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.
Table 1.
Release of metal ions from doped calcium phosphate cements.
Cell culture supernatants from cement samples (6mm diameter, 1 mm height) during direct cultivation with human PBMC/osteoclasts were analysed by ICP-MS.
Fig 3.
Release of PO43-, Ca2+ (d-f) and Cr3+ from Cr3+ doped CPC.
ICP-MS was used to quantify the release of PO43-(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 PO43- was subtracted as blank.
Fig 4.
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.
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.