Osteoclastic differentiation and resorption is modulated by bioactive metal ions Co2+, Cu2+ and Cr3+ incorporated into calcium phosphate bone cements.

Anne Bernhardt,Uwe Gbureck,Michael Gelinsky,Martha Schamel,Dominique Heymann

doi:10.1371/journal.pone.0182109

Abstract

Biologically active metal ions in low doses have the potential to accelerate bone defect healing. For successful remodelling the interaction of bone graft materials with both bone-forming osteoblasts and bone resorbing osteoclasts is crucial. In the present study brushite forming calcium phosphate cements (CPC) were doped with Co2+, Cu2+ and Cr3+ and the influence of these materials on osteoclast differentiation and activity was examined. Human osteoclasts were differentiated from human peripheral blood mononuclear cells (PBMC) both on the surface and in indirect contact to the materials on dentin discs. Release of calcium, phosphate and bioactive metal ions was determined using ICP-MS both in the presence and absence of the cells. While Co2+ and Cu2+ showed a burst release, Cr3+ was released steadily at very low concentrations (below 1 μM) and both calcium and phosphate release of the cements was considerably changed in the Cr3+ modified samples. Direct cultivation of PBMC/osteoclasts on Co2+ cements showed lower attached cell number compared to the reference but high activity of osteoclast specific enzymes tartrate resistant acid phosphatase (TRAP), carbonic anhydrase II (CAII) and cathepsin K (CTSK) and significantly increased gene expression of vitronectin receptor. Indirect cultivation with diluted Co2+ cement extracts revealed highest resorbed area compared to all other modifications and the reference. Cu2+ cements had cytotoxic effect on PBMC/osteoclasts during direct cultivation, while indirect cultivation with diluted extracts from Cu2+ cements did not provoke cytotoxic effects but a strictly inhibited resorption. Cr3+ doped cements did not show cytotoxic effects at all. Gene expression and enzyme activity of CTSK was significantly increased in direct culture. Indirect cultivation with Cr3+ doped cements revealed significantly higher resorbed area compared to the reference. In conclusion Cr3+ doped calcium phosphate cements are an innovative cement modification because of their high cytocompatibility and support of active resorption by osteoclasts.

Highlights

The development of synthetic calcium phosphate based bone graft materials includes the incorporation of bioactive molecules, like growth factors and drugs to generate osteoinductive properties
The following cement modifications were included into this study: 10 mmol Cu2+/g β-TCP (“Cu10”), 10 mmol Co2+/g β-TCP (“Co10”), 10 mmol Cr3+/g β-TCP (“Cr10”), 50 mmol Cr3+/g β-TCP (“Cr50”) cement without metal ions was prepared as a reference
After 16 days of cultivation activity of tartrate resistant acid phosphatase (TRAP), carbonic anhydrase II (CAII) and cathepsin K (CTSK) was detected on all cement modifications except Cu10 (Fig 2)

Summary

Introduction

The development of synthetic calcium phosphate based bone graft materials includes the incorporation of bioactive molecules, like growth factors and drugs to generate osteoinductive properties. Co2+ doping of calcium phosphate was shown to have proangiogenic effects [4,5]. Cu2+ modification of a calcium phosphate ceramic resulted in an enhanced and directed vascularization [6]. In a previous study we have analyzed the impact of Co2+, Cu2+ and Cr3+ doping of biomimetic calcium phosphate bone cement on materials properties, as well as on proliferation and osteogenic differentiation of human mesenchymal stromal cells in vitro [7]. A positive effect of doping with Cr3+ and, to a less extend, of Cu2+ on proliferation and osteogenic differentiation was found

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