Plasma-derived extracellular vesicle surface markers CD45, CD326 and CD56 correlate with the stage of osteoarthritis: a primary study of a novel and promising diagnostic tool of the disease.

Recently, there is a growing interest in the research based on extracellular vesicles (EVs) which represent paracrine factors secreted by almost all cell types. Both, normal and pathological cells are able to release various types of EVs with different physiological properties, functions and compositions. EVs play an important role in intercellular communication, mechanism and tissue repair. Moreover, EVs could help not only in the treatment of diseases but also in their diagnostics. This work focused on the evaluation of the potential of EVs being used as biomarkers for the diagnosis of osteoarthritis (OA) based on a comparison of the composition of EVs separated from platelet-poor plasma (PPP) of healthy donors and OA patients at different stages of OA. OA is established as a complex syndrome with extensive impact on multiple tissues within the synovial joint. It is a chronic disease of musculoskeletal system that mainly affects the elderly. Depending on the use of the Kellgren-Lawrence classification system, there are four grades of OA which have a negative impact on patients' quality of life. It is very difficult to detect OA in its early stages, so it is necessary to find a new diagnostic method for its timely detection. PPP samples were prepared from whole blood. PPP-EVs were separated from 3 groups of donors-healthy control, early stage OA, end-stage OA, and their content was compared and correlated. EVs from PPP were separated by size exclusion chromatography and characterized in terms of their size, yield and purity by NTA, western blotting, ELISA and flow cytometry. Detection of surface markers expression in EVs was performed using MACSPlex approach. Inflammatory and growth factors in EVs were analysed using MAGPix technology. Our study confirmed significant differences between EVs surface markers of patients and healthy controls correlating with the age of donor (CD63, CD31 and ROR1) and stage of OA (CD45, CD326 and CD56), respectively. Circulating EVs have been under extensive investigation for their capability to predict OA pathology diagnosis as potential targets for biomarker discovery. Taken together, obtained results indicated that PPP-EVs surface markers could be used as potential biomarkers in the early diagnosis of OA.

Transcriptomic Analysis of Macrophage Polarization Protocols: Vitamin D3 or IL-4 and IL-13 Do Not Polarize THP-1 Monocytes into Reliable M2 Macrophages.

Two main types of macrophages (Mφ) include inflammatory (M1) and anti-inflammatory (M2) macrophages. These cells can be obtained in vitro by polarization of monocytic cell lines using various stimuli. Since there is currently no consensus on the best method for the acquisition of reliable M1 and M2 macrophages from the THP-1 cell line, we decided to compare three different polarization protocols at the transcriptomic level. Whole transcriptomes of Mφ polarized according to the chosen protocols were analyzed using RNA-seq. Differential expression of genes and functional enrichment for gene ontology terms were assessed. Compared with other protocols, M1 macrophages polarized using PMA (61.3 ng/mL) and IFN-γ along with LPS had the highest expression of M1-associated regulatory genes and genes for M1 cytokines and chemokines. According to the GO enrichment analysis, genes involved in defensive and inflammatory processes were differentially expressed in these Mφ. However, all three chosen protocols which use Vit D3, IL-13/IL-4, and IL-4, respectively, failed to promote the polarization of macrophages with a reliable M2 phenotype. Therefore, optimization or development of a new M2 polarization protocol is needed to achieve macrophages with a reliable anti-inflammatory phenotype.

Soluble and EV-Associated Diagnostic and Prognostic Biomarkers in Knee Osteoarthritis Pathology and Detection.

Osteoarthritis (OA) is the most common degenerative disease of the connective tissue of the human musculoskeletal system. Despite its widespread prevalence, there are many limitations in its diagnosis and treatment. OA diagnosis currently relies on the presence of clinical symptoms, sometimes accompanied by changes in joint X-rays or MRIs. Biomarkers help not only to diagnose early disease progression but also to understand the process of OA in many ways. In this article, we briefly summarize information on articular joints and joint tissues, the pathogenesis of OA and review the literature about biomarkers in the field of OA, specifically inflammatory cytokines/chemokines, proteins, miRNA, and metabolic biomarkers found in the blood, synovial fluid and in extracellular vesicles.

Mesenchymal Stem Cells in the Treatment of Human Spinal Cord Injury: The Effect on Individual Values of pNF-H, GFAP, S100 Proteins and Selected Growth Factors, Cytokines and Chemokines.

At present, there is no effective way to treat the consequences of spinal cord injury (SCI). SCI leads to the death of neural and glial cells and widespread neuroinflammation with persisting for several weeks after the injury. Mesenchymal stem cells (MSCs) therapy is one of the most promising approaches in the treatment of this injury. The aim of this study was to characterize the expression profile of multiple cytokines, chemokines, growth factors, and so-called neuromarkers in the serum of an SCI patient treated with autologous bone marrow-derived MSCs (BM-MSCs). SCI resulted in a significant increase in the levels of neuromarkers and proteins involved in the inflammatory process. BM-MSCs administration resulted in significant changes in the levels of neuromarkers (S100, GFAP, and pNF-H) as well as changes in the expression of proteins and growth factors involved in the inflammatory response following SCI in the serum of a patient with traumatic SCI. Our preliminary results encouraged that BM-MSCs with their neuroprotective and immunomodulatory effects could affect the repair process after injury.

The Role of Synovial Membrane in the Development of a Potential In Vitro Model of Osteoarthritis.

