Revised method for preparation of simulated body fluid for assessment of the apatite-forming ability of bioactive materials: proposal of mixing two stock solutions

Bioactive glasses and glass-ceramics exhibit osteoconductivity, which is the ability to form a direct bond with living bone tissue. This property is typically assessed by observing the formation of a hydroxyapatite layer in vitro using simulated body fluid (SBF), a solution designed to mimic the inorganic constituents of human blood plasma. SBF was developed by Kokubo et al. (T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi and T. Yamamuro, J. Biomed. Mater. Res., 1990, 24, 721-734), and its preparation and storage procedures are precisely regulated according to ISO 23317. However, despite following the regulations, SBF may precipitate during storage owing to local ion concentration fluctuations during preparation because it is a supersaturated solution of hydroxyapatite. The present study is focused on designing a preparation process that enhances the stability of SBF-type biomimetic solutions. Solutions with a nominal composition that is the same as that of SBF were prepared by mixing stock solutions containing calcium or phosphate ions separately. Depending on the preparation procedure, two types of solutions-modified biomimetic solution (mBS) and evolved biomimetic solution (eBS)-were proposed. To evaluate the stability of these solutions, variants with 1.5 times the concentration of the original solutions were prepared, and the prepared solutions were denoted as xSBF, xmBS, and xeBS (where x = 1.0 or 1.5). It was found that the formation of calcium phosphate precipitates in 1.5mBS and 1.5eBS was slower than that in 1.5SBF. Additionally, the precipitate in 1.5eBS exhibited a different morphology from hydroxyapatite precipitate, which may be due to a higher hydrogencarbonate concentration than that in 1.5SBF. eBS demonstrated increased stability and a higher concentration of carbonic acid species (carbonate ions) than SBF, while mBS showed increased stability with a lower carbonate concentration than SBF. The newly proposed routes allow the production of an aqueous solution supersaturated with hydroxyapatite as a conventional SBF that has the potential to evaluate apatite formation via in vitro examination under high initial stability.