high-grade group showed a significantly higher regulated expression level of hsa-miR-1260a and lower expression levels for hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-148b-5p, hsa-miR-301a-3p, hsa-miR-451a, and hsa-miR-454-3p compared to the low-grade group. No significant differences were found between the low-grade and aseptic groups. When comparing the tissue samples, the high-grade group showed significantly higher expression levels for 23 different miRNAs and lower expression levels for hsa-miR-2110 and hsa-miR-3200-3p compared to the aseptic group. No significant differences were found in miRNA expression between the high- and low-grade groups, as well as between the low-grade and aseptic groups. Conclusion. With this prospective pilot study, we were able to identify a circulating miRNA signature correlating with high-grade PJI compared to aseptic patients undergoing hip arthroplasty revision. Our data contribute to establishing miRNA signatures as potential novel diagnostic and prognostic biomarkers for PJI. Cite this article: Bone Jt Open 2024;5(6):479–488

Objectives. Diabetes mellitus (DM) is known to impair fracture healing. Increasing evidence suggests that some microRNA (miRNA) is involved in the pathophysiology of diabetes and its complications. We hypothesized that the functions of miRNA and changes to their patterns of expression may be implicated in the pathogenesis of impaired fracture healing in DM. Methods. Closed transverse fractures were created in the femurs of 116 rats, with half assigned to the DM group and half assigned to the control group. Rats with DM were induced by a single intraperitoneal injection of streptozotocin. At post-fracture days five, seven, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture days five and 11. For further analysis, real-time polymerase chain reaction (PCR) analysis was performed at each timepoint. Results. Microarray analysis showed that there were 14 miRNAs at day five and 17 miRNAs at day 11, with a greater than twofold change in the DM group compared with the control group. Among these types of miRNA, five were selected based on a comparative and extended literature review. Real-time PCR analysis revealed that five types of miRNA (miR-140-3p, miR-140-5p, miR-181a-1-3p, miR-210-3p, and miR-222-3p) were differentially expressed with changing patterns of expression during fracture healing in diabetic rats compared with controls. Conclusions. Our findings provide information to further understand the pathology of impaired fracture healing in a diabetic rat model. These results may allow the potential development of molecular therapy using miRNA for the treatment of impaired fracture healing in patients with DM. Cite this article: S. Takahara, S. Y. Lee, T. Iwakura, K. Oe, T. Fukui, E. Okumachi, T. Waki, M. Arakura, Y. Sakai, K. Nishida, R. Kuroda, T. Niikura. Altered expression of microRNA during fracture healing in diabetic rats. Bone Joint Res 2018;7:139–147. DOI: 10.1302/2046-3758.72.BJR-2017-0082.R1

Bone regeneration is pivotal for the healing of fractures. In case this process is disturbed a non-union can occur. This can be induced by environmental factors such as smoking, overloading etc. Co-morbidities such as diabetes, osteoporosis etc. may be more intrinsic factors besides other disturbances in the process. Those pathways negatively influence the bone regeneration process. Several intrinsic signal transduction pathways (WNT, BMP etc.) can be affected. Furthermore, on the transcriptional level, important mRNA expression can be obstructed by deregulated miRNA levels. For instance, several miRNAs have been shown to be upregulated during osteoporotic fractures. They are detrimental for osteogenesis as they block bone formation and accelerate bone resorption. Modulating those miRNAs may revert the physiological homeostasis. Indeed, physiological fracture healing has a typical miRNA signature. Besides using molecular pathways for possible treatment of non-union fractures, providing osteogenic cells is another solution. In 5 clinical cases with non-union fractures with defects larger than 10 cm, successful administration of a 3D printed PCL-TCP scaffold with autologous bone marrow aspirate concentrate and a modulator of the pathogenetic pathway has been achieved. All patients recovered well and showed a complete union of their fractures within one year after start of the regenerative treatment. Thus, non-union fractures are a diverse entity. Nevertheless, there seem to be common pathogenetic disturbances. Those can be counteracted at several levels from molecular to cell. Compositions of those may be the best option for future therapies. They can also be used in a more personalized fashion in case more specific measurements such as miRNA signature and stem cell activity are applied

