Laboratory diagnosis of osteoarthritis

In most cases, patients with osteoarthritis do not have changes in blood and urine tests, except for cases of synovitis with significant effusion, when an increase in ESR, hypergammaglobulinemia, an increase in the level of acute phase indicators - CRP, fibrinogen, etc. may occur. When examining synovial fluid, no significant differences from normal indicators are revealed.

In recent years, there has been an intensive search for possible biological markers (BM) of degradation and reparation of joint tissues (mainly cartilage and bone). BM should reflect these dynamic changes, serve as predictors of osteoarthrosis prognosis and markers of the effectiveness of pathogenetic treatment. The discovery of new and more in-depth study of known biological markers will allow a better understanding of the mechanisms of osteoarthrosis pathogenesis. However, the main task of using biological markers of cartilage metabolism is to assess the chondroprotective properties of drugs and monitor treatment with drugs belonging to the DMO AD group - "disease modifying".

In osteoarthritis, pathological changes occur mainly in the articular cartilage, as well as in the subchondral bone, synovial membrane, and other soft tissues of the joint. Since our ability to directly examine these structures is limited, the most important sources for collecting biological markers are blood, urine, and synovial fluid.

Urine testing is the most preferable, since it does not involve any invasive procedures. In our opinion, the ideal material for testing is daily urine. Analysis of the morning portion of urine would be more appropriate, but the possibility of using it is based only on the fact that this type of analysis is used to determine biological markers of bone metabolism in osteoporosis: it is known that biological markers are subject to circadian rhythms, and the peak concentration of biological markers of bone metabolism occurs at night. At present, there is no information in the literature on the circadian rhythms of biological markers of soft tissues, cartilage, so the final decision on the choice of an adequate urine test will be made after conducting appropriate studies.

Blood tests are routine clinical tests. Some biological markers are already being determined in the blood, such as acute phase indices, while others may be included in the standard list of biochemical tests in the near future. For each biological marker, it is necessary to specify in which component of the blood it should be determined - plasma or serum. Research results indicate that the concentration of biological markers in blood plasma differs significantly from that in serum. Biological markers are usually determined in blood serum. According to V. Rayan et al. (1998), the concentrations of biological markers in blood taken from a vein near the affected joint and from a more distant vein are different. These data indicate the need to standardize blood sampling for studying biological markers.

According to LJ Attencia et al. (1989), the cartilage of the synovial joints of an adult makes up only 10% of the total mass of hyaline cartilage in the body, including intervertebral discs. Thus, the determination of biological markers in the blood and urine reflects systemic metabolism rather than local changes in the joint affected by osteoarthrosis. Synovial fluid is closest to the pathological focus in osteoarthrosis and probably most accurately reflects the processes occurring in the affected joint. The concentration of biological markers in the synovial fluid can be significantly higher than in the blood, which means it is easier to determine. Examples include epitope 846 of aggrecan - in synovial fluid it is 40 times more than in blood serum, cartilage oligomeric matrix proteins (COMP) - 10 times more than in blood serum. Degradation products in synovial fluid more accurately reflect catabolic processes in articular cartilage. Drainage of molecules from synovial fluid through the local lymphatic system can lead to a decrease in their size and even to their destruction.

Despite the invasiveness of the synovial fluid collection technique, associated with a number of possible complications, the value of determining biological markers in it is obvious. To avoid problems with the so-called dry joint, 20 ml of isotonic NaCl solution can be injected into the joint immediately before fluid collection. Immediately after the injection of the isotonic solution, the patient should flex and extend the limb in the joint 10 times, followed by rapid aspiration of the diluted synovial fluid. According to EM-JA Thonar (2000), such dilution of the synovium affects the metabolism in the articular cartilage. However, the results of the study by FC Robion et al. (2001) indicate that repeated lavage of the equine stifle joints does not cause significant changes in cartilage metabolism. These data certainly require confirmation. Therefore, for each biological marker, the effect of joint lavage on changes in its concentration must be determined at the stage of preclinical studies in animals.

The next important point is to determine the half-life time in synovial fluid and blood for each biological marker. Without such data, interpretation of test results will be difficult. Usually, the half-life of biologically active substances in blood is shorter than in other liquid media due to effective clearance by the liver and kidneys. Thus, for each biological marker, it is also necessary to determine the elimination pathway. Thus, the N-propeptide of type III collagen is excreted by the liver by receptor-mediated endocytosis, and non-glycosylated collagen fragments are excreted mainly by urine, as is osteocalcin. There are receptors for glycosaminoglycans on the endothelial cells of the sinuses of the liver lobules, so hyaluronic acid and proteoglycans are eliminated by the liver. The half-life of hyaluronic acid in the blood is 2-5 minutes. The presence of synovitis may accelerate the clearance of biological markers from joints, although a study in rabbits found no significant differences in proteoglycan clearance with or without synovitis. Thus, the effect of inflammation on changes in the concentration of biological markers in body fluids needs to be investigated.

