Campillo-Gimenez L.1, Frochot V.2, Bianchi A.3, Larédo J.-D.4, Chappard C.5, Combes C.6, Cohen-Solal M.1,7, Lioté F.1,7, Bazin D.2,8, Daudon M.2, Ea H.-K.1,7
1INSERM UMR1132, Paris, France. 2AP-HP, Tenon Hospital, Department of “Explorations fonctionnelles multidisciplinaires”, Paris, France. 3UMR7365, Lorraine University, Nancy, France. 4AP-HP, Lariboisière Hospital, Department of osteoarticular radiology, Paris, France. 5IMOSAR, PRES Paris Sorbonne Cité. 6ENSIACET, CIRIMAT, INPT-UPS-CNRS, Toulouse, France. 7AP-HP, Lariboisière Hospital, Rheumatology Department, Paris, France. 8LCMPC, UMR7574, Collège de France, Paris, France.
Background: Cartilage mineralization is commonly found in osteoarthritic cartilages. The two main types of calcium-containing (CaC) crystals are basic calcium phosphate (BCP) and calcium pyrophosphate (monoclinic and triclinic dihydrate, m- and t-CPPD) crystals. Meniscus calcification is frequently observed on conventional radiographs but the precise type of CaC crystals has never been assessed. We aimed to characterize meniscus calcification along with knee cartilages. In parallel, we assessed the expression of several proteins involved in the regulation of extracellular phosphate and pyrophosphate (Pi/PPi) concentrations.
Methods: Menisci, tibia (from femoro-tibia-FT- and peroneo-tibia-PT-) cartilages harvested from cadaveric donors (n=30, mean age 84 years old) and menisci harvested from patients undergoing total knee replacement (surgical menisci, n=18, mean age 74 years old) were subjected to digital-contact radiography, FTIR and scan electronic microscopy. Three different portions of cadaveric menisci (anterior, medium and posterior), superficial and deep tibia cartilages were analyzed. Surgical menisci were separated into 2 portions: one was used to isolate ARN and qPCR analysis and the other was paraffin-embedded for immunohistochemistry analysis. Crystal deposits were analyzed with the Spotlight 400 FTIR imaging System in the mid infrared spectral range to obtain infrared maps of tissue slides at high spatial resolution.
Results: In cadaveric samples, calcification, CPPD, BCP and both CaC crystals, respectively, were identified in lateral (85%, 3%, 38%, 44%) and medial (88%, 9%, 32%, 47%) menisci, FT cartilages (55%, 6%, 48%, 0%) and PT cartilages (87%, 43%, 20%, 23%). t-CPPD crystals were identified in 88% in menisci and 100% in FT/PT cartilages. m-CPPD crystals were only detected in menisci in 11%. Both m- and t-CPPD crystals were identified in 33% in menisci. In surgical meniscus samples, 42% were calcified containing in 100% CPPD crystals (t-CPPD 67%) and in 16% both CPPD and BCP crystals. Immunostaining and gene expression assessed by qPCR for ANK, PC-1, TNAP, MGP, Pit-1 and CD73 were similar between calcified and non-calcified menisci.
Conclusion: Calcification was widespread in knee joints involving meniscus, FT and PT containing both CPPD and BCP crystals. t-CPPD crystals were the most frequent phase. In surgical menisci, the expression of genes involved in the metabolism of Pi and PPi was unchanged whatever the calcification state.