Aims. To investigate the effects of senescent osteocytes on bone homeostasis in the progress of age-related osteoporosis and explore the underlying mechanism. Methods. In a series of in vitro experiments, we used tert-Butyl hydroperoxide (TBHP) to induce senescence of MLO-Y4 cells successfully, and collected conditioned medium (CM) and senescent MLO-Y4 cell-derived exosomes, which were then applied to MC3T3-E1 cells, separately, to evaluate their effects on osteogenic differentiation. Furthermore, we identified differentially expressed microRNAs (miRNAs) between exosomes from senescent and normal MLO-Y4 cells by high-throughput RNA sequencing. Based on the key miRNAs that were discovered, the underlying mechanism by which senescent osteocytes regulate osteogenic differentiation was explored. Lastly, in the in vivo experiments, the effects of senescent MLO-Y4 cell-derived exosomes on age-related bone loss were evaluated in male SAMP6 mice, which excluded the effects of oestrogen, and the underlying mechanism was confirmed. Results. The CM and exosomes collected from senescent MLO-Y4 cells inhibited osteogenic differentiation of MC3T3-E1 cells. RNA sequencing detected significantly lower expression of miR-494-3p in senescent MLO-Y4 cell-derived exosomes compared with normal exosomes. The upregulation of exosomal miR-494-3p by miRNA mimics attenuated the effects of senescent MLO-Y4 cell-derived exosomes on osteogenic differentiation. Luciferase reporter assay demonstrated that miR-494-3p targeted phosphatase and tensin homolog (PTEN), which is a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overexpression of PTEN or inhibition of the PI3K/AKT pathway blocked the functions of exosomal miR-494-3p. In SAMP6 mice, senescent MLO-Y4 cell-derived exosomes accelerated bone loss, which was rescued by upregulation of exosomal miR-494-3p. Conclusion. Reduced expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenic differentiation and accelerates age-related bone loss via PTEN/PI3K/AKT pathway. Cite this article: Bone Joint Res 2024;13(2):52–65

Bone tissue is known to possess an intrinsic regeneration potential. However, in cases of major injury, trauma, and disease, bone loss is present, and the regeneration potential of the tissue is often impaired. The process of bone regeneration relies on a complex interaction of molecules. MicroRNAs (miRNA) are small, non-coding RNAs that inhibit messenger RNAs (mRNA). One miRNA can inhibit several mRNAs and one mRNA can be inhibited by several miRNAs. Functionally, miRNAs regulate the entire proteome via the local inhibition of translation. In fact, miRNA modulation has been shown to be involved in several musculoskeletal diseases. 1. In those pathologies, they modulate the transcriptional activity of mRNAs important for differentiation, tissue-specific activity, extracellular matrix production, etc. Because of their function in inhibiting translation, miRNAs are being researched in many diseases and are already being used for interventional treatment. 2. Bone tissue and its related conditions have been widely investigated up to this day. 1,3. This talk will focus on the relevancy of miRNAs to bone tissue, its homeostasis, and disease. After, examples will be given of how miRNAs can be used in bone regeneration and diseases such as osteoporosis and osteosarcoma. The use of miRNAs in both, detection and therapy will be discussed

Hip and knee arthroplasty (HKA) are two of the most successful orthopaedic procedures. However, one major complication necessitating revision surgery is osteolysis causing aseptic loosening of the prosthesis. JAK-STAT has been demonstrated to influence bone metabolism and can be regulated by microRNA (miRNA). Adult patients with osteolysis or aseptic loosening undergoing revision HKA were recruited. Age and gender matched patients undergoing primary hip or knee arthroplasty were our controls. Samples of bone, tissue and blood were collected and RNA isolation was performed. The best quality samples were used for RNA-sequencing. Data analysis was performed using RStudio and Galaxy to identify differentially expressed genes. Western blotting of IL6 was used to confirm protein expression. Five circulating miRNA were identified which had 10 differentially expressed genes in bone and 11 differentially expressed genes in tissue related to the JAK-STAT pathway. IL6 in bone and EpoR in bone were highly significant and IL6 in tissue, MPL in bone, SOCS3 in tissue, JAK3 in bone and SPRED1 in bone were borderline significant. Western blot results demonstrated up-expression of IL6 in bone tissue of revision patients. Periprosthetic osteolysis and aseptic loosening can be attributed to miRNA regulation of the JAK-STAT pathway in osteoblasts and osteoclasts, leading to increased bone resorption. These findings can be used for further experiments to determine utility in the clinical setting for identifying diagnostic markers or therapeutic targets

