Introduction: The need for diagnostic markers in osteoarthritis (OA) is acute and immediate, as sensitive and precise tools that monitor disease activity and treatment response are lacking. Collagens—types I, II, and III—are the skeleton of the extracellular matrix of joint tissues. Joint collagens are generally turned over at a low rate, but the balance between formation and degradation is disturbed, leading to the loss of, for example, cartilage. Areas covered: We discuss the markers reflecting collagen turnover and provide examples of how they have been applied in OA research, as well as how we believe these should be used in the future. We have searched PubMed for full-text articles written in English using different combinations of the following terms: OA, biomarker, and collagen. The result is a narrative review that gives examples from the literature. Expert opinion: Collagen markers show promise, as they are direct measures of tissue balance. Until now, collagen markers have mainly been tested in observational cohorts, which may provide insights into the association between the candidate marker and clinical variables; however, these do not advance the development of qualified markers that can be used for drug development or in clinical practice.

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis. Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control. Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

(1) Background: Mechanical loading is an essential part of the function and maintenance of the joint. Despite the importance of intermittent mechanical loading, this factor is rarely considered in preclinical models of cartilage, limiting their translatability. The aim of this study was to investigate the effects of intermittent dynamic compression on the extracellular matrix during long-term culture of bovine cartilage explants. (2) Methods: Bovine articular cartilage explants were cultured for 21 days and subjected to 20 min of 1 Hz cyclic compressive loading five consecutive days each week. Cartilage remodeling was investigated in the presence of IGF-1 or TGF-β1, as well as a TGF-β receptor 1 (ALK5) kinase inhibitor and assessed with biomarkers for type II collagen formation (PRO-C2) and fibronectin degradation (FBN-C). (3) Results: Compression of cartilage explants increased the release of PRO-C2 and FBN-C to the conditioned media and, furthermore, IGF-1 and compression synergistically increased PRO-C2 release. Inhibition of ALK5 blocked PRO-C2 and FBN-C release in dynamically compressed explants. (4) Conclusions: Dynamic compression of cartilage explants increases both type II collagen formation and fibronectin degradation, and IGF-1 interacts synergistically with compression, increasing the overall impact on cartilage formation. These data show that mechanical loading is important to consider in translational cartilage models.

To examine the effect of dynamic compressive loading applied intermittently on bovine cartilage explants stimulated with proinflammatory cytokines over 21 days. Cartilage explants were cultured for 21 days with Oncostatin M and TNFα (O ​+ ​T) [10/5 ​ng/mL] or in culture medium alone (w/o). The explants were either left free-swelling or subjected to dynamic compressive loading of 20 ​min, at 1 ​Hz, with loads ranging between 0.1 and 1 ​MPa, 5 times weekly. Metabolic activity was measured once weekly using Alamar Blue and cartilage turnover was assessed with biomarkers targeting degradation fragments of aggrecan (AGNx1) and type II collagen (C2M). Glycosaminoglycan degradation was quantified was the DMMB assay. Furthermore, explant weight and histological analysis was used to assess the cartilage degradation. Dynamic compression of cartilage explants attenuated the O ​+ ​T-mediated C2M release on day 21 with 40% (p ​= ​0.0068) compared to the unloaded explants. Additionally, the change in explant weight from day −1 to day 21 showed that O ​+ ​T stimulation alone mediated a cartilage loss of 11%, whereas O ​+ ​T-stimulated explants subjected to compressive loading demonstrated a decreased cartilage weight loss of 6%, which was supported by the histological analysis. However, loading had no effect on aggrecan degradation. In cartilage explants cultured in a proinflammatory milieu, dynamic compressive loading confers anti-catabolic effects, inhibiting type II collagen degradation and reducing explant cartilage loss. These results demonstrate that compressive loading alters cartilage tissue turnover and enforces the need to include mechanical loading in a translation ex vivo cartilage model.

Background Osteoarthritis (OA) patients suffer from progressive degradation of articular cartilage, to which there is no available treatment to block or reverse the catabolic mechanisms. Articular cartilage confers a biomechanical function in the joints, and in concordance chondrocytes have shown to be mechanosensitive. The effect of dynamic compression on cartilage extracellular matrix (ECM) could prove to be crucial for translational research in development and screening of new OA drug candidates. Objectives This study investigates the effect of long-term dynamic compression of a bovine articular cartilage ex vivo model through quantification of cartilage associated biomarkers. Methods Full depth bovine cartilage explants were cultured for 2 weeks. The explants were treated 3 times a week with either OSM [10 ng/mL] and TNF-a [2 ng/mL] (O+T), or TGF-B1 [50 ng/mL]. Untreated samples were included as negative controls (w/o). For each condition two groups were established; an unloaded group and a group compressed 3 times a week. Compression was applied using Electroforce 5500 (TA Instruments), in a sine wave with a maximum load per cycle of 1 MPa, at 1 Hz frequency for 1200 cycles. Metabolic activity was measured once a week using AlamarBlue. Biomarkers released to the supernatant were assessed using the following well-described ELISAs: formation of type II collagen was measured by ProC2, and MMP-degradation of type II collagen was measured by C2M. Sulfated glycosaminoglycans (sGAG) released to media, and sGAG extracted from the explants on the last day using a 4 mM GuHCl solution were quantified using a 1.9-dimethylmethylene blue (DMB) assay. Results Compression of bovine explants significantly decreased release of C2M compared to unloaded samples in O-T and w/o groups with 42% and 48% respectively. ProC2 release was not affected by compression. Compression of w/o or TGF-b treated explants increased sGAG release with 195% and 152% relative to their unloaded controls. However, the total amount of sGAG extracted from the explants on the last day remained unchanged. Compression of the explants did not affect the metabolic activity. Conclusions Compression significantly reduced the degradation of type II collagen, which in combination with unaltered formation as measured by ProC2, may suggest suppression of the involved catabolic processes and increased deposition of type II collagen in the cartilage matrix. Furthermore, compression elevated the sGAG release without reduction in the explant GAG content, indicating an increased turnover of GAG. In conclusion, the cartilage ECM turnover is significantly modulated by dynamic compression. This method adds essential translational value to the continuous research addressing OA. Disclosure of Interest None declared