Total disc replacements, are valuable interventions for the spinal surgeon for the treatment of back pain associated with degeneration of the intervertebral disc. The longevity of these devices is compromised by wear and there are growing concerns within the neurosurgical community regarding the exposure of periprosthetic tissues to metal particles and/or ions. Considering the potential for metallic wear debris and ions to trigger inflammation, genotoxicity, cytotoxicity, hypersensitivity and pseudotumour formation, coupled with evidence that nanoscale metal particles can compromise the barrier function of the outer meningeal layer, it is imperative to determine the effects of metallic wear particles on cells of the spinal cord. It was hypothesised that, utilising a 3D type-I collagen gel, enabling glial cells to behave in a more physiologically relevant manner than when cultured in monolayer, the effects of increasing concentrations of metallic wear particles on glial cell viability, cellular reactivity, and cytokine release could be more accurately determined.
Clinically relevant cobalt chrome and stainless steel wear particles were generated using a six-station pin-on-plate wear simulator. Initially in 2D culture C6 glial, PC12 neuronal cells and primary astrocytes with microglia were cultured with increasing concentrations of metallic particles (0.05μm3-50μm3 debris per cell) and their effect on cell viability and DNA integrity assessed. Using a more physiologically relevant 3D culture environment the effects of increasing metallic particles (0.5μm3-50μm3 debris per cell) on cell viability, cellular activity and cytokine expression were investigated using live/dead staining, immunocytochemistry and an enzyme linked immunosorbent assay, respectively.
This study highlighted the necessity for appropriate cell culture environments in biomaterial biocompatibility testing. In 2D culture all cobalt chrome particle doses triggered significant reductions in primary astrocyte and microglia viability, however, in 3D culture, cobalt chrome particles (30-39nm in length) only adversely affected the viability of primary astrocytes and microglia in co-culture when cultured with the highest cobalt chrome particle dose (50μm3 debris per cell) after two and five days in culture (41.8% and 54.2% viable cells, respectively) and with 5μm3 debris per cell after five days in culture (70.5% viable cells). In 2D culture, after 24 hours in culture 0.5μm3, 5μm3 and 50μm3 stainless steel particles per cell caused significant reductions in cell viability (38.8%, 38.9% and 24.9% reductions respectively) however, no adverse effect on viability was observed in 3D culture. Ions released from cobalt chrome caused significant reductions in astrocyte viability (in isolation) at all doses after two days in culture, this effect was not as pronounced after five days. Ions from cobalt chrome particles only caused adverse effects on the viability of astrocytes and microglia after five days at the 5μm3 per cell ion concentration in 3D culture. Ions released from stainless steel caused significant reductions in astrocyte viability (in isolation) at all doses after five days in culture. Stainless steel ions caused adverse effects on the viability of astrocytes and microglia after five days with the 50μm3 per cell ion concentration. DNA damage was observed with both astrocytes and microglia and astrocytes in isolation with both biomaterials tested. Intriguingly, when glial cells were cultured with stainless steel wear particles, the DNA damage observed did not correlate with cell death. Increasing particle volumes of cobalt chrome did not trigger the release of TNF-a, however 50μm3 stainless steel debris per cell caused the release of significantly elevated levels of TNF-a after 48 hours in culture (29.9 pg.ml-1). Stainless steel wear particles did not stimulate astrocyte reactivity unlike cobalt chrome wear products, which had a dose dependent affect on astrocyte activation. The effect was more pronounced in the presence of microglia. Thus the use of 3D culture, whereby glial cells behaved in a more physiologically relevant manner, with a low baseline of reactivity and more representative of the in vivo cellular spatial arrangement was a more appropriate cell culture environment for determining the biological response of cells of the central nervous system to metal wear particles. The results from this study would suggest that stainless steel is more biocompatible than cobalt chrome.
