SUITABLE EVALUATION METHOD FOR IN VITRO ANTIBACTERIAL ACTIVITY OF BIOMATERIALS

Abstract

Bacterial infections related to orthopaedic implants is one of the serious types of complications. Recently, there has been a greater interest in antibacterial biomaterials. However, antibacterial evaluations of each material are inconsistant, so intercomparison of the antibacterial performance is difficult. This study focused on the Japanese Industrial Standards test (JIS Z2801), which is used for antibacterial evaluation of commodities. The study investigated a suitable evaluation method for in vitro antibacterial activity of biamaterials. In 2007, JIS Z2801 test was approved as international standard ISO 22196.

Hydroxyapatite (HA) powder containing 3 wt % of silver oxide (Ag) was sprayed on the surface of titanium disks with the thermal spraying method, using an acetylene torch. This coating has been proved to generate strong antibacterial activity in previous studies. The antibacterial activity was examined with the JIS Z 2801 test and modified JIS Z2801 test. The bacterial strains used in JIS Z2801 test were Escherichia coli (E.coli), Staphylococcus aureus (S.aureus). Bacterial culture medium was instilled onto the surface of the test disks (about 106 cells/ml) and covered with polystyrene films. After cultivation in 1/500 Nutrient Broth for 24 h at 35°C, the bacteria was washed out with the broth. The numbers of viable bacteria in the broth were counted with the agar plate culture method. Additionally, Modified JIS Z2801 test was performed. Modified points were added to the bacterial strain of biofilm-forming methicillin-resistant S.aureus (BF-MRSA), using Fetal Bovine Serum (FBS) as a culture medium, and cultivated at 37°C.

In the JIS Z2801 test, Antibacterial activity values of the HA-Ag disk were composed against E.coli 4.1 and S.aureus 5.0. In the modified JIS Z2801 test, antibacterial activity values against E.coli, S.aureus and BF-MRSA were 8.2, 5.5, and 7.1. When this value is greater than 2.0, it shows there is antibacterial activity. The titanium disk coated with HA-Ag showed antibacterial activity in both tests.

The JIS Z2801 test is designed to evaluate comodities in poor nutritional environment. However, the environment in the body is eutrophic. It is easy to make bacterial growth. For this reason, it is necessary to consider evaluating for biomaterials with suitable method considered in vivo. In this study, to examine the condition like that found in the body, we cultivated FBS at 37°C. In addition, the antibacterial activity against BF-MRSA was examined to consider the bacterial infection related to orthopaedic implants. The modified JIS Z2801 test showed that it is a suitable evaluation method for in vitro antibacterial activity of biomaterials.