The Buffer Illusion: How TRIS and HEPES Enhance Bioactivity of Bioglass 45S5

In vitro tests of glass, glass-ceramic, or ceramic biomaterials such as synthetic hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) are performed using simulated body fluid (SBF), in which the pH is maintained using TRIS buffer (ISO standard 23317:2014; recently updated as ISO standard 23317:2025). When evaluating the experiments, we not only looked at the tested material itself but also analyzed the SBF leachate. We have found out that TRIS buffer, as well as other tested buffers (Good's buffers), actively interacts with glass and ceramic materials. They significantly accelerate their dissolution (up to 3× higher compared to unbuffered SBF). This increases the local supersaturation of biogenic ions near the surface and promotes the formation of the expected hydroxyapatite (the newly formed hydroxyapatite layer is labeled as HAp). In this work, we focused on bioglass 45S5 to observe the corrosion rate of the glass surface on samples in the form of bulk, in SBF solutions buffered with (i) TRIS and (ii) HEPES, and we used (iii) SBF without any buffer for comparison. The glass corrosion rate and the growth kinetics of the new HAp layer (SEM/EDS, XRD, FTIR) were compared with the results of the leachate analyses (pH, ICP-OES). The concentration of Si^IV in the leachates showed the corrosive effects of each of the solutions, while the concentrations of Ca²⁺ and (PO₄)^3- confirmed precipitation of a new Ca-P phase. The results confirmed earlier findings and showed that the TRIS and HEPES buffering systems are not inert and are strongly corrosive to bioglass 45S5. Moreover, their parts incorporate into the newly formed Ca-P layer and change its quality and crystallinity. These findings suggest that the current approach to in vitro testing of these materials requires reconsideration.