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

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.

Fig 1. Osteoclast formation on the surface of the cements.

CLSM images of osteoclasts differentiated from human PBMC for 16 days under stimulation with M-CSF and RANKL on brushite cement as reference, and on Cu10, Co10, Cr10 and Cr50 modifications. Nuclei are stained in blue, and actin cytoskeleton is stained in green. Scale bars represent 50 μm.

Fig 2. DNA content, TRAP, CAII and CTSK activities of osteoclasts after direct cultivation on CPC.

Osteoclasts were differentiated for 16 days directly on the surface of the CPC (reference) and Cu10, Co10, Cr10 and Cr50 modifications. PBMC of 5–9 donors were included into the analysis. Each box shows the 25th to 75th percentile of the measured data. Squares (□) represent mean values, horizontal bars inside the box show the median value, while upper and lower bars indicate the upper and lower values within 1.5 times the inter-quartile range from the upper and lower quartile. Individual data points are shown on the left side of each box. * statistically significant differences (p<0.05) to all other sample types.

Fig 3. Release of PO43-, Ca²⁺ (d-f) and Cr³⁺ from Cr³⁺ doped CPC.

ICP-MS was used to quantify the release of PO₄^3-(a-c) Ca²⁺ (d-f) and Cr³⁺ (g, h) into cell culture medium in the absence of cells (passive) and during cultivation of PBMC/osteoclasts (active). For passive release 6 samples of the respective cement modification were incubated in each 200 μl of cell culture medium. For active release PBMC of three different donors were seeded on 18 cement samples per modification in total (6 samples per donor). Medium of all samples was changed at days 1, 2, 6, 9, 13 and 16. Three medium samples per donor, modification and cultivation day were pooled to get enough volume for analysis. Cell culture medium concentration of Ca²⁺and PO₄^3- was subtracted as blank.

Fig 4. Gene expression analysis.

Expression of osteoclast-specific genes was analysed after 16 days of osteoclast cultivation on CPC (reference) and Co10, Cr10 and Cr50 modifications A) TRAP, B) CAII, C) VNR and D) CTSK. Six cement samples of each modification were seeded with cells and lysates of three samples were pooled to obtain two data points per modification and donor. Cells of two donors were included into the analysis. PCR products were visualized in agarose gels; the intensities of the gel bands was analysed using the open source software Fiji and related to the intensity of the GAPDH bands. Furthermore, the band intensities of the reference cement were set to 1 for each individual donor. * statistically significant differences (p<0.05) to the reference.

Fig 5. SEM analysis of resorption pits.

Representative SEM images of resorption pits after 16 days cultivation of PBMC derived human osteoclasts on dentine slices in the presence of different cement extracts (reference = non-modified CPC) compared to cell culture medium (control). A, B, C represent cells of three different PBMC donors, scale bar represents 500 μm.

Fig 6. Average resorbed area on dentin slices.

Osteoclasts were differentiated in the presence of different cement extracts (reference = non-modified CPC) compared to cell culture medium (control). Image J software was used to calculate resorbed area of six to eight SEM images with 500 x magnification, A, B, C represent cells of three different donors.

Fig 7. TRAP staining.

Osteoclasts were differentiated in the presence of different cement extracts (reference = non-modified CPC) compared to cell culture medium (control) on TCPS for 16 days. TRAP activity stained in purple, nuclei are counterstained in blue. Scale bars represent 200 μm.