Bacterial infection related to prosthetic replacement is one of the serious types of complications. Recently, there has been a greater interest in antibacterial biomaterials. In order to reduce the incidence of replacement-associated infections, we developed a novel coating technology of Hydroxyapatite (HA) containing silver (Ag). We reported the Ag-HA coating showed high antibacterial activity against E. coli, S. aureus and methicillin-resistant S. aureus (MRSA) under static condition. However, human bodies have a circulating body fluid, which is not a static condition. And the growth and the maturation of biofilm, which is said that a common course of persistent infections at a surgical site, are enhanced by the flow of broth in culture environment. Therefore, we evaluated whether the Ag-HA coating inhibits the biofilm formation on its surface or not by a biofilm-forming test under flow condition in this study.

Ag-HA or HA powder was sprayed onto the commercial pure titanium disks using a flame spraying system. The HA coating disks were used as negative control. The biofilm-forming methicillin sensitive S. aureus (BF-MSSA; Seattle 1945) strain and the BF-MRSA (UOEH6) strain were used. The pre-culture bacterial suspension (about 10⁵ colony forming units; CFU) was inoculated onto the Ag-HA and HA coating disks. After cultivation at 37 °C for 1 h, the disks were rinsed twice with 500 μL sterile PBS (-) to eliminate the non-adherent bacteria. The number of the adherent bacteria on these disks was counted using culture method. After rinsing, the disks were transferred into petri-dish containing Trypto–Soy Broth (TSB) + 0.25% glucose with a stirring bar on the magnetic stirrer and they were cultured at 37°C for 7 days. In the meantime, the stirring bar was spun at 60 rounds per minute. Then, the disks were immersed in a fluorescent reagent to stain the biofilm. Finally, the biofilm on each disk was observed by a fluorescence microscope and the biofilm-covered rate on the surfaces of them was calculated using the NIH image software.

The number of the bacteria on these disks was not so different between Ag-HA and HA coating after rinsing. After biofilm-forming test, the coverage of the biofilm of BF-MSSA was 2.1% and 81.0% on the Ag-HA and HA coatings, respectively. Similarly, in the case of BF-MRSA, it was 7.7% and 72.0% on the Ag-HA and HA coatings, respectively. Though bacteria slightly adhered, biofilm was hardly observed on the Ag-HA coating. The biofilm on the HA coating was extensive and mature. The inhibition effect of biofilm formation on the Ag-HA coating might be ascribed to the antibacterial effect by Ag ions released from the coating. Because Ag ions have a broad spectrum of antibacterial activity against pathogens, including biofilm forming bacteria, they inhibited the biofilm formation on the Ag-HA coating by killing adherent bacteria. Even in a flow condition, it was suggested that the AgHA shows the antibacterial activity, though the conditions in this work are different from those in living body.