Monitoring the stability of therapeutic ions in SBF

For the assessment of potentially bioactive materials, in vitro tests are performed in simulating body fluid (SBF) to prove its hydroxyapatite-forming ability and its biodegradability based on ions released into the solution. Information on ion release is essential for understanding the biological activity of both the ions and the materials as a whole. To understand these processes, it is important to accurately determine the concentration of elements in a complex SBF matrix. Most published studies are not performed consistently, do not take into account the specifics of the method, and possible interactions, or do not provide information on the protocols applied to achieve stability of the solutions analysed. In our study, we performed model experiments aimed at verifying the determination of ions in SBF when the sample is and is not suitably treated for its storage.

The first experiment investigated the simulation of the long-term stability of ions in SBF samples collected after in vitro test of apatite formation (often incorrectly stated as bioactivity test). SBF solutions were spiked with Al, B, Ce, Cu, Ga, Sr, Y, and Zn at 0.5, 2.5, and 10 mg/L concentrations and determined within 1 h. The concentration of spiked solutions was analyzed again after 7 and 14 days. Elemental recovery was evaluated in comparison with solutions containing the same amounts of these elements but treated with nitric acid to achieve pH ∼ 2. The second experiment focused on stratifying the ion concentrations in the tubes during the ICP-OES measurement. The difference between the analysis performed immediately after the addition of the elements of interest to the solution and the analysis performed 3 h later was determined in the three positions of the tubes.The changes in the measured concentration significantly different than the nominal values for the Al, Ce, Y, and Zn were detected. If the concentration of these ions decreases, phosphorus and calcium have been removed from the solution and vice versa. This indicates that these ions get trapped in the precipitates and complexes formed. A protocol of recommended sample handling procedures was developed to avoid potential errors and inconsistencies when storing the SBF sample after leaching tests (also known as ion release test) before ICP-OES analysis.