2.9. In vitro biomineralization

The bioactivities of the 3D printed scaffolds were assessed through immersion in SBF. The scaffolds were immersed in an SBF solution having a composition similar to what Rodriguez and Gatenholm reported[16], to determine the effect of increasing HAp powder loading to their biomineralization activity as a function of time. A liter of SBF solution was prepared by dissolving the analytic grade reagents (< 99%) in distilled water in the following order shown in Table 3.

Reagents and composition of the simulated body fluid solution.

In preparing the SBF solution, each reagent was added after the previous reagent has dissolved completely. The solution was prepared at 36.5°C under constant stirring. The pH of the solution was also adjusted to pH 7.4 using 1 M HCl solution and was kept refrigerated at 4°C before usage. The SBF is similar to the human blood plasma ionic concentration and composition. The samples were immersed in 15 mL of SBF solution and placed inside a dedicated oven set at 37°C for 24, 48, and 72 h to assess the growth and deposition of apatite species on the scaffold[17,18]. The SBF-immersed samples were retrieved from the solution and dried in the oven overnight, and finally characterized through digital microscopy, gravimetric analysis, and XRD.