Review
Biomarkers of cartilage turnover. Part 1: Markers of collagen degradation and synthesis

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Abstract

Type II collagen is a major component of articular cartilage and its breakdown is a key feature of osteoarthritis. Products of cartilage collagen metabolism can be detected in the blood, synovial fluid and urine. Several biomarker assays have been developed which can be used to measure the synthesis and degradation of collagen, and therefore provide information regarding cartilage turnover. This is the first part of a two-part review and describes the need for accurate, reliable information regarding collagen turnover, the processes by which the biomarker epitopes are generated, their application to the study of both healthy and diseased cartilage and the results of currently published studies, with particular reference to the veterinary species. The second part of the review considers the non-collagenous biomarkers of cartilage matrix turnover.

Introduction

Osteoarthritis (OA) is a common, debilitating joint disease that affects all mammals including the horse, dog and man (Poole, 1996). Degradation of articular cartilage in the affected joint is a major feature of the disease (Evans et al., 1982, Spiers et al., 1994) and OA can be defined as a process of aberrant repair with gradual and progressive loss of articular cartilage through degradative mechanisms. Progressive deterioration of the articular cartilage leads to loss of function and, ultimately, to failure of the joint.

The dry-weight of cartilage is predominantly composed of collagen (Eyre, 2004) and the remainder is proteoglycan and glycoprotein (Todhunter, 1996). Since adult cartilage contains neither a vascular nor a neural supply, chondrocytes metabolise largely anaerobically, obtaining their nutrition via the synovial fluid (SF), by means of diffusion. Since type II collagen is specific for hyaline cartilage and is in high abundance in this tissue, the major biomarkers of collagen turnover in cartilage are epitopes derived from type II collagen.

Over the past three decades, one focus of OA research has been the study of candidate molecular biomarkers for the early detection of OA, as well as for monitoring disease or prediction of progression. The ability to detect cartilage loss is highly desirable, not only from a diagnostic, prognostic and therapeutic perspective, but also as surrogate outcome measures in trials of candidate structure-modifying agents (Felson and Lohmander, 2009). The current imaging modalities of radiography, arthroscopy and magnetic resonance imaging (MRI) are costly, require general anaesthesia in some instances, and typically yield results only when gross damage to the cartilage has already occurred. The aim of biomarker research is to reduce or alleviate these disadvantages.

Cartilage destruction leads to an accumulation of breakdown products in the SF. These are released into the circulation and ultimately filtered and excreted, or broken down in vivo. Analysis of body fluids (such as plasma, serum, urine or SF) can provide information regarding the health, or turnover of the cartilage prior to the development of gross pathology, or can highlight any metabolic changes attributable to the treatment being studied.

The perfect biomarker would be specific to the diseased tissue and pathology, sensitive to changes in disease progression or therapeutic intervention, and predictive of disease outcome. Although the discovery of a single ‘golden marker’ of OA is unlikely, the search continues for a marker which is both sensitive to cartilage damage, and specific to the damaged joint. If such a screening tool was available, it would be of enormous value in early diagnosis. It is unlikely that a single biomarker will ever meet all the necessary criteria for the complex diagnosis and observation of OA, and that a profile of several markers will emerge as a more accurate and discriminatory watermark.

When considering the measurement of biomarkers in serum or urine, it is important to remember that the greatest proportion of cartilage in the body is located in the spine and respiratory system, and that contribution from a single diseased joint may be relatively small. In addition, experimental induction of joint instability, performed with the aim of inducing experimental OA (for example, transection of the cranial cruciate ligament) often stimulates degeneration of soft tissues associated with those joints which may result in the release of significant quantities of biomarker. As a result, the epitope of interest could have originated from sources other than the specific cartilage of interest. Biomarkers may therefore be considered ‘non-specific’ not only to the cartilage itself, but to OA, as other diseases of articular cartilage may also result in alterations in their concentrations. Additionally, conflicting conclusions have sometimes been drawn when comparing studies of naturally-occurring with experimentally-induced OA.

