The effect of the antimicrobial peptide, Dhvar-5, on gentamicin release from a polymethyl methacrylate bone cement.

The objective of this study was to investigate the release mechanism and kinetics of the antimicrobial peptide, Dhvar-5, both alone and in combination with gentamicin, from a standard commercial polymethyl methacrylate (PMMA) bone cement. Different amounts of Dhvar-5 were mixed with the bone cement powders of Osteopal and the gentamicin-containing Osteopal G bone cement and their release kinetics from the polymerized cement were investigated. Additionally, the internal structure of the bone cements were analysed by scanning electron microscopy (SEM) of the fracture surfaces. Secondly, porosity was investigated with the mercury intrusion method and related to the observed release profiles. In order to obtain an insight into the mechanical characteristics of the bone cement mixtures, the compressive strength of Osteopal and Osteopal G with Dhvar-5 was also investigated. The total Dhvar-5 release reached 96% in the 100 mg Dhvar-5/g Osteopal cement, whereas total gentamicin release from Osteopal G reached only 18%. Total gentamicin release increased significantly to 67% with the addition of 50mg Dhvar-5/g, but the Dhvar-5 release was not influenced. SEM showed an increase of dissolved gentamicin crystals with the addition of Dhvar-5. The mercury intrusion results suggested an increase of small pores (< 0.1 microm) with the addition of Dhvar-5. Compressive strength of Osteopal was reduced by the addition of Dhvar-5 and gentamicin, but still remained above the limit prescribed by the ISO standard for clinical bone cements. We therefore conclude that the antimicrobial peptide, Dhvar-5, was released in high amounts from PMMA bone cement. When used together with gentamicin sulphate, Dhvar-5 made the gentamicin crystals accessible for the release medium presumably through increased micro-porosity (< 0.1 microm) resulting in a fourfold increase of gentamicin release.

Release of antimicrobial peptide Dhvar-5 from polymethylmethacrylate beads.

Osteomyelitis is still a major cause of morbidity and remains a difficult complication to treat in orthopaedic surgery. The treatment of choice is a combination of systemic and local antibiotics. The insertion of gentamicin-loaded polymethylmethacrylate (PMMA) beads into the bone results in high local concentrations of gentamicin and low systemic concentrations. However, the effectiveness of this treatment is being hampered by the emergence of antimicrobial resistance. New antimicrobial agents are therefore needed. One new class of promising antibiotics is antimicrobial peptides (AMP). Derived from natural human peptides, these have a low tendency to induce antimicrobial resistance. Dhvar-5 is an antimicrobial peptide based on histatin-5, which is found in human saliva and consists of 14 amino acids. It has demonstrated bactericidal activity in vitro. In order to develop a new local treatment using Dhvar-5 for osteomyelitis, we investigated its release from PMMA beads and its antimicrobial activity against a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) before and after release from PMMA beads. Specific amounts of Dhvar-5 were incorporated into PMMA mini beads, containing 120, 600 and 1200 microg of Dhvar-5, respectively. Dhvar-5 was released from the beads in all three groups. Total release from the 120 microg beads was 9 microg per bead after 7 days. However, the release per bead in the 600 and 1200 microg beads was far more, respectively, 416 and 1091 microg over a 28 day period. After release, the Dhvar-5 also retained its antimicrobial activity against MRSA. On the basis of these data we conclude that the amount of Dhvar-5 release from PMMA beads is not proportionate to the amount incorporated; instead, it demonstrated an exponential relationship to the amount of total peptide released. Furthermore, the released peptide remained biologically active against a clinical isolate of MRSA.

Prevention of infection with tobramycin-containing bone cement or systemic cefazolin in an animal model.

We investigated in an animal model the efficacy of tobramycin-containing bone cement and systemic cefazolin for infection prophylaxis. In 18 female rabbits, the femoral cavity was inoculated with Staphylococcus aureus before injection of bone cement. The first group of six rabbits received tobramycin-containing Simplex-P bone cement. Two other groups of six rabbits received plain Simplex-P bone cement. Preoperatively, in one of the two latter groups cefazolin was administered intravenously. The other group served as untreated controls. The rabbits were monitored for clinical signs of infection. At 7 days' follow-up, the femora were harvested and cultures from the bone adjacent to the cement plug were quantified. Cultures from the rabbits which received antibiotic prophylaxis (either cefazolin systemically or tobramycin-containing bone cement) were all negative. In contrast, all rabbits in the untreated control group had positive cultures. These rabbits also had other signs of infection such as an elevated erythrocyte sedimentation rate and loss of body weight. Culture results were confirmed by the absence of bacterial DNA in the polymerase chain reaction hybridization assay. In conclusion, we found that both tobramycin-containing bone cement and systemic cefazolin are effective in preventing implant bed infection in rabbits up to 7 days after contamination with S. aureus.