The effect of calcium crystals on chondrocyte phenotype and signalling in osteoarthritis and chondrocalcinosis

 

Sina Stücker, Jennifer Gehring, Christoph H. Lohmann, Prof. Dr., Jessica Bertrand, Prof. Dr.

Otto-Von-Guericke University Magdeburg

 

Status : Osteoarthritis (OA) and chondrocalcinosis (CC) are common arthropathies associated with the accumulation of calcium-containing crystals in the cartilage and synovial membrane. Basic calcium phosphate (BCP) crystals are commonly present in OA, whereas calcium pyrophosphate dehydrate (CPP) crystals are a key feature of CC. Further, BCP crystals have been linked to wnt signaling and hypertrophic differentiation of chondrocytes, whereas CPP crystals may be responsible for chondrocyte senescence. Despite their association with different types of calcium crystals, OA and CC share similar clinical phenotypes including a progressive degeneration and calcification of articular cartilage.

Objective : The aim of this study is to investigate the differential role of calcium crystals in OA and CC regarding morphogen binding, matrix sulfation and signal transduction in chondrocytes.

Methodology : Based on plain radiographs OA and CC was assessed in the knee joints of 141 OA patients. Calcium crystals were visualized using scanning electron microscopy and von kossa staining of cartilage tissue. To assess the effect of calcium crystals on the chondrocyte phenotype, human chondrocytes were incubated with BCP or CPP crystals followed by rt-qpcr for various phenotype marker genes. The expression of calcification-mediating enzymes in OA and CC cartilage was analyzed by rt-qpcr. Morphogen binding to calcium crystals was assessed via incubation with different proteins, followed by western blot analysis. To determine the effect of protein lipidation in crystal binding, proteins were delipidated before incubation with calcium crystals. Cartilage matrix sulfation was analysed using alcian pas staining and rt-qpcr. In order to assess the effect of matrix sulfation on signal transduction in chondrocytes, proteoglycan sulfation was reduced via naclo3 treatment and western blot for β-catenin.

Findings : In a cohort of 141 OA patients, 35 patients showed radiologically detectable CC. However, histological signs of CC were more frequent. Interestingly, BCP and CPPd crystals were present in cartilage and synovial membrane of OA and CC patients, respectively. Calcium crystals increased the expression of hypertrophy markers genes including mmp-13 and collagen 10 in vitro. In line with this, our preliminary data suggests that calcium crystals may sequester morphogens. Both BCP and CPP crystals were able to bind wnt3a and il1-b in vitro but CPP showed a stronger binding affinity. Green fluorescent protein (gfp) did not bind to the crystals and delipidation decreased protein binding, suggesting protein lipidation as a mediating factor in morphogen binding to calcium crystals. Both OA and CC cartilage showed similar expression levels of sulfation-mediating enzymes. Reducing proteoglycan sulfation decreased β-catenin-dependent wnt activation in primary human chondrocytes in vitro.

Conclusion : Our preliminary data suggests that CPP and BCP crystals affect the chondrocyte phenotype in vitro and share a similar role in morphogen binding and matrix sulfation in cartilage. Further, OA and CC cartilage show similar matrix sulfation which may promote aberrant signal transduction in chondrocytes. However, the differential effects of calcium crystals and matrix sulfation on signalling pathways and the chondrocyte phenotype require further investigation.

 

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