In vitro studies on the effect of particle size on macrophage responses to nanodiamond wear debris

Abstract

Nanostructured diamond coatings improve the smoothness and wear characteristics of the metallic component of total hip replacements and increase the longevity of these implants, but the effect of nanodiamond wear debris on macrophages needs to be determined to estimate the long-term inflammatory effects of wear debris. The objective was to investigate the effect of the size of synthetic nanodiamond particles on macrophage proliferation (BrdU incorporation), apoptosis (Annexin-V flow cytometry), metabolic activity (WST-1 assay) and inflammatory cytokine production (qPCR). RAW 264.7 macrophages were exposed to varying sizes (6, 60, 100, 250 and 500 nm) and concentrations (0, 10, 50, 100 and 200 μg ml(-1)) of synthetic nanodiamonds. We observed that cell proliferation but not metabolic activity was decreased with nanoparticle sizes of 6-100 nm at lower concentrations (50 μg ml(-1)), and both cell proliferation and metabolic activity were significantly reduced with nanodiamond concentrations of 200 μg ml(-1). Flow cytometry indicated a significant reduction in cell viability due to necrosis irrespective of particle size. Nanodiamond exposure significantly reduced gene expression of tumor necrosis factor-α, interleukin-1β, chemokine Ccl2 and platelet-derived growth factor compared to serum-only controls or titanium oxide (anatase 8 nm) nanoparticles, with variable effects on chemokine Cxcl2 and vascular endothelial growth factor. In general, our study demonstrates a size and concentration dependence of macrophage responses in vitro to nanodiamond particles as possible wear debris from diamond-coated orthopedic joint implants.

Fig. 1

XRD and Raman spectral characterization of different nanodiamond particles.

Fig. 2

FT-IR spectral characterization of different nanodiamond particles: 6 nm nanodiamond before (a) and after (b) acid treatment indicating appearance of peak for –COOH group; (c–f) respectively 60, 100, 250 and 500 nm diamond after acid treatment.

Fig. 3

Representative TEM photomicrographs of ~6 nm nanodiamonds and ~8 nm TiO₂ nanoparticles dispersed in PBS.

Fig. 4

Photomicrographs (400×) showing the morphology of Raw 264.7 cells cultured in the presence of nanodiamonds of various sizes (6–500 nm) and TiO₂ nanoparticles (50 μg ml⁻¹) in comparison with cells cultured in PBS and human serum (controls). Scale bar = 60 μm.

Fig. 5

Effect of concentration of 6 nm nanodiamonds on cell metabolic activity measured by WST-1 assay after culturing the cells (~40,000 Raw 264.7 cells per well) in the presence of varying concentrations of nanodiamonds (10–200 μg ml⁻¹) for 24 h. *P < 0.05 vs. other concentrations at same time point.

Fig. 6

Effect of human serum incubated nanodiamonds at 50 μg ml⁻¹ for 24 h on cell metabolic activity by WST-1 assay. P = PBS; S = Serum; T = TiO₂ NP; 6, 60, 100, 250, 500 are sizes of nanodiamonds in nm. All nanoparticles (both titanium dioxide and nanodiamond) significantly differ from PBS and serum.

Fig. 7

Effect of human serum incubated nanodiamonds at 50 μg ml⁻¹ for 24 h on cell proliferation by BrdU immunoassay. P = PBS; S = Serum; T = TiO₂ NP; 6, 60, 100, 250, 500 are sizes of nanodiamonds in nm. *P < 0.05 vs. corresponding serum-only (S) or PBS (P) control.

Fig. 8

Apoptosis and cell viability as estimated by flow cytometry after culturing. 200,000 cells per well with 50 μg ml⁻¹ nanodiamonds in MEM containing 10% FCS were cultured for 24 h on a 24-well plate. After washing with 250 μl PBS, cells were labeled with propidium iodide and Annexin V-FITC (1:100). Among the apoptotic dye-positive cells, Annexin V-FITC labeled cells were plotted as early apoptotic cells and propidium iodide + Annexin-V-FITC labeled cells were plotted as late apoptotic cells. Unlabeled (non-apoptotic) cells were plotted for cell viability. *P < 0.05 vs. serum-only or PBS control.

Fig. 9

TEM images showing the internalization of TiO₂ nanoparticles (~8 nm) and nanodiamond particles (6, 60 and 250 nm). Higher-magnification images of the corresponding marked area are given on the right side of each image.

Fig. 10

Gene expression of selected cytokines and growth factors. P = PBS; S = Serum; T = TiO₂ NP; *P < 0.05 vs. serum control.