In order to enhance implant survival, bioactive coatings have moved into the focus of research and development. Due to the increasing risk of implant infections from multiresistant bacteria such as MRSA (multiresistant Staphylococcus aureus
] or ESBL (enterobacteria producing extended spectrum beta-lactamases) [24
], different antimicrobial coatings [17
] are being developed. However, the mechanical, biological and chemical properties of such innovative coatings have to be investigated thoroughly. Cu ions can be an effective antimicrobial agent to inhibit bacterial growth and biofilm formation on endoprosthetic surfaces [10
]. Analyses of the ion release are strictly necessary for the determination of relevant Cu concentrations required in order to regulate both antimicrobial effects and compatibility to human cells. Furthermore, the release kinetics of copper-doped surfaces needs to be investigated in standardised tests in order to ensure effective and valid ion concentrations. In this context, however, standardised test conditions have not been established so far.
The AAS is a suitable device to measure Cu concentrations in supernatants. Uncoated samples did not show any Cu in any of the analysed supernatants, whereas the Cu-treated samples revealed differences in Cu concentration. Nevertheless, using an AAS, both Cu+
ions are assessed at the same time without any distinction. However, only Cu2+
ions cause an antimicrobial effect [30
]. Hence, the concentration alone is not enough to predict the effectiveness of the coating and should be supported by microbiological tests.
Investigations of the ion release of an antimicrobial coating should coincide with cell biological tests using human cells to study biocompatibility of the coating. Furthermore, test fluid volumes should be the same for all studies, ensuring similar Cu concentrations. A test fluid volume of 700μ
L was chosen to represent in vivo
conditions. After conventional implantation technique, Wu et al. [31
] observed that 40% of an uncemented femoral stem showed no bone contact with an average gap width between the bone and the femoral stem of 0.77
mm. In relation to the test samples deployed in this study, a volume of approximately 200μ
L would be adequate to simulate the gap volume at the uncemented stem. However, 200μ
L is not enough to cover the test samples completely, which would make in vitro
testing impossible. Therefore, a volume of 700μ
L was chosen as a compromise, which was the smallest possible volume that assured proper cell and bacterial growth in the supernatant.
The results of this study show that the amount of Cu ions released into the supernatant depends on various factors. The supernatant and its properties to dissolve Cu ions plays an important role. Surprisingly, the Cu concentration in double-distilled water for the Cu-PIII coating after 24 hours did not differ from the Cu concentration after 5 days. Therefore, double-distilled water showed an early Cu saturation for both tested coatings, whereas human serum and DMEM revealed a much higher Cu concentration in the supernatant. In fact, the highest concentration was obtained in DMEM for the Ti-Cu coating. The presence of serum proteins increases the solubility of Cu in the supernatants significantly compared to the double-distilled water because of the complex compounds between copper and amino groups found in the proteins in human serum and DMEM. Wu et al. [32
] showed a higher concentration of Ag+
ions after 24
h in cell culture medium as in simulated body fluid, which coincides with the Cu concentrations found in our present study. However, based on the current results, it is not possible to appoint a Cu ion saturation level for neither human serum nor DMEM. Repeated and cumulative investigations at different time periods need to be carried out and could provide a precise saturation level.
Surface roughness increases the surface area in contact with the supernatant. With rising surface roughness, an increase of the Cu concentration in the supernatant was observed. Compared to a polished surface, the corundum-blasted surface released approximately three times as many Cu ions within 24
h. In double-distilled water, the Cu concentration levels remained constant after 5 days regardless of the surface roughness, since the Cu saturation has set in within the first 24
Temperature may also influence the ion release characteristic of an implant material. Therefore, the release studies should be carried out at 37°C body temperature. A time-dependant Cu release behaviour, cumulative and non cumulative, of the analysed coatings is currently under investigation. First results show that most of the Cu is released during the first 24
h, followed by a highly reduced release rate during the succeeding days. Furthermore, the effect of copper ions on human cells and tissue is currently under investigation provided by cell biological and microbiological tests as well as animal studies using an infection model.