Tendons and tendon-to-bone entheses don't usually regenerate after injury, and the hierarchical organization of such tissues makes them challenging sites of study for tissue engineers. In this study, we have tried a novel approach using miRNA and a bioactive bioink to stimulate the regeneration of the enthesis. microRNAs (miRNAs) are short, non-coding sequences of RNA that act as post-transcriptional regulators of gene and protein expression [1]. Mimics or inhibitors of specific miRNAs can be used to restore lost functions at the cell level or improve healing at the tissue level [2,3]. We characterized the healing of a rat patellar enthesis and found that miRNA-16-5p was upregulated in the fibrotic portion of the injured tissue 10 days after the injury. Based on the reported interactions of miRNA-16-5p with the TGF-β pathway via targeting of SMAD3, we aimed to explore the effects of miRNA-16-5p mimics on the tenogenic differentiation of adipose-derived stem cells (ASCs) encapsulated in a bioactive bioink [4,5]. Bioinks with different properties are used for the 3D printing of biomimetic constructs. By integrating cells, materials, and bioactive molecules it is possible to tailor the regenerative capacity of the ink to meet the particular requirements of the tissue to engineer [5]. Here we have encapsulated ASCs in a gelatin-methacryloyl (GelMa) bioink that incorporates miR-16-5p mimics and magnetically responsive microfibers (MRFs). When the bioink is crosslinked in the presence of a magnetic field, the MRFs align unidirectionally to create an anisotropic construct with the ability to promote the tenogenic differentiation of the encapsulated ASCs. Additionally, the obtained GelMA hydrogels retained the encapsulated miRNA probes, which permitted the effective 3D transfection of the ASC and therefore, the regulation of gene expression, allowing to investigate the effects of the miR-16-5p mimics on the tenogenic differentiation of the ASCs in a biomimetic scenario

Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network. hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting. Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics. We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases

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

Introduction and Objective. Intervertebral disc (IVD) degeneration is one of the major contributors to low back pain, the leading cause of disability worldwide. This multifactorial pathological process involves the degradation of the extracellular matrix, inflammation, and cell loss due to apoptosis and senescence. While the deterioration of the extracellular matrix and cell loss lead to structural collapse of the IVD, increased levels of inflammation result in innervation and the development of pain. Amongst the known regulators of inflammation, toll-like receptors (TLRs) and more specifically TLR-2 have been shown to be specifically relevant in IVD degeneration. As strong post-transcriptional regulators, microRNAs (miRNAs) and their dysregulation has been connected to multiple pathologies, including degenerative diseases such as osteoarthritis and IVD degeneration. However, the role of miRNAs in TLR signalling in the IVD is still poorly understood and was hence investigated in this study. Materials and Methods. Human Nucleus pulposus (hNP) and Annulus fibrosus (hAF) cells (n=5) were treated with the TLR-2/6 specific agonist PAM2CSK4 (100 ng/mL for 6 hours) in order to activate the TLR2 signalling pathway. After the activation both miRNA and mRNA were isolated, followed by next-generation sequencing and qPCR analysis of proinflammatory cytokines respectively. Furthermore, cell supernatants were used to analyze the secretion of proinflammatory cytokines with enzyme-linked immunosorbent assay. TLR-2 knockdown (siRNA) cells were used as a control. Statistical analysis was conducted by performing Kolmogorov-Smirnov test and a two-tailed Student's t-test using GraphPad Prism version 9.0.2 for Windows (GraphPad Software, La Jolla California USA). Results. TLR-2 activation resulted in the induction of an inflammatory cell response, with a significant increase in gene expression of interleukin (IL)-6 (525 ± 180 fold change, p < 0.05) and IL-8 (7513 ± 1907 fold change, p < 0.05) and protein secretion of IL-6 (30.5 ± 8.1 pg/mL) and IL-8 (28.9 ± 5.4 pg/mL). TLR-2 activation was furthermore associated with changes in the miRNA profile of hNP and hAF cells. Specifically, we identified 10 differentially expressed miRNAs in response to TLR-2 activation, amongst which were miR-335–3p (1.45 log2 FC, p < 0.05), miR-125b-1–3p (0.55 log2 FC, p < 0.05), and miR-181a-3p (−1.05 log2 FC, p < 0.05). Conclusions. The identified miRNAs are known to be associated with osteoarthritis (miR-335-3p), inflammation and IVD degeneration (mir-125-1-3p and miR-181a-3p), but the link to TLR signalling has not been previously reported. Experiments to validate the identified miRNAs and elucidate their functional role are undergoing. The identification of these miRNAs provides an opportunity to further investigate miRNAs in the context of TLR activation and inflammation and to enhance our understanding of underlying molecular mechanisms behind disc degeneration, inflammation, and TLR dysregulation

