There is increasing evidence that titanium ions are released from orthopedic implants by biocorrosion. The aim of this study was to investigate titanium uptake by human T-lymphocytes and its effects on phenotype and proliferation. Freshly isolated human nonadherent peripheral blood mononuclear cells (NA-PBMC), were exposed to TiCl 4 [Ti(IV)]. Bioavailability and distribution of Ti(IV) in T-lymphocytes was determined by energy-filtered electron microscopy (EFTEM). The effects of Ti(IV) challenge on nonactivated and PHA-activated cells were assessed by flow cytometric analysis of surface markers, RANK-L production, and proliferation assays. EFTEM colocalized Ti(IV) with phosphorus in the nucleus, ribosomes, cytoplasmic membranes, and the surface membrane of T-lymphocytes. Ti(IV) increased significantly the expression of CD69, CCR4, and RANK-L in a concentration-dependent manner. Titanium enters T-lymphocytes through a currently unknown mechanism and binds to phosphorus-rich cell structures. Titanium influences phenotype and function of T-lymphocytes, resulting in activation of a CD69þ and CCR4þ T-lymphocyte population and secretion of RANK-L. These results strongly suggest the involvement of titanium ions challenged T-lymphocytes in the complex pathophysiological mechanisms of aseptic loosening of orthopedic implants. ß

Orthopaedic metal implants composed of titanium are routinely used in bone fracture repair and for joint replacement therapies. A considerable fraction of implant recipients are unable to benefit due to implant failure resulting from aseptic loosening, while others may experience cutaneous sensitivity to titanium after implantation. An adaptive immune reactivity towards titanium ions, originating from the biocorrosion of the implants, could play a role. As an initiator of the adaptive immune response, dendritic cells (DC) were studied for uptake and characteristics after titanium exposure. Energy filtered transmission electron microscopy showed uptake of titanium(iv) (Ti(iv)) ions by DCs in vitro and co-localisation with phosphorus-rich cell structures of the DC membranes (phospholipids), cytoplasm (ribosomes and phosphorylated proteins) and the nucleus (DNA). DC maturation and function were investigated by measuring cell surface marker expression by flow cytometry. After exposure, DCs showed a decrease in MHC class II (HLA-DR), co-stimulatory molecules (CD40, CD80 & CD86) and chemokine receptors (CCR) 6 and CCR7 but an increase in CCR4 after Ti(iv) treatment. However, Ti(iv) treated DCs had an increased stimulatory capacity towards allogenic lymphocytes. A Ti(iv) concentration dependant increase of IL-12p70 was observed amidst decrease of the other measured cytokines (TGF-β1 and TGF-β2). Hence, Ti(iv) alters DC properties, resulting in an enhanced T lymphocyte reactivity and deviation towards a Th1 type immune response. This effect may be responsible for the inflammatory side effects of titanium implants seen in patients.

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Titanium is increasingly used for implanted bio‐medical devices, including bone fracture treatment and joint replacement therapies. Bio‐corrosion of implants and accumulation of titanium in immune cells, lymph nodes and spleen has been reported. Inflammatory reactions against titanium implants have been reported in up to 15% of cases. However, little is known about the effect of titanium on immune cells. The aim of this study was to investigate the effect of Ti(IV) ions (1 to100μM) on human T‐lymphocytes, freshly isolated from buffy coats or peripheral venous blood of healthy individuals (n=10). No toxic effect was detected in viability assays for the applied titanium concentrations. Using energy filtered transmission electron microscopy, uptake and accumulation of titanium in T‐lymphocytes was documented. Titanium induced cell proliferation (BrdU incorporation assays) in a concentration dependent manner in 40% of the tested individuals. Cytokine measurement in the culture supernatants revealed increased IL‐6 and TNFα production at 12.5μM titanium concentration. Measurement of surface marker expression with flow cytometry showed increased expression of CD3, CD4, CD45 and the chemokine receptor CCR4.This study indicates that titanium affects human T‐lymphocytes by enhancing their inflammatory properties.