Pro-inflammatory Potential of Titanium Species in the Peri-implant Environment

Objectives: Investigations using high resolution synchrotron x-ray fluorescence mapping have demonstrated free titanium (Ti) distributions within soft tissues adjacent to craniofacial and orthopaedic Ti-implants. Characterization of the soft tissues using XANES revealed multiple Ti species including anatase and rutile. This study aimed to: 1) determine the pro-inflammatory potential of both species by studying neutrophil oxidative reactivity; 2) demonstrate intracellularisation of Ti by neutrophils using synchrotron x-ray techniques.

Methods: Human peripheral blood neutrophils from healthy volunteers (n=20) were purified by density centrifugation, challenged with anatase and rutile powders dispersed in PBS at 2000, 200 and 20ppm. Neutrophils were challenged with opsonised S.aureus (MOI: 300:1) and F.nucleatum (MOI: 100-1) as positive controls. Total and extracellular reactive oxygen species (ROS) were measured by luminol/isoluminol chemiluminescence. Data were analysed by Wilcoxon test. Subsequently, neutrophils exposed to anatase (2000ppm) for 2hrs were washed in PBS (x10), mounted on ultrapure silica and air-dried. Ti fluorescence mapping and speciation using XANES was performed with the micro-focus capability (5µm spot size) of the I-18 beamline at the Diamond Light Source (Oxfordshire, UK).

Results: Significant increase in total ROS generation were observed following stimulation with 2,000ppm (p<0.001), 200ppm (p<0.001) and 20ppm (p<0.05) anatase. Extracellular ROS generation was significantly increased following 2000ppm and 200ppm (p<0.001) anatase exposures. Stimulation with rutile also resulted in significantly increased total and extracellular ROS. Rutile exposure resulted in significantly increased extracellular ROS at 2000ppm and 200ppm compared with anatase (p<0.05). XANES analyses of neutrophils exposed to 99.9% purity anatase demonstrated intracellular modification of Ti speciation.

Conclusions: ROS-release following stimulation with Ti degradation products confirms the pro-inflammatory potential of Ti. The differential ROS-release responses to the two Ti species emphasizes the need to understand further the physical and chemical mechanisms leading to the titanium dispersal within peri-implant tissues and their potential to exacerbate neutrophilic inflammation.