There is a lack of in vitro models able to plausibly represent the inflammation microenvironment of knee osteoarthritis (OA). We analyzed the molecules released from OA tissues (synovial membrane, cartilage, infrapatellar fat pad) and investigated whether the stimulation of human synovial fibroblasts (SFs), with synthetic cytokines (IL-1ß and TNF-α or IFN-γ) or conditioned media (CM) from OA tissues, influence the SFs' response, in the sense of pro-inflammatory cytokines, chemokines, growth factors, and degradative enzymes modulation. Human SFs were obtained from OA synovial membranes. SFs and their CM were analyzed for biomarkers, proliferation rate, protein profile and gene expression, before and after stimulation. Real-time PCR and multiplex assays quantified OA-related gene expression and biomolecule production. Unlike other activators, CM from OA synovial membrane (CM-SM), significantly up-regulated all genes of interest (IL-6, IL-8, MMP-1, MMP-3, RANTES, MCP-1, TSG-6, YKL-40) in SFs. Multiplex immunoassay analysis showed that levels of OA-related cytokines (IL-6, IL-8, MCP 1, IL-1Ra), chemokine (RANTES) and growth factor (VEGF), produced by CM-SM stimulated SFs, increased significantly compared to non-stimulated SFs. Molecules released from the SM from OA patients induces OA-like changes in vitro, in specific OA synovial populations (SFs). These findings promote the use and establish a compelling in vitro model that simulates the versatility and complexity of the OA disease. This model, in the future, will allow us to study new cell therapies or test drugs by reducing or avoiding animal models.

Small Extracellular Vesicles Derived from Human Chorionic MSCs as Modern Perspective towards Cell-Free Therapy.

Mesenchymal stem cells (MSCs) are of great interest to scientists due to their application in cell therapy of many diseases, as well as regenerative medicine and tissue engineering. Recently, there has been growing evidence surrounding the research based on extracellular vesicles (EVs), especially small EVs (sEVs)/exosomes derived from MSCs. EVs/exosomes can be secreted by almost all cell types and various types of EVs show multiple functions. In addition, MSCs-derived exosomes have similar characteristics and biological activities to MSCs and their therapeutic applications are considered as a safe strategy in cell-free therapy. The aim of this study was the characterization of MSCs isolated from the chorion (CHo-MSCs) of human full-term placenta, as well as the isolation and analysis of small EVs obtained from these cells. Accordingly, in this study, the ability of small EVs' uptake is indicated by synovial fibroblasts, osteoblasts and periosteum-derived MSCs. Improvement in the understanding of the structure, characteristics, mechanism of action and potential application of MSCs-derived small EVs can provide new insight into improved therapeutic strategies.

A Preliminary Study of Combined Detection of COMP, TIMP-1, and MMP-3 in Synovial Fluid: Potential Indicators of Osteoarthritis Progression.

OBJECTIVE: Osteoarthritis (OA) commonly affects weight-bearing joints and is characterized by articular cartilage breakdown combined with osteophyte formation at the joint margins and chronic nonspecific inflammation of synovium. Understanding the profile of inflammation in a patient population is an essential starting point to predict or prevent OA progression. The aim of this study was to identify the profile of selected biomolecules in synovial fluid (SF) and investigate the correlation according to gender, age, and severity of the disease within patients from among the general knee OA population. DESIGN: In our study SF samples were aspirated from the knees of 65 OA patients (46 patients with early knee OA and 19 patients with end-stage knee OA according to the Kellgren-Lawrence grading scale). The concentration of interleukins (IL-6, IL-8), matrix metalloproteinases (MMP-1, MMP-3, MMP-13), MMPs inhibitors (TIMP-1, TIMP-2), cartilage oligomeric matrix protein (COMP), and adiponectin was analyzed using a multiplex ELISA-based approach. CONCLUSIONS: Our results indicate significant linear correlation of MMP-13 and COMP concentration with age (P < 0.05), but not with OA severity. In fact, 3 of the examined biomolecules, MMP-3 (P < 0.01), TIMP-1 (P < 0.01), and COMP (P < 0.05) significantly correlate with the grade of knee OA and might be associated with OA severity.

Study of bilateral elbow joint osteoarthritis treatment using conditioned medium from allogeneic adipose tissue-derived MSCs in Labrador retrievers.

Canine elbow dysplasia is a common cause of forelimb lameness in dogs and can lead to development of osteoarthritis (OA). A potential alternative to pain management is the use of a safe cell-free based therapy through trophic and paracrine factors of mesenchymal stem cells (MSCs). The aim of study was to identify the profile of selected mediators of potential clinical relevance in synovial fluid (SF) samples of dogs with elbow OA and analyse the range of motion (ROM) before and after cell-free MSCs-based treatment. In this study, conditioned medium from allogeneic canine adipose tissue - derived MSC (CM-AD-MSC) was prepared and administered into both elbow joints with OA in six Labrador retriever dogs (n = 6) on day 0 and 14 without creating a control group with a placebo. The SF of the elbow joints was analysed for the presence of several biomolecules (IL-6, IL-10, IL-8, IL-2, IL-12, TNF-αIFN-γ, MMP-3TIMP-1) before and after intraarticular applications of CM-AD-MSC. Kinematic analysis was used to assess the clinical effect of CM-AD-MSC. Analyses of SF and ROM were performed on days 0, 14 and 42. Concentration levels of MMP-3, TIMP-1, IL-6 and TNF-α in SF showed significant differences before and after the treatment (P < .05). There was a significant improvement in ROM between day 0 and 42 (P < .001). No severe adverse events were observed during the study. Results support the potential supportive effect of CM-AD-MSC as a noninvasive therapeutic tool for pain management of OA elbow joints in dogs.