Orthopaedic cobalt chromium particles and ions can induce indirect DNA damage and chromosome aberrations in human cells on the other side of a cellular barrier in tissue culture. This occurs by intercellular signalling across the barrier. We now show that the threshold for this effect depends on the metal form and the particle composition.

Ionic cobalt and chromium induced single strand breaks at concentrations equivalent to those found in the blood of patients with well functioning metal on metal hip prostheses. However, they only caused double strand breaks if the chromium was present as chromium (VI), and did not induce chromosome aberrations. Nanoparticles of cobalt chromium alloy caused DNA double strand breaks and chromosome aberrations, of which the majority were tetraploidy. Ceramic nanoparticles induced only single strand breaks and/or alkaline labile sites when indirectly exposed to human fibroblasts.

The assessment of reproductive risk from maternal exposure to biomaterials, especially those liberated by orthopaedic implants, is not yet possible with epidemiology. Whilst the barrier model used here differs from the in vivo situation in several respects, it may be useful as a framework to evaluate biomaterial induced damage across physiological barriers.

Purpose

Metal-on-Metal (MoM) hip bearings are being implanted in ever-increasing numbers and into ever-younger patients. The consequence of chronic exposure to metal ions is a cause for concern. Therefore, using cytogenetic biomarkers, we investigated a group of patients who have had MoM bearings in situ for in excess of 30 years.

Increasing numbers of young people receive metal on metal (CoCr on CoCr) total hip replacements. These implants generate nano-particles and ions of Co and Cr. Previous studies have shown that micro-particles, nano-particles and ions of CoCr cause DNA damage and chromosomal abberrations in human fibroblasts in tissue culture, and in lymphocytes and bone marrow cells in patients with implants. Several surgeons have used these implants in women of child-bearing age who have subsequently had children. Significantly elevated levels of cobalt and cromium ions have been measured in cord blood of pregnant women with CoCr hip implants. The MHRA (Medicines and Healthcare products Regulatory Agency) subsequently stated that there is a need to determine whether exposure to cobalt and chromium represents a health risk during pregnancy.

In an attempt to investigate this risk, we used a well established in vitro model of the placental barrier comprised of BeWo cells (3 cells in thickness) derived from the chorion and exposed this barrier to nanometer (29nm) and micron (3.4μm) sized CoCr particles, as well as ions of Co2+ and Cr6+ individually or in combination. We monitored DNA damage in BJ fibroblasts beneath the barrier with the alkaline gel electrophoresis comet assay and with γH2AX staining.

The results showed evidence of DNA damage after all types of exposure. The indirect damage (through the barrier) was equal to the direct damage at the concentrations tested. The integrity of the barriers was checked with measurements of electrical resistance (TEER values) and permeability to sodium fluorescein (376Da) and found to be intact.

In light of these results and with the knowledge that BeWo cells express the transmembrane protein Connexin 43, we tested the theory that a damaging signal was being relayed via gap junctions or hemi channels in the BeWo cells to the underlying fibroblasts. We used the connexin mimetic peptides Gap19 and Gap26 (known to selectively block hemichannels and gap junctions respectively) and 18α-glycyrrhetinic acid (non-selective gap junction blocker). All of these compounds completely obliterated the indirect damaging effect seen in our previous experiments.

We conclude that CoCr particles can cause DNA damage through a seemingly intact barrier, and that this damage occurs via a bystander mechanism. It would be of interest to test whether this is simply a tissue culture effect or could be seen in vivo.

Background: Metal-on-Metal (MoM) hip bearings are being implanted in ever increasing numbers and into ever-younger patients. The consequence of chronic exposure to metal ions is a cause for concern. Therefore, by using cytogenetic biomarkers, we investigated a group of patients who have had MoM bearings in-situ for in excess of 30 years.

Method: Whole blood specimens were obtained from an historical group of patients who have had MoM bearings in-situ for in excess of 30 years. Blood was also obtained from an age and sex matched control group and from patients with Metal-on-Polyethylene (MoP) components of the same era.

The whole blood was cultured with Pb-Max karyotyping medium and harvested for cytogenetics after 72 h. The 24 colour FISH (Fluorescent In Situ Hybridisation) chromosome painting technique was performed on the freshly prepared slides allowing chromosomal mapping. Each slide was evaluated for chromosomal aberrations (deletions, fragments and translocations) against the normal 46 (22 pairs and two sex) chromosomes. At least 20 metaphases per sample were scored and the number of Aberrations per cell calculated.

Results: Chromosomal aberrations, including deletions, fragments and translocations were only detected in the peripheral blood lymphocytes isolated from the group that had MoM bearings. These changes were not present in the age and sex matched control group. The chromosomal aberrations were also detected in the patients previously exposed to MoM bearings who have been revised to a MoP articulation.

Conclusion: We have detected dramatic chromosomal aberrations in peripheral blood lymphocytes in a group of patients chronically exposed (over 30 years) to elevated metal ions. It is not known whether these aberrations have clinical consequences or whether they are reproduced in other cells in the body. The results emphasise the need for further investigations into the effect of chronic exposure to elevated metal ions produced by Orthopaedic implants.