Impact of mechanical loading of osteoblasts on chondrocytes: a novel murine model for assessing bone/cartilage communication

Purpose: Osteoarthritis (OA) is characterized by joint degeneration, leading to a progressive loss of articular cartilage in response to mechanical and biochemical factors. However, OA is a complex disease not limited to cartilage degeneration. Indeed, several sets of in vitro and in vivo experiments suggest that subchondral bone remodeling could initiate and/or contribute to cartilage loss in OA through a bone/cartilage interplay. The aim of this study was to demonstrate the ability of soluble mediators released by loaded osteoblasts to shift the articular chondrocyte phenotype from a resting to a pro-degradative phenotype through a novel and unique bone/cartilage communication model. Methods: Thanks to a three dimensional (3D) culture model, murine calvaria osteoblasts were submitted to compression in Biopress Flexercell plates (1.7 MPa, 1Htz during 24h). Then, media from loaded or unloaded osteoblasts were used to treat primary mouse articular chondrocytes for 24h. Chondrocyte expression and secretion of matrix metalloproteinases (MMP-3 and -13), MMPs inhibitors (TIMP-1, -2 and -3) and extracellular matrix proteins (type II collagen, aggrecan) were analyzed by real time RT-PCR, ELISA and immunoblotting. Osteoblast conditioned media were then analyzed by iTRAQ (isobaric tags for relative and absolute quantification), a quantitative proteomic approach. Results: Media from compressed osteoblast (CM) strongly induced MMP-3 and -13 chondrocyte mRNA expression (Table). Consistently, CM also significantly stimulated the releases of MMP-3 and -13 by chondrocytes (respectively 10.6±0.75 fold, p<0.001, and 6.5±2.1 fold, p<0.01 compared to control). In addition, CM enhances TIMP-1 expression whereas it strongly inhibited TIMP-2 and -3 expressions (Table). Osteoblast conditioned media also affected cartilage matrix proteins expressions. Indeed, CM downregulated aggrecan and type II collagen mRNA levels (Table). Effects of CM on cytosolic type II collagen protein amounts were confirmed by western blot (a decrease of 31±9%, p<0.01). Finally, in order to identify osteoblast soluble mediators responsible for this chondrocyte phenotype, osteoblast conditioned media were analyzed by iTRAQ®. This advanced and sophisticated new proteomic technique allowed identification of 105 proteins secreted by osteoblasts among which only 10% were modified in response to compression. Conclusions: These results support the hypothesis that soluble mediators released by bone cells submitted to a mechanical stress promote chondrocyte activation leading to the release of pro-degradative mediators. Analysis of these soluble mediators will open a new area of novel biomarkers relying on abnormal bone-cartilage communication.TableEffects of media from uncompressed (UCM) or compressed (CM) osteoblasts on MMPs, TIMPs and matrix proteins chondrocytes expression. Results are expressed in fold compared to control (non-stimulated chondrocytes).MMP-3MMP-13TIMP-1TIMP-2TIMP-3Collagen IIAggrecanUCM5.9±3 (ns)3.4±0.4 (ns)1.5±0.4 (ns)0.7±0.2 (p<0.01)0.6±0.1 (p<0.001)0.7±0.3 (ns)0.7±0.3 (ns)CM50.9±14.5 (p<0.001)18.3±10.9 (p<0.01)5.2±1.6 (p<0.001)0.5±1.6 (p<0.001)0.1±0.05 (p<0.001)0.4±0.1 (p<0.01)0.2±0.1(p<0.001) Open table in a new tab