Characterisation of wear particles
The particles of CoCr generated in this study were similar in terms of size and morphology to previous studies [
20,
22], and comparable to those generated in metal-on-metal total disc replacement devices [
2]. The stainless steel particles were similar in morphology to the CoCr particles; however, their size range was larger, which is in line with the different material properties of the two metals, particularly with respect to chemistry, hardness and microstructure, e.g. grain size [
23].
Effects of wear particles on cell viability and DNA integrity in 2D culture
When CoCr wear particles were cultured with primary rat astrocytes and microglial cells in 2D culture, cell viability was adversely affected by all doses of particles. This is in line with previous studies that exposed monocytes and fibroblasts to CoCr wear particles [
20]. When cells were exposed to stainless steel particles in 2D culture, initially an adverse effect was observed; however, this effect was only consistent at the highest particle concentration at the 5-day time point. Other studies have observed variable cellular toxicity associated with stainless steel particles, with some cell types, e.g. monocytes adversely affected [
24] and others not affected, e.g. osteoblasts [
25]. The different effects are postulated to be due to different sensitivities of cell lines and different compositions of the two metals, with CoCr having higher chromium content than stainless steel (27–30% compared to 16–18%) and the absence of cobalt in stainless steel [
26].
When glial cells were exposed to CoCr and SS particles and DNA integrity analysed, again there were differences between the two biomaterials. High doses of CoCr particles significantly affected DNA integrity of glial cells. In isolation, astrocytes were more sensitive to CoCr particles. These results suggested that when microglial cells were present, these cells influenced the effects that the particles had on the astrocyte cells, often with the microglial cells experiencing adverse effects in the form of DNA damage, whilst appearing to protect the astrocytes from these effects in the co-culture. After culture with SS particles, adverse effects on glial cell DNA integrity were observed after 24 h with all concentrations of particles. In contrast, astrocytes in isolation were not affected by stainless steel particles.
Effects of wear particles and ions on cell viability and cell reactivity in 3D culture
When primary astrocytes and microglia were cultured with increasing particle volumes of CoCr, a dose-dependent effect on cell viability was observed. When primary astrocytes were cultured in isolation with increasing particle volumes of CoCr, only the highest particle dose significantly reduced cell viability. Interestingly, the removal of microglia from the culture environment appeared to reduce the sensitivity of primary astrocytes to CoCr wear particles, suggesting the resident macrophage cell type, the microglia, influences the effects that the particles have on astrocytes. When cells were exposed to CoCr ions, the results suggested that astrocytes in isolation were more sensitive than when glial cells were co-cultured. We postulate that this may be due to either active uptake (phagocytosis) of large volumes of particles into microglia, followed by the release of cytokines or signalling factors that trigger subsequent cell death in the astrocytes. When astrocytes were cultured in isolation with particles, uptake may occur via pinocytosis and hence be much slower, resulting in lower levels of toxicity [
15,
16].
In contrast, there were no adverse effects on cell viability when glial cells were cultured with increasing particle volumes of stainless steel at any particle dose, or at any time point. These results echo the results in the 2D culture system, indicating that differences in elemental composition between the two biomaterials may be responsible for the different effects on cell viability. In contrast, adverse effects on cell viability were observed when glial cells were exposed to ions from stainless steel particles, particularly when astrocytes were cultured in isolation, indicating that in the absence of microglia, astrocytes were more sensitive to ions from implant biomaterials.
Upregulation of the expression of glial fibrillary acidic protein (GFAP) to a variety of stimulants including injury [
21] and biomaterial particles [
27] has been widely reported in the literature. CoCr particles caused increased expression of GFAP at low particle concentrations by the 5-day time point; however, often the higher concentrations of particles did not. We postulate that these higher particle concentrations caused significant reductions in cell viability, and with a large proportion of the cells dead or dying, this masked the detection of GFAP. Stainless steel particles had no effect on the GFAP expression in glial cells at any dose or any time point.
In summary, CoCr particles adversely affected glial cell viability and triggered increased GFAP expression, indicating that astrocytes developed a reactive phenotype and were subjected to significant DNA damage. Cobalt chromium ions also had adverse effects on cell viability. In contrast, stainless steel particles rarely affected cell viability and did not elicit increased expression of GFAP. However, SS particles caused significant DNA damage and glial cells appeared to be more sensitive to stainless steel ions compared to CoCr ions, where the former were found to adversely affect cell viability at low concentrations and early time points. The results indicated that DNA damage was the result of interaction with ionic species rather than the particles themselves.
The particles used in the present study were generated using a simple configuration pin-on-plate wear simulator using water as a lubricant. In a previous study [
2], the biocompatibility of CoCr wear particles generated from CoCr alloy on CCr alloy TDRs in serum and water was compared with particles generated in water using the same pin-on-plate set-up used in the present study. In L929 fibroblast cells, Pasko [
2] found that there was no difference in the viability of L929 cells exposed to the different types of particles, with higher volumes of particles (5 and 50 µm
3 of particles per cell) causing adverse effects on cell viability in a similar way that CoCr particles affected neural cells in the present study. The use of particles from joint simulators using serum lubricants causes issues with microbial contamination, endotoxin contamination and degraded proteins, which all affect the cellular responses, making it difficult to determine the effects of the particles alone. Simple configuration pin-on-plate simulators provide a quick, cost-effective way to generate large volumes of debris for cell culture easily avoiding these issues.
This study has revealed for the first time that clinically relevant wear particles from metallic biomaterials have numerous adverse effects on the cells of the spinal cord, and caution should be exercised when using these materials in spinal devices and instrumentation.