Introduction
Osteoarthritis (OA) of the joint is one of the most common diseases of the musculoskeletal system. It is accompanied by various types of manifestations from pain, synovial membrane inflammation (synovitis) and cartilage degeneration to enlargement (hyperplasia) of the subchondral bone [1]. OA has long been thought to be a disease caused primarily by mechanical wear of cartilage. The basic pillar of this theory was the fact that cartilage is made up of a single type of cell - chondrocytes, which have low metabolic activity and therefore are unable to regenerate damaged cartilage. In addition, this type of tissue is not physiologically innervated and vascularized, which also reduces regenerative capacity. However, with increasing scientific knowledge, this theory has been abandoned and replaced by the so-called inflammatory environment theory. This is based on the action of molecules such as cytokines and prostaglandins, which significantly affect the production of matrix metalloproteinases by chondrocytes, and thus contribute to the gradual degradation of articular cartilage [2]. Cytokines are secreted by chondrocytes, synovial cells and a number of other cells present in the joint and are detectable in synovial fluid (SF) in OA patients. Studies from previous years have confirmed the significant effect of the synovial membrane on pathological cartilage changes through the autoimmune system, leading to the progression of OA [3]. Extracellular vesicles (EVs) are particles naturally released from the cells that are bounded by a lipid bilayer and cannot replicate, i. e. they do not contain a functional nucleus. They are produced by a number of cells types and can be found in several body fluids (plasma, serum, urine, cerebrospinal fluid and SF). In the past, EVs were considered to be waste products of cell metabolism. Today, studies have shown that they have a number of important functions. EVs contain biologically active molecules such as non-coding RNA (ncRNA), proteins, microRNA (miRNA), messenger RNA (mRNA), etc. Due to the specific "cargo", EVs are thought to be able to affect the microenvironment and provide intercellular communication [4]. Activated synovial fibroblasts have been shown to have the ability to directly affect OA pathology through these small transporters. There are several methods by which EVs can be separated/isolated, either from conditioned media from cells, tissues or from biofluids [5]. Such methods include: differential centrifugation, density gradient centrifugation, particle size chromatography, filtration, precipitation, and immunomagnetic separation.
Basics in characterisation and classification of EVs
In recent decades, the study of EVs has expanded into several areas of research [6,7]. Therefore, it was necessary to propose a general uniform nomenclature and categorization of these particles. Researchers studying EVs have taken on this role and developed a manual called: Minimum experimental requirements for the definition of extracellular vesicles and their functions in 2014 [8]. According to current recommendations, EVs should therefore be named either according to size (small EVs <200nm, large EVs ˃200nm), density (low, medium, high), biochemical composition (expression of surface markers), or according to the source of the cells from which they originate (e. g. mesenchymal stem cell-derived EVs) [8]. In 2018, the original criteria were supplemented and another manual called: Minimal Information for Studies of Extracellular Vesicles 2018 - MISEV2018 was published [4]. In addition to the nomenclature of EVs, the criteria contain a wealth of information on the various options for separating and characterizing EVs. For each experiment, the number of cultured cells must be reported, the total initial volume of biofluid or weight/volume/tissue size at the time of collection. The presence of at least three positive EVs protein markers (at least one of the transmembrane – CD9, CD63, CD81, CD82 and cytosolic – TSG101, FLOT1/2, ALIX, HSC70) must be confirmed using either Western blot or flow cytometry (FACS). One negative protein marker (APOB100, albumin etc.) must also be analysed to demonstrate the nature of the EVs and the degree of purity of the EVs preparation. Visualization of individual EVs can be achieved using atomic force microscopy (AFM) or transmission electron microscopy (TEM). Using these methods, it is possible to determine the shape and amount of EVs. The methods used to determine the number and average size of EVs are based on the principle of measurement by resistive pulse sensing, light scattering properties (NTA) or fluorescence properties (FCS, FACS) [4]. By adhering to these basic rules, the results can be compared between individual laboratories without misinterpretation or inconsistency in the presented research results in the field of EVs.
Synovial fluid - source of EVs
Since blood plasma is a source of information from the whole organism and it is not possible to determine exactly where the information from plasma-derived EVs comes from, ways are being sought for a more accurate diagnosis of OA [9]. Nowadays, the diagnostics of OA itself takes place on the basis of visualization techniques (e. g. X-ray, MRI, USG) and the patient's clinical history, which does not always correlate exactly with the symptoms themselves (pain, stiffness, swelling and others) [10]. One of the possibilities is a diagnostic method based on the analysis of SF and EVs isolated from it. SF is a suitable source of information in monitoring the pathogenesis of OA, because it directly connects the tissues present in the joint capsule, such as the synovial membrane, cartilage or Hoff's fat pad [11]. The advantage of SF is that changes in the OA process are more pronounced in it than in other biological fluids [12]. Compared to visualization methods, aspiration of SF is an invasive diagnostic tool. In healthy joints, physiological soft cartilage and SF work together to create as little friction as possible within the connective tissue. The fluid itself provides mechanical shock absorption, lubrication and cartilage nutrition [13]. SF is produced by the inner membrane of the joint (synovial membrane) and contains mainly serum albumin, hyaluronan, lubricin and globulin [14]. During friction, which occurs in the joint during physiological movement, there are changes in the composition of the SF. A naturally thin layer of homogeneous fluid changes into a rough heterogeneous mixture of fluid and precipitates by frictional forces [15]. Current research is focused on the characterization of proteins and miRNAs as potential biomarkers of OA present in SF [9]. Since SF is in direct contact with OA tissues, it is expected that a potential biomarker can be detected in it, which could ideally correlate with its plasma levels. Thus, based on SF-derived biomarker research, we would reach the stage of less invasive diagnostics of OA (from an ordinary blood sample).
miRNA and lncRNA as a diagnostic biomarker of OA
The key assumption for successful treatment of most diseases is their early diagnosis. EVs provide potential information in the form of microRNA (miRNA), long non-coding RNA (lncRNA), mRNA, etc. [16]. These could be used in the diagnosis of OA or in determining the specific degree of the disease [17]. Only a small part of the transcriptome encodes proteins and the rest belongs to the so-called non-coding RNA (ncRNA), which is not translated into proteins in the translation process [18]. One of many types of such ncRNA molecules are miRNAs composed of 19-23 nucleotides. These molecules can bind partially complementarily to the 3'UTR of the target mRNA and thus regulate many biochemical reactions involved in the pathogenesis of several diseases [19]. The heterogeneity of lncRNA molecules is due to their size, which ranges from several hundred to several thousand nucleotides [20]. They play an important regulatory role in the processes of cell development, differentiation, proliferation, apoptosis and metabolism [21]. However, it seems that not their size but their secondary and tertiary structure are essential for the proper performance of the functions of these molecules. In recent years, miRNAs and lncRNAs have been among the most studied molecules in EVs in association with the research of potential diagnostic biomarkers of OA [20]. A recent study compared EV-associated miRNAs from serum and SF in OA patients [22]. There were observed 31 "upregulated" and 33 "downregulated" EV-associated miRNAs in SF compared to serum (Fig.1).
