Erwin Chan

Metal is not inert: Role of metal ions released by biocorrosion in aseptic loosening—Current concepts

Metal implants are essential therapeutic tools for the treatment of bone fractures and joint replacements. The metals and metal alloys used in contemporary orthopedic and trauma surgery are well tolerated by the majority of patients. However, complications resulting from inflammatory and immune reactions to metal implants have been well documented. This review briefly discusses the different mechanisms of metal implant corrosion in the human body, which lead to the release of significant levels of metal ions into the peri-implant tissues and the systemic blood circulation. Additionally, this article reviews the effects of the released ions on bone metabolism and the immune system and discusses their involvement in the pathophysiological mechanisms of aseptic loosening and metal hypersensitivity in patients with metal implants.

Bio-corrosion of stainless steel by osteoclasts—in vitro evidence

Most metals in contact with biological systems undergo corrosion by an electrochemical process. This study investigated whether human osteoclasts (OC) are able to grow on stainless steel (SS) and directly corrode the metal alloy leading to the formation of corresponding metal ions, which may cause inflammatory reactions and activate the immune system. Scanning electron microscopy analysis demonstrated long‐term viable OC cultures and evident resorption features on the surface of SS discs on which OC were cultured for 21 days. The findings were confirmed by atomic emission spectrometry investigations showing significantly increased levels of chromium, nickel, and manganese in the supernatant of OC cultures. Furthermore, significant levels of pro‐inflammatory cytokines IL‐1β, IL‐6, and TNF‐α, which are considered to be major mediators of osteolysis, were revealed in the same cultures by cytometric bead array analysis. Within the present study, it was shown that human osteoclast precursors are able to grow and differentiate towards mature OC on SS. The mature cells are able to directly corrode the metal surface and release corresponding metal ions, which induce the secretion of pro‐inflammatory cytokines that are known to enhance osteoclast differentiation, activation, and survival. Enhanced corrosion and the subsequently released metal ions may therefore result in enhanced osteolytic lesions in the peri‐prosthetic bone, contributing to the aseptic loosening of the implant.

Titanium induced production of chemokines CCL17/TARC and CCL22/MDC in human osteoclasts and osteoblasts

There is increasing evidence that titanium (Ti(IV)) ions are released from orthopedic implants and play a role in aseptic loosening. This study aimed to investigate whether titanium induces expression of chemokines and cytokines that are important in osteoclastogenesis in human osteoclasts and osteoblasts. Incubation of those cells with 1 muM Ti(IV) significantly upregulated expression of CCL17/TARC and CCL22/MDC, RANK-L, M-CSF and pro-inflammatory cytokines as determined by quantitative real-time PCR and ELISA assays. Additionally, flow cytometry was used to show Ti(IV) related increased expression of CCR4, the cognate receptor for CCL17 and CCL22 in challenged osteoclast precursors. These results strongly suggest that Ti(IV) ions play a role in the recruitment of osteoclast precursors to the bone-implant interface by increasing CCL17 and CCL22 expression and by upregulating their cognate receptor. Moreover the increased expression of RANK-L and M-CSF by osteoblasts together with increased levels of pro-inflammatory cytokines may enhance osteoclast differentiation and activity, and subsequently contribute to the pathomechanism of aseptic loosening.

Titanium IV ions induced human osteoclast differentiation and enhanced bone resorption in vitro.

