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Calcium pyrophosphate dihydrate crystal deposition disease: characterization and IgG binding properties of monoclinic calcium pyrophosphate dihydrate crystals Winternitz, Charles Ilderton


Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease is caused by CPPD crystals which are released into the synovial fluid of affected joints. These crystals are highly surface active and are quickly coated by proteins present in the joint cavity, particularly immunoglobulin G (IgG). Through a series of events the coated crystals interact with neutrophils causing their lysis and the resultant release of lysosomal enzymes into the joint cavity, which leads to inflammation and pain for the affected patient. Calcium pyrophosphates have been reported to exist in a variety of hydrate and polymorphic forms including triclinic, monoclinic and hexagonal dihydrates (t-CPPD, m-CPPD and h-CPPD respectively), and an orthorhombic tetrahydrate (o-CPPT). It was reported that h-CPPD formed from the dehydration of o-CPPT. At room temperature and pressure the stable crystal form is t-CPPD and only t-CPPD and m-CPPD crystals have ever been isolated from joints. Previous efforts to study the crystals which cause this disease have concentrated on the stable t-CPPD form even though m-CPPD has also been shown to have inflammatory potential. The purpose of this work was to synthesize m-CPPD, o-CPPT and h-CPPD crystals and characterize their physicochemical properties and then to begin studying the role of m-CPPD crystals in CPPD crystal deposition disease by comparing the adsorption of IgG protein onto the surface of m-CPPD and t-CPPD crystals. A reliable method for the synthesis of m-CPPD crystals was obtained. Synthesis of orthorhombic calcium pyrophosphate tetrahydrate (o-CPPT) was also achieved but the method proved unreliable and only a limited supply of o-CPPT crystals was available. As a result h-CPPD crystals were not synthesized. The t-CPPD, m-CPPD, and o-CPPT crystals were characterized and compared using X-ray powder diffraction, light and scanning electron microscopy, and Fourier transform infrared spectroscopy. Different methods of calculation failed to confirm the unit cell dimensions of m-CPPD crystals. Both the m-CPPD and o-CPPT crystals were needleshaped while the t-CPPD crystals appeared as parallelograms under the microscope. The m-CPPD crystals had a smaller particle size compared to t-CPPD by a factor of about 10, meaning they had a larger surface area per unit volume than the t-CPPD crystals. Thermal analysis using differential scanning calorimetry confirmed the hydration level of the crystals. Zeta potentials for m-CPPD and t-CPPD crystals in deionized water were found to be -18.8 mV and -35.3 mV respectively. The solubilities of triclinic and monoclinic CPPD were measured in terms of Ca2+ concentration in solution which was measured using either an ion selective calcium electrode, atomic absorption spectroscopy, or complexiometric titration with EDTA. It was found that m-CPPD crystals were 10 times more soluble at low pH than at neutral pH. The solubility of both m-CPPD and t-CPPD crystals was found to increase with temperature. Throughout the entire temperature range studied m-CPPD crystals had a higher solubility than t-CPPD crystals confirming that m-CPPD is the metastable form of CPPD. Through extrapolation of the data using a Van't Hoff plot the transition temperature between the two polymorphic forms was estimated to be 163° C. Adsorption of IgG to m-CPPD and t-CPPD crystals surfaces was studied using fluorescent labelled FITC-IgG. The binding isotherms produced were shown to fit the Freundlich equation and the binding constants were explained in terms of heterogeneous binding.

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