Chronic inflammatory events have been associated to almost every chronic disease, including cardiovascular-, neurodegenerative- and autoimmune- diseases, cancer, and host-implant rejection. Given the toll of chronic inflammation in healthcare and socioeconomical costs developing strategies to resolve and control chronic states of inflammation remain a priority for the significant benefit of patients. Macrophages (Mφ) hold a central role both in the initiation and resolution of inflammatory events, assuming different functional profiles. The outstanding features of Mφ counting with the easy access to tissues, and the extended networking make Mφ excellent candidates for precision therapy. Moreover, sophisticated macrophage-oriented systems could offer innovative immune-regulatory alternatives to effectively regulate chronic environments that traditional pharmacological agents cannot provide. We propose magnetically assisted systems for balancing Mφ functions at the injury site. This platform combines polymers, inflammatory miRNA antagonists and magnetically responsive nanoparticles to stimulate Mφ functions towards pro-regenerative phenotypes. Strategies with magnetically assisted systems include contactless presentation of immune-modulatory molecules, cell internalization of regulatory agents for functional programming via magnetofection, and multiple payload delivery and release. Overall, Mφ-oriented systems stimulated pro-regenerative functions of Mφ supporting magnetically assisted theranostic nanoplatforms for precision therapies, envisioning safer and more effective control over the distribution of sensitive nanotherapeutics for the treatments of chronical inflammatory conditions. Acknowledgements: ERC CoG MagTendon No.772817; FCT Doctoral Grant SFRD/BD/144816/2019, and TERM. RES Hub (Norte-01-0145-FEDER-022190)

Osteoarthritis (OA) of the equine distal interphalangeal joint (DIPJ) is a common cause of lameness. MicroRNAs (miRNAs) from biofluids such as plasma and synovial fluid make promising biomarker and therapeutic candidates. The objectives of this study are (1) Identify differentially expressed (DE) miRNAs in mild and severe equine DIPJ OA synovial fluid samples and (2) Determine the effects of DE miRNAs on equine chondrocytes in monolayer culture. Synovial fluid samples from five horses with mild and twelve horses with severe DIPJ OA were submitted for RNA-sequencing; OA diagnosis was made from MRI T2 mapping, macroscopic and histological evaluation. Transfection of equine chondrocytes (n=3) was performed using the Lipofectamine® RNAiMAX system with a negative control and a miR-92a mimic and inhibitor. qPCR was used to quantify target mRNA genes. RNA-seq showed two miRNAs (miR-16 and miR-92a) were significantly DE (p<0.05). Ingenuity Pathway Analysis (IPA) identified important downstream targets of miR-92a involved in the pathogenesis of osteoarthritis and so this miRNA was used to transfect equine chondrocytes from three donor horses diagnosed with OA. Transfection was successfully demonstrated by a 1000-20000 fold increase in miR-92a expression in the equine chondrocytes. There was a significant (p<0.05) increase in COMP, COL3A1 and Sox9 in the miR-92a mimic treatment and there was no difference in ADAMTS-5 expression between the miR-92 mimic and inhibitor treatment. RNA-seq demonstrated miR-92a was downregulated in severe OA synovial fluid samples which has not previously been reported in horses, however miR-92a is known to play a role in the pathogenesis of OA in other species. Over expression of miR-92a in equine chondrocytes led to significantly increased COMP and Sox9 expression, consistent with a chondrogenic phenotype which has been identified in human and murine chondrocytes