There is increasing evidence that titanium (Ti) ions are released from orthopedic implants, with concentrations in the range of 1 microM in tissue and blood, and may play a role in aseptic loosening of orthopedic implants. This study investigated whether Ti(IV) ions induce differentiation of monocytic osteoclast precursors into osteo-resorptive multinucleated cells and influence the activation and function of in vitro generated osteoclasts. Human monocytes and in vitro generated osteoclasts were exposed to 1 microM Ti(IV) ions for 10 days. Thereafter, osteoclast differentiation, activation, and function were evaluated. Transcription of specific osteoclastic genes was measured using quantitative reverse transcription polymerase chain reactions, which showed increased expression of tartrate-resistant acid phosphatase (TRAP) in approximately 20% of Ti(IV)-treated monocytes. Detection and quantification of intracellular TRAP activity using ELF97 as a fluorescent substrate revealed a significant increase of TRAP-positive cells in Ti(IV)-treated monocytes. Additionally, as demonstrated on dentin slide cultures, Ti(IV)-treated monocytes became functional bone resorbing cells, significantly increasing their osteo-resorptive activity to similar levels as osteoclasts in vitro. These results suggest that Ti(IV) ions released by biocorrosion from orthopedic implants induce differentiation of monocytes toward mature, functional osteoclasts, which may well contribute the pathomechanism of aseptic loosening.

Titanium uptake, induction of RANK-L expression, and enhanced proliferation of human T-lymphocytes

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 TiCl4 [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 CD69+ and CCR4+ 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.

Effects of titanium(IV) ions on human monocyte-derived dendritic cells

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.

Effect of vanadium on human dendritic cells

Vanadium is a heavy metal that has no known biological role in humans. However, increasing exposure through advances in medical applications – orthodontic/orthopedic metal implants, vanadium-containing drugs and environmental exposure have raised questions on the influence of vanadium on the immune system. As initiators of the adaptive immune response, dendritic cells (DC) are responsible for the differentiation of effector T-lymphocytes and the resulting immune reactivity. This study assessed the differences in immune reactivity towards various vanadium compounds and investigated the influence of vanadium (III), (IV), and (V) ions on peripheral blood monocytes (PBMC) and DC. Exposure to vanadium (III) and (IV) concentrations above 125 µM reduced the proliferation of lymphocytes. Mitochondrial DC activity was reduced in the presence of low concentrations of vanadium. Flow cytometry analysis of cell surface molecules showed slight alterations of MHC class II (antigen presenting molecule) and reduction of CD54 (adhesion molecule) with increasing vanadium concentrations. Secreted cytokines and chemokines produced by DC were measured through a cytometric bead assay, which showed no significant difference after exposure to various vanadium ions. Finally, the ability of vanadium-treated DC to interact with lymphocytes was measured through proliferation of allogenic non-adherent PBMC and showed retention of proliferative capacity. These results indicate that vanadium exerts a weak influence on the maturation and function of DC, which suggests that alterations in immune reactivity by vanadium is mediated directly on effector lymphocytes.

Immune response deviation and enhanced expression of chemokine receptor CCR4 in TBI patients due to unknown serum factors

BACKGROUND Severe brain trauma leads to an activation of the immune system. To this date, neither the exact perturbation of the specific immune reaction induced by the traumatic brain injury (TBI), nor the interactions leading to the infiltration of peripheral immune cells into the brain are fully understood. PATIENTS AND METHODS Serum was collected from 17 patients with TBI and a long bone fracture, 24 patients with an isolated long bone fracture and from healthy individuals. The effect of the serum on normal human monocytes and T-lymphocytes was tested in vitro by assessing proliferation and expression of surface markers, chemokine receptors and cytokines. RESULTS Serum collected from patients with a TBI and a long bone fracture increased the expression of the chemokine receptor CCR4 in monocytes when compared to patients with an isolated long bone fracture. Extending this comparison to T-lymphocytes, the serum from TBI patients induced lower proliferation rates and decreased expression of the pro-inflammatory cytokine TNF-alpha, while simultaneously increasing the secretion of immune-modulatory cytokines (IL-4, IL-10 and TGF-beta) (p<0.05). CONCLUSION Patients with a TBI release currently unknown soluble factors into the circulating blood that up regulate expression of chemokine receptor CCR4 in peripheral blood monocytes whilst concurrently inducing expression of immunosuppressive cytokines by activated T-lymphocytes.