Background/Purpose: Calcification of cartilage is a common finding during osteoarthritis (OA) and is directly linked to the severity of cartilage degradation. We have found in a previous study that NPP1 is an important player in OA associated cartilage calcification in mouse and humans. The observed cartilage changes resemble aspects of endochondral ossification. In this study we aim to investigate the matrix changes and calcification of cartilage during the development of OA- like changes in mice. 

Methods: The tip-toe walking (ttw/ttw) mouse that carries a mutation in the enpp1 gene encoding for NPP1 was used as a natural model of OA. Using von Kossa staining in combination with Safranin-orange staining of knee sections we assessed the calcification of articular cartilage and the severity of OA using the Mankin-Score over a time course from 4 to 22 weeks. We analysed the compostion of cartilage matrix using toluidine blue, alcian blue/ PAS staining. To investigate cartilage matrix remodelling we performed immunohistological stainings for collagen II, collagen X and the aggrecan cleavage marker BC-3. We performed the same stainings in knee joint sections of wild type mice with induced OA (DMM model). The influence of basic calcium phospahe (BCP) crystals on neonatal chondrocytes was investigated in micro mass cultures with alcian blue and alizarin red staining. Using Western Blot for β- catenin and pCamKII we investigated the activation of WNT signalling. 

Results: We found a loss of proteoglycans in the joint cartilage of ttw/ttw mice already at an age of 8 weeks, whereas no changes of cartilage matrix were detectable in wild type mice. An increased Mankin Score in ttw/ttw compared to wild type mice also reflected these changes. Early matrix remodelling in ttw/ttw cartilage was followed by an increased calcification starting at the border to the subchondral bone. The straight border of the tide mark gets broken down by multiple calcified chondrocytes that give the border a wave-like appearance. Looking at the matrix changes in more detail we found an increase in sulphated proteoglycans in the ttw/ttw cartilage as well as in the induced OA cartilage. The same pattern was observed in the growth plate in the area of hypertrophic chondrocytes. We found an increased expression of collagen X in the cartilage and collagen II was down-regulated with increasing age and OA severity. Using neonatal chondrocytes of wild type and ttw/ttw mice we found that BCP crystals induce production of highly sulphated matrix and reduce proliferation of chondrocytes. Chondrocytes of ttw/ttw mice show an increase in β- catenin, which was also observed in wild type chondrocytes stimulated with BCP crystals. 

Conclusion:

We conclude from our data that matrix remodelling precedes cartilage calcification. The calcification seems to be associated with activation of canonical WNT signalling. Taken together, the data support the notion that OA is characterized by the re-initiation of developmental programms associated with endochondral ossification.

---

« Back to 2013 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/cartilage-matrix-remodeling-and-activation-of-canonical-wnt-signaling-precedes-calcification-and-oa-like-changes-in-mice/