Elevated clearance, prior loss of cartilage, or dilution of the biomarker (particularly in the case of joint effusion), may all render inaccurate the absolute concentration of SF concentrations of a biomarker (Lohmander et al., 1994). Synovial inflammation, which often accompanies joint disease, may partly explain differences in measurements from blood and SF from the same patient (Meyers et al., 1995). Expressing a biomarker concentration as a ratio to another marker (Saxna and Heinegard, 1992) may avoid this problem.

Further criticisms levelled at studies measuring serum or SF biomarker concentrations include reports that since the concentration of several biomarkers have been shown to increase with exercise, the activity level of a subject may influence an association (Roos et al., 1995, Neidhart et al., 2000, Mündermann et al., 2005, Frisbie et al., 2008), and that some species differences are apparent when considering the value of different biomarkers as a diagnostic or prognostic indicator of disease (Sharif et al., 1995, Petersson et al., 1998). Furthermore, the highly concentrated samples may require dilution to fit with the standard curve of an assay, increasing the potential for error.

Another, as yet unresolved issue, concerns that of epitope stability, since further enzymatic cleavage of biomarker fragments may occur following release into the circulation. In addition, the rate of degradation of cartilage and progression of subsequent OA is considerably faster in an experimental model than occurs in natural disease, leading to earlier production of peak levels, which may also be of higher concentration. Whilst a more rapid disease progression is essential for laboratory study, it may explain discrepancies between associations arising from experimental studies, and studies concerning the same biomarkers in populations with natural disease. This area deserves further study.

A variety of methods used to quantify collagen destruction and repair from cartilage have been developed. However, while these methods provide important information about disease mechanisms in OA, they have left many questions unanswered. For a biomarker to be appropriate for use as a measure of the joint’s response to the test treatment, it ‘must reliably predict the overall effect on the clinical outcome’ (Fleming and DeMets, 1996); a standard which many biomarkers have failed to meet. OA is a complex process and numerous interdependent factors can affect the clinical outcome; the requirement for a biomarker to correlate with the clinical outcome, but also capture the net effect of treatment, has proved challenging (Felson and Lohmander, 2009).

In this first part of a two-part review we focus on the current status of collagen biomarkers. The second article considers the non-collagenous biomarkers of cartilage matrix turnover (Garvican et al., 2010).

Section snippets

Production of type II collagen

Type II collagen is synthesised as a pre-propeptide with C- and N-terminal globular domains (Fig. 1). Final aggregation of procollagens and formation of collagen fibrils requires the removal of these domains to give tropocollagen. This cleavage (by the extracellular proteinases C- and N-proteinase) ensures that intracellular helix formation cannot occur. The C- and N-terminal propeptides are present in the highest proportion in fetal cartilage, are rapidly reduced at birth, and are present only

Breakdown of type II collagen

It is thought that the structural design of mature collagen aids its longevity and provides an inherent degree of protection from proteolysis, because enzymatic cleavage sites in and between molecules are limited. Breakdown and turnover of cartilage collagen is largely mediated by a family of degradative enzymes called matrix metalloproteinases (MMPs), named for the metal ion (generally zinc) present at the active site (Fig. 2). Collagenolysis must (for reasons of functional continuation) be

Conclusions

The processes which result in the catabolism of collagen, the subsequent synthesis in an attempt to maintain tissue integrity, and the ensuing detectable metabolites, are numerous and complex and, as yet, not fully understood. Measurement of a combination of biomarkers, often including more than one marker for degradation or synthesis, often provides complementary information, not necessarily a redundant replication of data. In addition, further methods for the quantification of these processes

Conflict of interest statement

None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

Acknowledgements

Elaine Garvican was funded by a Pfizer Global Research studentship. This review is dedicated to the memory of Dr. Anne Vaughan-Thomas, a dear colleague and friend.

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