A Novel High Sensitivity Type II Collagen Blood-Based Biomarker, PRO-C2, for Assessment of Cartilage Formation.

Anne-Christine Bay-Jensen,Ali Mobasheri,Ditte Reker,Yunyun Luo,Morten Karsdal,Christoph Ladel,Ole Simonsen,Yi He,Martin Michaelis,Natasja Gudmann,Kim Henriksen

doi:10.3390/ijms19113485

Abstract

N-terminal propeptide of type II collagen (PIINP) is a biomarker reflecting cartilage formation. PIINP exists in two main splice variants termed as type IIA and type IIB collagen NH2-propeptide (PIIANP, PIIBNP). PIIANP has been widely recognized as a cartilage formation biomarker. However, the utility of PIIBNP as a marker in preclinical and clinical settings has not been fully investigated yet. In this study, we aimed to characterize an antibody targeting human PIIBNP and to develop an immunoassay assessing type II collagen synthesis in human blood samples. A high sensitivity electrochemiluminescence immunoassay, hsPRO-C2, was developed using a well-characterized antibody against human PIIBNP. Human cartilage explants from replaced osteoarthritis knees were cultured for ten weeks in the presence of growth factors, insulin-like growth factor 1 (IGF-1) or recombinant human fibroblast growth factor 18 (rhFGF-18). The culture medium was changed every seven days, and levels of PIIBNP, PIIANP, and matrix metalloproteinase 9-mediated degradation of type II collagen (C2M) were analyzed herein. Serum samples from a cross-sectional knee osteoarthritis cohort, as well as pediatric and rheumatoid arthritis samples, were assayed for PIIBNP and PIIANP. Western blot showed that the antibody recognized PIIBNP either as a free fragment or attached to the main molecule. Immunohistochemistry demonstrated that PIIBNP was predominately located in the extracellular matrix of the superficial and deep zones and chondrocytes in both normal and osteoarthritic articular cartilage. In addition, the hsPRO-C2 immunoassay exhibits acceptable technical performances. In the human cartilage explants model, levels of PIIBNP, but not PIIANP and C2M, were increased (2 to 7-fold) time-dependently in response to IGF-1. Moreover, there was no significant correlation between PIIBNP and PIIANP levels when measured in knee osteoarthritis, rheumatoid arthritis, and pediatric serum samples. Serum PIIBNP was significantly higher in controls (KL0/1) compared to OA groups (KL2/3/4, p = 0.012). The hsPRO-C2 assay shows completely different biological and clinical patterns than PIIANP ELISA, suggesting that it may be a promising biomarker of cartilage formation.

Highlights

Osteoarthritis (OA) is generally characterized by a slowly progressive degeneration of articular cartilage [1,2]
There are several biomarkers available in the field of OA which assess cartilage degradation in the serum or urine of patients; there are very few that detect the formation of cartilage
The specificity of the antibody to PIIBNP was further confirmed by Western blot using recombinant PIIBNP, recombinant PIIANP, and human articular cartilage tissue

Summary

Introduction

Osteoarthritis (OA) is generally characterized by a slowly progressive degeneration of articular cartilage [1,2]. The extracellular matrix (ECM) in articular cartilage consists primarily of proteoglycans and type II collagen [3,4]. The ECM is characterized by a low turnover of collagens, whereas in OA, the homeostasis is disrupted, leading to an imbalance between cartilage formation and degradation [5]. There are no disease-modifying OA drugs (DMOADs) available. Those in development are either preventing cartilage breakdown (anti-catabolic pathways) or targeting cartilage formation (anabolic pathways). According to the list of DMOADs in past and present clinical development registered in clinicaltrials.gov, the majority of these trials are based on anti-catabolic or anti-inflammatory approaches [8]. There is an unmet need in DMOAD development, where non-invasive biomarkers of cartilage formation can provide an early indication of drug efficacy

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