Sina Stücker, Franziska Kosslowski, Christoph H. Lohmann, Jessica Bertrand
Department of Orthopaedic Surgery
Status: Calcification is a hallmark in osteoarthritis caused by the accumulation of intra-articular basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP) crystals. While BCP crystals are associated with osteoarthritis (OA), CPP crystal deposition is characteristic for chondrocalcinosis (CC), radiographically presenting as dense calcifications of the joint cartilage or menisci. On a cellular level, BCP crystals have been linked to Wnt signalling and chondrocyte hypertrophy, while CPP crystals may rather be associated with a senescent chondrocyte phenotype. However, the process of calcification is not well understood. Therefore, this study aims to characterize calcification of cartilage and synovial membrane in a cohort of OA patients with and without CC, particularly focussing on location, crystals type and potential mechanisms leading to calcification.
Methodology: OA and CC were determined based on radiological assessment of the knee joint. Von Kossa staining was used to visualize and quantify calcifications of cartilage and synovial membrane. Safranin Orange and H&E stainings were performed to assess histological cartilage destruction and synovial inflammation, using OARSI and Krenn-Scores. The type of calcification was determined using Raman spectroscopy. To investigate the mechanisms of calcification, the gene expression of enzymes regulating extracelluar phosphate and pyrophosphate levels were analysed via RT-qPCR.
Findings: In a cohort of 97 OA patients undergoing knee replacement surgery, 50 patients were radiologically diagnosed with CC. However, histological calcification was more frequent and did not always coincide with the radiological diagnosis. Heavy calcifications occurred in cartilage and synovial membrane of both OA and CC patients. Therefore, we quantified the von Kossa-stained calcified area in both tissues and classified patients based on the grade of calcification. In general, cartilage was more calcified than synovial membrane, particularly in CC patients. Cartilage destruction and synovial inflammation did not correlate with the grade of calcification in the respective tissue. BCP was the predominant crystal type in cartilage and synovial membrane. In CC joints, both CPP and BCP could be identified, sometimes coexisting in the same joint or even the same tissue. CC joints also show decreased TNAP expression compared to OA joints as well as decreased synovial membrane expression of CD73. Gene expression of ENPP1 and ANKH increased with general calcification in cartilage. However, we could not observe any correlation between gene expression and the type of calcification as determined by Raman spectroscopy.
Significance: We show that calcifications occur in cartilage and synovial membrane, independently from the radiological diagnosis, suggesting that X-rays alone may not allow a reliable detection of joint tissue calcification. BCP was the predominant crystal type, whereas CPP crystals were only found in a minority of patients, sometimes co-existing with BCP crystals within the same joint or tissue. Cartilage destruction and synovial inflammation did not correlate with the grade or type of calcification in the respective tissue. Further, we could not correlate gene expression of ENPP1, TNAP, ANKH and CD73 in cartilage and synovial membrane with the grade or type tissue calcification, highlighting the need for further investigation into the mechanisms of joint tissue calcification.