# Nanostructured Strontium-Doped Calcium Phosphate Cements: A Multifactorial Design
## 2 Materials and Methods  
Cement Preparation. Sr-doped \(\alpha\)-tricalcium phosphate (\(\alpha\)-TCP) powders with different strontium content (Sr/(Ca + Sr) = 0, 2, 4 mol%, henceforth coded as Sr0, Sr2, Sr4, respectively) were prepared by solid-state reaction of CaHPO\({}_{4}\), CaCO\({}_{3}\), and SrCO\({}_{3}\) (Merck KGaA, Darmstadt, Germany) [44; 45] at three different temperatures (1200 \({}^{\circ}\)C, 1400 \({}^{\circ}\)C, and 1600 \({}^{\circ}\)C) and dwell times (20, 60, 180 min) in a Pt crucible, followed by rapid quenching in air. A more detailed preparation route of similar powders, together with the chemicophysical characterizations proving the substitution of calcium with strontium ions, has been previously reported [42]. Then, the powders were milled in ethanol using a planetary mono mill (Pulverisette 6 classic line, Fritsch, Idar-Oberstein, Germany) at 400 rpm [36; 37]into a zirconia jar with three different milling times (15, 50, and 90 min) and zirconia milling ball diameters (1, 2, and 5 mm) (Nikkato corporation, Osaka, Japan). Afterward, aqueous solutions containing three different amounts of Na2 HPO4 (0, 2.5, and 5 wt%) (Merck KGa, Darmstadt, Germany) and sodium alginate (0, 1, and 2 wt%) (Alginic Acid Sodium Salt from Brown Algae, Merck KGa, Darmstadt, Germany) were prepared and mixed by hand with a spatula for about 30 s according to three different liquid-to-powder mass ratios (0.50, 0.55, and 0.60). The sodium alginate was chosen to improve the injectability, cohesion, and compression strength of the cements, without affecting the hardening process [42]. In addition, cylindrical models (diameter = 6 mm, height = 2 mm) were filled with the as-obtained pastes and kept at 37 \({}^{\circ}\)C in saturated humidity conditions to obtain consolidated samples to be tested for their compressive strength. Cement characterization. The phase composition of the precursor powders and the cements was investigated by X-ray diffraction (20 range = 10-80\({}^{\circ}\), scan step = 0.02\({}^{\circ}\), step time = 0.5 s) (Bruker D8 Advance Diffractometer equipped with LINXYE detector, Karlsruhe, Germany); the domain size of the \(\alpha\)-TCP crystals was also investigated by means of Rietveld analysis by using the crystal models of \(\alpha\)-TCP [46], \(\beta\)-TCP [47], and HA [48] phases. The particle size of the powders (D50) was analyzed with Sedigraph 5100 (Micromerities, Norcross, GA, USA). The morphology of cements was investigated by scanning electron microscopy (FEG-SEM, Sigma NTS GmbH, Carl Zeiss, Oberkochen, Germany). The cement injectability was qualitatively evaluated by measuring the amount of cement in the syringe after extrusion [29]. The initial and final setting times of the cements were evaluated with Gillmore apparatus, according to standard ASTM C266-99, by testing 5 specimens for each formulation. The compressive strength and [PERSON]'s modulus of the cements were evaluated by testing 5 cylindrical specimens (diameter = 8 mm, height = 17 mm) after 3 days of immersion in Simulated Body Fluid (SBF) [49] at 37 \({}^{\circ}\)C, followed by drying for 24 h in a vacuum chamber. The tests were performed in agreement with ISO 9917 with a universal testing machine (MTS Insight 5, Eden Prairie, MN, USA). [PERSON]'s criterion was applied to exclude outliers [50]. [PERSON]'s modulus was calculated by evaluating the slope of the linear regression of the stress-strain curves until the failure stress of the specimen. The porosity was evaluated on the cylindrical specimens as P = 1 - \(\rho\)/\(\rho\)0, where \(\rho\) is the cement density determined as a weight-to-volume ratio and \(\rho\)0 refers to the actual phase composition, as obtained by the Rietveld analysis.