The kidneys selectively filter biological markers. Thus, glycosaminoglycans, which carry a large negative charge, may not penetrate the renal basement membrane, whereas glycosaminoglycans such as chondroitin-6-sulfate and chondroitin-4-sulfate are detected in the urine.

In addition to pathology (in particular, osteoarthritis), a number of factors can influence the concentration of biological markers in the body's fluids:

1. Circadian rhythms have been studied only for a small number of biological markers. They have been studied for bone metabolism markers. Thus, the peak concentration of osteocalcin occurs at night, and that of collagen cross-links in the morning - at 8 o'clock. In rheumatoid arthritis, the peak activity of IL-6 also occurs at night (about 2 o'clock), and earlier than that of osteocalcin. These data are of some interest in relation to the participation of IL-6 in inflammation and physiology of bone tissue. TNF-a, on the contrary, does not have circadian rhythms. However, the receptors of this cytokine can obey them.
2. Peristalsis. Hyaluronic acid is synthesized by synovial cells (as well as many other cells) and is a potential marker of synovitis in osteoarthritis and rheumatoid arthritis. However, the highest concentration of hyaluronate is found in the intestinal lymphatic system. Not surprisingly, the concentration of circulating hyaluronic acid can increase after eating. Therefore, blood sampling for determining biological markers should be done on an empty stomach or 3 hours after eating. And the effect of peristalsis on the level of biological markers in the blood requires study.
3. Physical activity in the morning after sleep leads to an increase in the concentration of hyaluronic acid in the blood, MMP-3 and the epitope of keratan sulfate in healthy individuals. Physical activity can change the concentration of some markers in both synovial fluid and blood serum. Such an increase is more pronounced in patients with rheumatoid arthritis, moreover, the concentration of biological markers correlates with the clinical condition of these patients.
4. Liver and kidney diseases. Liver cirrhosis causes a significant increase in serum hyaluronic acid levels and probably affects proteoglycan elimination. Kidney diseases are known to affect osteocalcin concentrations. This issue also requires more in-depth study.
5. Age and gender. During growth, the activity of growth plate cells increases, which is accompanied by an increase in the concentration of skeletal biological markers in the blood serum. An example is the increase in the concentration of aggrecan fragments and type II collagen in the peripheral blood and urine of growing animals. Thus, the interpretation of biological marker analyses in children and adolescents with musculoskeletal diseases is difficult. For many biological markers, an increase in concentration was found with aging. In men, the concentration of biological markers significantly exceeds that in women in cartilage and bone tissue. In addition, in women in the menopausal and postmenopausal periods, changes in the concentration of biological markers of cartilage metabolism can be expected, similar to what is observed in bone tissue.
6. Surgical procedures can also affect the levels of biological markers, and this effect can last for several weeks.

The concept of biological markers of osteoarthrosis is based on the assumption that they reflect certain aspects of metabolic processes in joint tissues. However, the relationship between the concentrations of biological markers in the body's fluids and the metabolism of cartilage, synovial and other tissues has proven to be very complex.

For example, the concentration of markers of articular cartilage ECM degradation in synovial fluid may depend not only on the degree of degradation of the matrix itself, but also on other factors, such as the degree of elimination of molecular fragments from the synovium, which was already mentioned above, as well as on the amount of cartilage tissue remaining in the joint.

Despite the above facts, the concentration of biological markers in synovial fluid generally correlates with the metabolism of ECM molecules of articular cartilage. For example, changes in the concentration of aggrecan fragments, epitope 846, COMB, and C-propeptide of collagen II in synovial fluid after joint injury and during the development of osteoarthrosis are consistent with changes in the intensity of aggrecan, COMB, and collagen II metabolism in experimental models of osteoarthrosis in animals/and in vivo and in articular cartilage of patients with osteoarthrosis/and in vitro.

Identification of specific sources of molecular fragments is a complex process. Increased release of molecular fragments can occur both due to a general increase in degradation processes that are not compensated by synthetic processes, and due to increased degradation with a simultaneous increase in the intensity of synthesis of the same ECM molecules; in the latter case, the concentration of ECM molecules does not change. Thus, it is necessary to search for markers specific for degradation and synthesis. An example of the former is fragments of aggrecan, and the latter is the C-propeptide of collagen 11.

