Cartilage turnover reflected by metabolic processing of type II collagen: a novel marker of anabolic function in chondrocytes

Abstract

The aim of this study was to enable measurement of cartilage formation by a novel biomarker of type II collagen formation. The competitive enzyme-linked immunosorbent assay (ELISA) Pro-C2 was developed and characterized for assessment of the beta splice variant of type II procollagen (PIIBNP). This is expected to originate primarily from remodeling of hyaline cartilage. A mouse monoclonal antibody (Mab) was raised in mouse, targeting specifically PIIBNP (QDVRQPG) and used in development of the assay. The specificity, sensitivity, 4-parameter fit and stability of the assay were tested. Levels of PIIBNP were quantified in human serum (0.6-2.2 nM), human amniotic fluid (163-188 nM) and sera from different animal species, e.g., fetal bovine serum (851-901 nM) with general good linearity (100% (SD 7.6) recovery) and good intra- and inter-assay variation (CV% < 10). Dose (0.1 to 100 ng/mL) and time (7, 14 and 21 days) dependent release of PIIBNP were evaluated in the conditioned medium from bovine cartilage explants (BEX) and human cartilage explants (HEX) upon stimulation with insulin-like growth factor (IGF-1), transforming growth factor (TGF)-β1 and fibroblastic growth factor-2 (FGF-2). TGF-β1 and IGF-1 in concentrations of 10-100 ng/mL significantly (p < 0.05) induced release of PIIBNP in BEX compared to conditions without treatment (WO). In HEX, IGF-1 100 ng/mL was able to induce a significant increase of PIIBNP after one week compared to WO. FGF-2 did not induce a PIIBNP release in our models. To our knowledge this is the first assay, which is able to specifically evaluate PIIBNP excretion. The Pro-C2 assay seems to provide a promising and novel marker of type II collagen formation.

Figure 1

It was found that the NB443-3-2-1 antibody showed great potential for development of a competitive ELISA assay. The antibody was very specific towards peptides of the targeted PIIBNP sequence of which 120 ng/mL peptide was enough to displace the signal, whereas it did not recognize the PIIANP or the rat/mouse splice variant.

Figure 2

PIIBNP evaluated in supernatant of BEX cultured for 3 weeks in the presence 0.1–100 ng/mL of IGF-1, FGF-2 or TGF-β1. BEX cultured without treatment (WO) were applied for comparison of treatment effect. The experiment was performed twice, shown as (A) and (B) respectively to investigate if the results were reproducible. * p < 0.05, ** p < 0.005.

Figure 3

Pro-C2 measured in supernatant of BEX cultured for 3 weeks in presence of catabolic stimuli Oncostatin M + TNF-α (O+T) compared to unstimulated (WO) explants. The experiment was performed twice (A) and (B) respectively to investigate if the results were reproduceable. * p < 0.05, ** p < 0.005.

Figure 4

Western blot of the NB443-3-2-1 antibody detected fragments in supernatant of the BEX cultures.

Figure 5

Pro-C2 evaluated in supernatant from HEX cultured in presence 0.1–100 ng/mL of IGF-1, FGF-2 or TGF-β1. HEX cultured without treatment (WO) was applied for comparison of treatment effect. The red line indicates the average background level of 10 nM originating from the FBS of the media. Error bars are shown as SEM. * p < 0.05.

Figure 6

Pro-C2 evaluated in supernatant from HEX cultured with or without presence of 100 ng/mL IGF-1 for 3 weeks. (A) The PIIBNP concentration in supernatants from HEX stimulated by 100 ng/mL IGF-1 3 times per week; (B) The concentration in supernatants of HEX stimulated once a week. Error bars are shown as SEM. * p <0.05.