Osteoclasts (OCs) are multinucleated cells that play a pivotal role in skeletal development and bone remodeling. Abnormal activation of OCs contributes to the development of bone-related diseases, such as osteoporosis, bone metastasis and osteoarthritis. Restoring the normal function of OCs is crucial for bone homeostasis. Recently, RNA therapeutics emerged as a new field of research for osteoarticular diseases. The aim of this study is to use non-coding RNAs (ncRNAs) to molecularly engineer OCs and modulate their function. Specifically, we investigated the role of the microRNAs (namely miR-16) and long ncRNAs (namely DLEU1) in OCs differentiation and fusion. DLEU1/DLEU2 region, located at chromosome 13q14, also encodes miR-15 and miR-16. Our results show that levels of these ncRNA transcripts are differently expressed at distinct stages of the OCs differentiation. Specifically, silencing of DLEU1 by small interfering RNAs (siDLEU1) and overexpression of miR-16 by synthetic miRNA mimics (miR-16-mimics) led to a significant reduction in the number of OCs formed per field (OC/field), both at day 5 and 9 of the differentiation stage. Importantly, time-lapse analysis, used to track OCs behavior, revealed a significant decrease in fusion events after transfection with siDLEU1 or miR-16-mimics and an alteration in the fusion mode and partners. Next, we investigated the migration profile of these OCs, and the results show that only miR-16-mimics-OCs, but not siDLEU-OCs, have a lower percentage of immobile cells and an increase in cells with mobile regime, compared with controls. No differences in cell shape were found. Moreover, mass-spectrometry quantitative proteomic analysis revealed independent effects of siDLEU1 and miR-16-mimics at the protein levels. Importantly, DLEU1 and miR-16 act by distinct processes and pathways. Collectively, our findings support the ncRNAs DLEU1 and miR-16 as therapeutic targets to modulate early stages of OCs differentiation and, consequently, to impair OC fusion, advancing ncRNA-therapeutics for bone-related diseases. Acknowledgements: Authors would like to thank to AO CMF / AO Foundation (AOCMFS-21-23A). SRM and MIA are supported by FCT (SFRH/BD/147229/2019 and BiotechHealth Program; CEECINST/00091/2018/CP1500/CT0011, respectively)

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

Summary Statement. Using the latest Next Generation Sequencing technologies, we have investigated miRNA expression profiles in human trabecular bone from total hip replacement (THR) revision surgery where wear particle associated osteolysis was evident. Introduction. A major problem in orthopaedic surgery is aseptic loosening of prosthetic implants caused by wear particle associated osteolysis. Wear debris is known to impact on a variety of cellular responses and genes in multiple pathways associated with the development of the periprosthetic osteolysis. MicroRNAs (miRNAs) act as negative regulators of gene expression and the importance of miRNAs in joint pathologies has only recently been addressed. However, miRNA profiles in osteolytic bone are largely unknown. Using the latest Next Generation Sequencing technologies, we have investigated miRNA expression profiles in human trabecular bone sourced from bone discarded during total hip replacement (THR) revision surgery where wear particle associated osteolysis was evident. Patients and Methods. Three groups of gender and age-matched patients (n=9 per group) were recruited for this study including patients undergoing revision surgery, primary THR patients and healthy subjects. Total RNAs were prepared from trabecular bone specimens. The cDNA libraries were constructed using a TruSeq Small RNA Sample Preparation kit, and then sequenced on an Illumina HiSeq2000 sequencer. All good quality tags were aligned against the reference sequences containing human chromosomal sequences and 18s and 28s rRNA sequences were analysed using Bowtie software. We used miRBase v19 to identify the start positions of all mature miRNA and the edgeR package to analyse differential expression. Osteogenesis pathway-related gene expression was also investigated using RT-qPCR Array assay. Results. We observed a significant difference in expressed miRNAs between revision and primary THR groups, including upexpressed miR127, miR-409, miR-211 and miR-146a. Importantly, the miR-127 (3.1 fold, p=0.005) and miR-146a (3.5 fold, p=0.001) were not only upexpressed in the revision group vs primary group, but also upexpressed in the revision group vs the healthy group. Thus, miR-127 and miR-146a may have potential as both biomarkers to predict osteolysis and as therapeutic targets. The miR-127 and miR-146a are critical in bone diseases because some of their target genes play an important role in osteogenesis. We have thus studied osteogenic genes and confirmed that SMAD4, RUNX2, FGFR1, TGFβ1, COL1A1 and WNT4 were downregulated. Our data also revealed that miR-93 and miR-204a were downexpressed (−3.7 fold, p=0.023; −2.5, p=0.003 respectively) and t IL-6 and IL-6R, which had been reported as miR-204 target genes, were upexpressed. Discussion and Conclusion. Our results showed that upexpressed miR-127, miR-146a, miR-204a and miR-93 in trabecular bone from revision THR may be the key negative regulators in either osteogenic genes involved in osteogenic differentiation of bone formation or inflammatory genes involved in osteoclastogenesis. Aberrant miRNA expressions identified in the revision THR group may also suggest the existence of genetic risk factors favouring the development of osteolysis in certain specific subgroups of patients. An in-depth understanding of the roles of these regulatory miRNAs in the skeleton warrants further investigation