Even if a biological marker is associated with a specific aspect of metabolism, it is necessary to take into account the specific features of this process. For example, the identified fragments may be formed as a result of degradation of a de novo synthesized molecule that has not yet integrated into the functional ECM, a molecule that has just been integrated into the ECM, and finally a permanent ECM molecule that is an important functional part of the mature matrix. Another problem is the definition of the specific matrix zone (pericellular, territorial, and interterritorial matrix) that served as a source of biological markers detected in synovial fluid, blood, or urine. In vitro studies indicate that the intensity of metabolism in individual zones of the articular cartilage ECM may be different. The study of certain epitopes associated with chondroitin sulfate sulfation may help to identify the population of de novo synthesized aggrecan molecules.

It can be assumed that the appearance of fragments of molecules normally present in the cartilage ECM in the synovial fluid is associated with the metabolism of the cartilage matrix. However, this is not always the case, since it depends on a number of factors, in particular on how much the concentration of a given molecule in the articular cartilage exceeds that in other joint tissues and how much the intensity of its metabolism in the cartilage exceeds that in other joint tissues. Thus, the total mass of aggrecan in the articular cartilage significantly exceeds that, for example, in the meniscus of the knee joint, while the total mass of COMB in the meniscus is practically no different from that in the articular cartilage. Both chondrocytes and synovocytes produce stromelysin-1, but the total number of cells in the synovial membrane exceeds that in the cartilage, so a significant part of the stromelysin-1 found in the synovial fluid is most likely of synovial origin. Thus, identification of the specific source of biological markers is extremely difficult and often impossible.

When studying biological markers in blood serum and urine, the problem of determining its possible extra-articular source arises. In addition, in case of monoarticular damage, biological markers secreted by the affected joint may mix with markers secreted by intact joints, including contralateral ones. Articular cartilage comprises less than 10% of the total mass of hyaline cartilage in the body. Thus, the determination of biological markers in blood and urine may be justified rather in polyarticular, or systemic, diseases (in relation to osteoarthrosis - in generalized osteoarthrosis).

The requirements for biological markers depend on whether they are used as a diagnostic, prognostic, or evaluative test. For example, a diagnostic test determines differences between healthy individuals and patients with osteoarthritis, which is expressed in terms of sensitivity and specificity of the test. A prognostic test identifies individuals in a cohort who are most likely to rapidly progress the disease. Finally, an evaluative test is based on the ability of the marker to monitor changes over time in an individual patient. In addition, biological markers can be used to determine the sensitivity of patients to a particular drug.

Initially, it was assumed that biological markers could serve as diagnostic tests that would help distinguish a joint affected by osteoarthrosis from an intact one, as well as conduct differential diagnostics with other joint diseases. Thus, determining the concentration of keratan sulfate in the blood serum was considered as a diagnostic test for generalized osteoarthrosis. However, subsequent studies showed that this biological marker can only reflect the degradation of cartilage proteoglycans in some situations. It turned out that the concentrations of biological markers in the blood serum depend on the age and sex of the person being examined.

Putative biological markers of joint tissue metabolism in synovial fluid and blood serum of patients with osteoarthritis

