Dissolution of calcium pyrophosphate crystals by polyphosphates: an in vitro and ex vivo study

Abstract

Objective: To determine the dissolving ability (DA) of linear pentasodium tripolyphosphate (PSTP), cyclic trisodium metaphosphate (TSMP), polymeric sodium metaphosphate (SMP) on synthetic crystals of calcium pyrophosphate dihydrate (CPPD) and on crystalline aggregates of menisci from patients with chondrocalcinosis (CC).

Methods: Synthetic CPPD crystals were mixed with phosphate buffered saline (PBS), which contained the different polyphosphates, for one hour at 37 degrees C. The calcified menisci were obtained from the knees of four female patients with CPPD disease who underwent total arthroscopic meniscectomy for degenerative meniscal lesions. Meniscal cryosections and fragments were incubated in SMP (15 mg/ml PBS) at 37 degrees C for one hour and 24 hours, respectively. Histological evaluation on meniscal samples after polyphosphate incubation was carried out by ordinary transmitted light microscopy and polarised light microscopy. The dissolution of CPPD crystals by polyphosphates was assessed by atomic absorption spectroscopy, which determined the amount of calcium liberated from synthetic crystals and meniscal fragments. Cytotoxicity of SMP was evaluated by tetrazolium salt assay and by an ultrastructural study on cultured chondrocytes.

Results: SMP and PSTP showed higher DA on CPPD crystals than TSMP. Analysis of the DA values at increasing concentrations of SMP showed that a concentration of 15 mg/ml completely dissolved 2.0 mg CPPD crystals. The solution of meniscal CPPD crystals showed a significant increase of calcium concentration after three hours and 24 hours of SMP incubation (p=0.0001; Kruskal-Wallis analysis of variance) compared with fragments incubated in PBS control solution. Macroscopic and microscopic evaluation of meniscal specimens showed a notable reduction of CPPD deposits. A 50% inhibitory dose on cultured chondrocytes was reached at the maximum concentration of SMP used in this work (15 mg/ml); ultrastructural analysis did not show morphological alterations in the treated cells.

Conclusion: The results of this study indicate that linear polyphosphates are effective in dissolving both synthetic and ex vivo CPPD crystal aggregates. This suggests a potential therapeutic use for these molecules in the treatment of symptomatic CC.

Figure 1

Structural formulas for (A) pyrophosphate, O(PO₃ )₂⁴⁻; (B) a linear tripolyphosphate, O(PO₃ )₃⁵⁻; (C) cyclic trimetaphosphate, (PO₃ )₃³⁻; (D) polymeric metaphosphate (PO₃ )_n.

Figure 2

Effect of increasing concentrations of polymeric sodium metaphosphate (SMP) on the solubilisation of synthetic CPPD crystals (2 mg) after one hour of incubation at 37°C. DA = dissolving ability. The bars show the mean (SD) of DA values obtained by three separate experiments. Values in brackets show the percentage of dissolution.

Figure 3

A calcified human meniscal section (7 µm) (A) before treatment; (B) after one hour of treatment with 15 mg sodium metaphosphate (SMP)/ml phosphate buffered saline (PBS), the crystalline aggregates of calcium pyrophosphate dihydrate (CPPD) are dissolved; (C) before treatment; (D) after one hour of treatment with PBS solutions, the crystalline aggregates of CPPD are not dissolved. (Ordinary transmitted light microscopy original magnification ×10.)

Figure 4

Calcified human meniscus fragments (A) before treatment; (B) after 24 hours of incubation with 15 mg sodium metaphosphate (SMP)/ml phosphate buffered saline (PBS), the crystalline aggregates of calcium pyrophosphate dihydrate (CPPD) are significantly decreased.

Figure 5

Variations of calcium concentrations after one hour, three hours, and 24 hours by incubating meniscal samples (n=4) in sodium metaphosphate (SMP) (15 mg/ml) and phosphate buffered saline (PBS) (p=0.0001 Kruskal-Wallis analysis of variance). *p<0.05: SMP 24 hours v SMP three hours; SMP 24 hours v SMP one hour; SMP three hours v SMP one hour; NS = not significant.

Figure 6

Transmission electron microscopy of human chondrocytes cultured in vitro: before (A, C) and after (B, D) treatment (24 hours of treatment with 15 mg sodium metaphosphate (SMP)/ml phosphate buffered saline (PBS)); cells did not shown structural abnormalities, including the capsule, the pericellular matrix, the cellular membrane, the mitochondria, the inclusions, the smooth and rough reticulum, the Golgi, the lysosomes, and the nucleus. (Original magnification figs A, C ×6000; figs B, D ×20 000.)