Pro-inflammatory potential of Titanium products in peri-implant soft tissues

Objectives: Advanced biophysical imaging techniques have demonstrated accumulation and speciation of titanium (Ti) ions and particles in soft tissues adjacent to skin/mucosa penetrating implants. Neutrophils are co-localised with Ti-debris and are central in the pathogenesis of peri-implantitis. We aimed to determine the potential of relevant Ti-debris, to modify the peri-implant inflammatory response through interactions with human neutrophils.

Methods: Neutrophils isolated from heparinised blood (n=10-20/experiment) were challenged with Ti (as anatase/rutile/metallic or peroxide-complex) in different particle sizes (30nm to 20μm) at concentrations in PBS from 0 to 2000ppm. Opsonised S.aureus (MOI:300-bacteria/neutrophil) and F.nucleatum (MOI:100-1) acted as positive controls. Total/extracellular Reactive Oxygen Species (ROS) release was measured by luminol/isoluminol chemiluminescence. Ti-debris' capacity to perturb neutrophil ROS release to peri-implant pathogens was assessed by priming cells with Ti-oxide species (50ppm in PBS) prior to exposure to Ops S.aureus or F.nucleatum and IFN-α was the positive control. Data were analysed by Wilcoxon tests. Cytokine (IL-1β,IL-6) and elastase release were detected by Enzyme Linked Immunosorbent Assays in 2.5x10⁶ neutrophil suspensions stimulated with Ti or LPS (control) at time-points from 0-24hrs. Cellular interactions with Ti particles were imaged using Transmission Electron Microscopy.

Results: A significant increase in ROS generation was observed following stimulation with Ti-debris in all the forms examined (speciation and sizes) for concentrations >20ppm (total ROS(p<0.05)) and >200ppm (extra-cellular ROS (p<0.001)). ROS levels varied with both particle size and speciation. Ti priming down-regulated responses to Ops S.aureus (p<0.05) but not to F.nucleatum. TEM demonstrated cell-membrane bound and intra-phagasome Ti. IL-1β and IL-6 were significantly upregulated after 4hrs (p<0.05).

Conclusions: Ti in forms identified in per-implant soft tissues was demonstrated to directly stimulate ROS-release, perturb responses to peri-implant pathogens and up-regulate pro-inflammatory cytokine release. Findings emphasise the need to characterise the mechanisms leading to Ti dispersal in peri-implant tissues and its potential to exacerbate inflammation.