Sina Stücker, Franziska Koßlowski, Adrian Buchholz, Christoph H. Lohmann, Jessica Bertrand
Affiliation(s):
Department of Orthopaedic Surgery, Otto-von-Guericke-University, Magdeburg, Germany
Objective: Calcification of articular cartilage and synovium is a common symptom in osteoarthritic knee joints and is closely related to the progression of the disease. Involved crystal types are difficult to identify and differentiate, resulting in inconsistent data on prevalence and distribution of calcified deposits. Extracellular phosphate and pyrophosphate levels play an important factor in the formation of different crystal types and are regulated by various metabolizing enzymes. This study analyses BCP and CPP calcifications in cartilage and synovium of OA patients undergoing knee replacement surgery.
Methodology: Combining multiple imaging methods including conventional radiography, histology and Raman spectroscopy, this study provides a comprehensive analysis of BCP and CPP based calcification, its frequency and distribution in cartilage and synovial membrane samples of 94 OA patients undergoing knee replacement surgery. To elucidate the process of BCP and CPP formation, we investigated the regulation of the phosphate metabolism by measuring the gene expression, protein expression and activity of various phosphatases and nucleotidases using qRT-PCR, immunohistochemistry and colorimetric activity assays.
Findings: 35% of patients displayed radiologically detectable calcifications. Histological analysis revealed calcifications in 88% of cartilage and 57% synovial samples. Here, BCP crystals usually formed variably sized deposits of brittle appearance, located on the cartilage surface or within synovial tissue. In contrast, CPP crystals accumulated in larger needle-shaped clusters, condensing to circular pockets below the cartilage surface or within synovial tissue. Raman spectroscopy detected BCP crystals in 75% of cartilage and 43% of synovial samples. Merely 18% of cartilage and 15% of synovial samples contained CPP crystals, often in vicinity to BCP deposits. Gene expression levels of Alkaline phosphatase (ALP), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), ankylosis homologue (ANKH) and 5′-nucleotidase (CD73) was increased in BCP calcified compared to non-calcified cartilage. However, we could not detect differences in gene expression in BCP- versus CPP-calcified cartilage. Expression levels in synovium did not differ either. On protein level, we could not detect significant differences in ALP, ENPP1, ANKH or CD73 expression in BCP- compared to CPP-calcified cartilage or synovium. Activity level of NPP1 and ALP were decreased in calcified compared to non-calcified cartilage, whereas inorganic phosphate levels were increased.