Biological marker	Process	In synovial fluid (links)	In blood serum (links)
1. Cartilage
Aggrecan
Core protein fragments	Aggrecan degradation	Lohmander LS. et al., 1989; 1993	Thonar EJMA et al., 1985; Campion GV et al., 1989; MehrabanF. et al., 1991; Spector TD et al., 1992; Lohmander LS., Thonar EJ-MA, 1994; Poole AR et al., 1994) t (Poole AR et al., 1994)
Core protein epitopes (cleavage zone specific neoepitopes)	Aggrecan degradation	Sandy JD et al., 1992; LohmanderLS. et al., 1993; LarkM.W. et al., 1997
Epitopes of keratonic sulfates	Aggrecan degradation	Campion GV et al., 1989; Belcher C et al., 1997
Epitopes of chondroitin sulfates (846, ЗВЗ, 7D4 and DR.)	Aggrecan synthesis/degradation	Poole AR et al., 1994; HazellP.K. et al., 1995; Slater RR Jr. et al., 1995; Plaas AHK et al., 1997; 1998; Lohmander LS. et al., 1998
The ratio of chondroitin-6 and chondroitin-4 sulfates	Aggrecan synthesis/degradation	Shinme iM. et al. 1993
Small proteoglycans	Degradation of small proteoglycans	Witsch-PrehmP. et al., 1992
Matrix proteins of cartilage
HOMP	Degradation of HOMP	Saxne T., Heinegerd D., 1992"; LohmanderLS. et al., 1994; Petersson IF etal., 1997	Sharif M. et al., 1995
Cartilage collagens
C-propeptide of type II collagen	Collagen II synthesis	ShinmeiM. et al., 1993; YoshiharaY. et al., 1995; LohmanderLS. et al., 1996
Fragments of the alpha chain of type II collagen	Collagen II degradation	Hollander AP et al., 1994; Billinghurst RC et al., 1997; AtleyLM. et al., 1998
MMPs and their inhibitors	Synthesis and secretion	From synovium or articular cartilage?
II. Menisci
HOMP	Degradation of HOMP	From articular cartilage, menisci or synovium?
Small proteoglycans	Degradation of small proteoglycans
III. Synovial membrane
Hyaluronic acid	Synthesis of hyaluronic acid		Goldberg RL et al., 1991; HedinP.-J. et al., 1991; Sharif M. et al., 1995
MMPs and their inhibitors
Stromelysin (MMP-3)	MMP-3 synthesis and secretion	LohmanerLS et al., 1993	ZuckerS. et al., 1994; YoshiharaY. et al., 1995
Interstitial collagenase (MMP-1)	MMP-1 synthesis and secretion	Clark IM et al., 1993; LohmanderLS. et al., 1993	Manicourt DH et al., 1994
TIMP	Synthesis and secretion of TIMP	Lohmander LS. et al., 1993; Manicourt DH et al., 1994	Yoshihara Y. et al., 1995
N-propeptide of type III collagen	Collagen III synthesis/degradation	Sharif M. et al., 1996	Sharif M. et al., 1996

A number of studies have demonstrated differences in the concentrations of aggrecan fragments, HOMP and MMP and their inhibitors in the joint fluid of the knee joints of healthy volunteers, patients with rheumatoid arthritis, reactive arthritis or osteoarthrosis. Despite the fact that the authors demonstrate significant differences in the average concentrations of biological markers, the interpretation of the data is difficult, since the comparative analysis was profile and retrospective. The prognostic properties of these tests need to be confirmed in prospective studies.

Biological markers can be used to assess the severity of the disease or the staging of the pathological process. In the case of osteoarthrosis, the severity of the disease and its stages are judged by the results of X-ray examinations, arthroscopy, as well as by the severity of the pain syndrome, limitation of the function of the affected joints and the functional capacity of the patient. L. Dahlberg et al. (1992) and T. Saxne and D. Heinegard (1992) proposed using some molecular markers of articular cartilage metabolism for additional characterization of the stages of osteoarthrosis. However, further research in this direction is necessary to introduce such biological markers into medical practice.

There are reports on the possible use of biological markers as prognostic tests. For example, it was shown that the concentration of hyaluronic acid (but not keratan sulfate) in the serum of patients with knee osteoarthritis at the beginning of the study indicates the progression of gonarthrosis during 5 years of observation. In the same population of patients, it was shown that an increased content of COMB in the serum of patients with gonarthrosis during the first year after the beginning of the study was associated with radiographic progression during 5 years of observation. Studies of biological markers in patients with rheumatoid arthritis have shown that the concentration of COMB, epitope 846, chondroitin sulfate in the serum is associated with more rapid disease progression. These results, obtained in small groups of patients, often do not demonstrate the strength of the relationship between the level of biological markers and disease progression, i.e., further studies, prospective and on larger cohorts of patients, are needed.

TD Spector et al. (1997) found a slight increase in serum CRP in patients with early osteoarthritis and reported that CRP may be a predictor of osteoarthritis progression. In this case, the increase in CRP reflects the processes of joint tissue damage and may be associated with an increase in hyaluronic acid, which also indicates disease progression. It is possible that the synovial membrane is responsible for most of the hyaluronic acid determined in the serum, which indicates the presence of mild synovitis. Increased concentrations of stromelysin MMP in synovial fluid and serum of patients with osteoarthritis and after joint injury may also be associated with mild synovitis.

Finally, biological markers can be used as efficacy criteria in clinical trials of drugs, as well as for monitoring pathogenetic treatment. However, there are two interrelated problems: the lack of drugs with proven "structure-modifying" or "disease-modifying" properties is largely due to the lack of reliable biological markers, and vice versa, the lack of specific markers of joint tissue metabolism is largely due to the lack of controlled studies of drugs in these groups.

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