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

Osteosarcoma (OS) is the most prevalent bone tumor in children and young adults. Most tumors arise from the metaphysis of the long bones and easily metastasize to the lungs. Current therapeutic strategies of osteosarcoma are routinely surgical resection and chemotherapy, which are limited to the patients suffering from metastatic recurrence. Therefore, to investigate molecular mechanisms that contribute to osteosarcoma progression is very important and may shed light on targeted therapeutic approach to improve the survival of patients with this disease. Several miRNAs have been found expressed differentially in osteosarcoma (OS), In this study, we found that miR-144 significantly suppresses osteosarcoma cell proliferation, migration andinvasion ability in vitro, and inhibited tumor growth and metastasisin vivo. The function and molecular mechanism of miR-144 in Osteosarcoma was further investigated. Tissue samples from fifty-one osteosarcoma patients were obtained from Shanghai Ninth People's Hospital. The in vitro function of miR-144 in Osteosarcoma was investigated by cell viability assay, wound healing assay, invasion assay, the molecular mechanism was identified by Biotin-coupled miRNA capture, Dual-luciferase reporter assays, etc. the in vivo function of miR-144 in osteosarcoma was confirmed by osteosarcoma animal model and miR-144−/− zebrafish model. Mechanically, we demonstrated that Ras homolog family member A (RhoA) and its pivotal downstream effector Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) were both identified as direct targets of miR-144. Moreover, the negative co-relation between downregulated miR-144 and upregulated ROCK1/RhoA was verified both in the osteosarcoma cell lines and clinical patients' specimens. Functionally, RhoA with or without ROCK1 co-overexpression resulted a rescue phenotype on the miR-144 inhibited cell growth, migration and invasion abilities, while individual overexpression of ROCK1 had no statistical significance compared with control in miR-144 transfected SAOS2 and U2-OS cells. This study demonstrates that miR-144 inhibited tumor growth and metastasis in osteosarcoma via dual-suppressing of RhoA and ROCK1, which could be a new therapeutic approach for the treatment ofosteosarcoma

Aim. The recent discovery of small non-coding RNAs, so-called micro RNAs (miRNAs), has provided new insights into cancer diagnosis. Several studies have shown that profiles of miRNA expression differ between normal tissue and tumour tissue and vary among tumour types. To exploit this difference, we evaluated the feasibility of using muscle-specific miRNAs (miR-1, 133a, 133b, 206) as biomarkers of rhabdomyosarcoma (RMS). Method. Total RNA was extracted from 16 cell lines (7 RMS, 4 neuroblastoma, 3 Ewing sarcoma and 2 malignant rhabdoid tumour) and 21 tumour specimens (7 RMS, 1 Ewing sarcoma, 4 undifferentiated sarcoma, 1 osteosarcoma, 1 alveolar soft part sarcoma, 2 neuroblastoma, 2 Wilms tumour, 1 malignant rhabdoid tumour, 1 adrenal carcinoma and 1 retinoblastoma). miRNA was quantified by real-time RT-PCR. The expression levels of miRNAs were calculated utilizing the delta-delta Ct method, normalised to the level of miR-16, and compared using the Mann-Whitney U test. Results. The expression levels of muscle-specific miRNAs in the RMS cell lines were significantly higher (p<0.01) than those in neuroblastoma, Ewing sarcoma and malignant rhabdoid tumour cell lines. miR-206 was most abundantly expressed and miR-1 was least abundantly expressed among muscle-specific miRNAs in RMS cell lines. The expression levels of musclespecific miRNAs in RMS tumour specimens were significantly higher (p<0.01) than those in other pediatric tumours. The difference in the expression levels between RMS and other tumours was largest in miR-206. Conclusion. The expression levels of muscle-specific miRNAs were significantly elevated in RMS cell lines and tumour specimens. Muscle-specific miRNAs, especially miR-206, can be potential biomarkers for RMS diagnosis