Biomimetic, antibacterial bone implants for the prevention of local infections

Hospital-acquired bone infections, for example, as a result of surgery, are a serious and costly problem, making the prevention of such infection a matter of the highest priority. Calcium phosphate (CaP) apatites are highly suitable candidates for the preparation of biomaterials for bone repair. However, although CaP compounds have been the subject of the extensive industrial research and development, there are still no technological concepts for equipping calcium phosphates with antibacterial properties. Since the use of antibiotics is problematic, due to the development of bacterial resistance, other strategies must be found, compared and developed.

The binational BioCapabili project involves the collaboration between the Fraunhofer IGB and the CIRIMAT Carnot Institute in France, in order to equip bioactive, biomimetic CaP apatite with various antimicrobial compounds and fully investigate them. Antibiotics were not used. CaP apatites were developed in the CIRIMAT “Phosphates, Pharmacotechnics and Biomaterials“ working group, with a focus on their synthesis, characterization and surface reactivity. Alternative surface modifications as well as biological characterization were carried out at the Fraunhofer IGB.

Calcium phosphates crystallized in the apatite structure have a similar construction to the mineral content of bone. In order to produce new formulations of nanocrystalline biomimetic apatites with antibacterial properties we modified the composition of the apatite. A second approach was to functionalize the hydrated surface layer of nanoscale CaP apatite with active agents. Various methods were employed to modify preferably the surface of the nanocrystals (e.g. using a dialysis membrane), or the entire crystal. The surface adsorption of organic or organic-inorganic compounds on the surface of the CaP crystals was also investigated. We were able to show antibacterial effects for various different pathogens and with various test parameters. We compared the concentration-dependency of the antibacterial effect with regard to cytotoxicity and antibacterial properties. The best formulations were subsequently tested in vivo for osteoconduction.

Several new formulations of chemically-modified (with or without surface modifications) nanocrystalline apatites were synthesized and fully characterized. The conditions enabling us to obtain single-phased apatite systems were retained and tests for antibacterial effects and cytotoxicity were carried out. Non-doped systems served as a reference. The apatite nanocrystals obtained showed high surface reactivity, especially through a hydrated surface layer. The influence of synthesis parameters, particularly the amount of antibacterial agent used per formulation and subsequent treatments was thoroughly investigated, especially in view of future possible areas of application.

All the systems developed in the project were screened for antibacterial properties and cytotoxicity and the results were compared to determine the most promising formulations for future development to an industrial level. The left figure shows several selected screening results. The figure shows the reduction factor R_F, which is calculated from the starting cell count used and the re-cultivable cells (R_F=log (starting cell count) – log (number of re-cultivable cells)). A high starting cell count of between 10⁷ cells/ml for Staphylococci and 10⁹ cells/ml for E. coli and P. aeruginosa was reached on silver and bismuth-doped CaP apatite in high-concentration screening. The reduction factor R_F shown in the diagram specifies the reduction in viable and reproductive cells in logarithm form. The maximum possible value corresponds to the number of starting cells used and therefore the total inactivation of the cells was accomplished. It was possible to fully inactivate the Staphylococci with the levels of doping shown. E. coli and P. aeruginosa show no impairment at 0.1 percent silver (R_F = 0), while at only 0.5 percent silver both bismuth concentrations were equally fully inactivated.

Preliminary in vivo implantation tests were launched at the end of the project to investigate the potential impact of the best formulations on osteogenesis.

With a view to future developments, we were able to establish contact with surgeons and industrial companies during the course of the project and a patent for one of the new formulations has been filed. A number of industrial companies have shown serious interest in this patent and in the French-German BioCapabili research alliance. Both institutes have identified several further novel applications for the antibacterial materials – as the foundation for long-term collaboration in the development of antibacterial materials in Europe.

We would like to thank the German Federal Ministry of Education and Research (BMBF) and the French National Research Agency (ANR) for funding the project “BioCapabili” within the joint project “Inter Carnot Fraunhofer PICF 2009”.

• CIRIMAT Carnot Institut, Prof. Dr. Christophe Drouet