Nalgene filter units

Amorphous Ca-polyP microparticles were prepared as outlined before [14 ]. To obtain this phase, the weight concentration ratio between Ca [as CaCl₂] and P [as Na-polyP] was set to ≈2. In turn, 2.8 g of CaCl₂ •·2 H₂O (#223506; Sigma-Aldrich, Taufkirchen; Germany) were dissolved in 50 ml ethanol solution (96%) and added drop-wise to 1 g of Na-polyP, dissolved in 50 ml distilled water (room temperature; the pH was adjusted to 9–10). The suspension formed was kept at pH 10 (using 1 N NaOH) and stirred for 5 h. During this period microparticles assembled; they could be collected by filtration (Nalgene Filter Units [pore size 0.45 μm]; Cole-Parmer, Kehl/Rhein; Germany). Then, the sample was washed three times with ethanol and dried at 60°C. Subsequently, the material was sieved through a sieve shaker AS 200 (mesh size 100 μm; Retsch GmbH, Haan; Germany). These microparticles were termed “Ca-polyP-MP”.
In one control experiment “Ca-P particles” were prepared in the same way: 2.8 g of CaCl₂ •·2 H₂O were dropped into 1 g of Na-phosphate (Na₃PO₄; Sigma #342483). The resulting material that precipitated was filtrated and dried as described for polyP.

The polyP particles were prepared as described before [15 (link)]. In brief, Na-polyP (2 g) was dissolved in 100 mL of distilled water; the resulting pH value was increased from pH 3.4 to 10 with 2 N NaOH. Then 60 mL of a CaCl₂ solution (5.6 g CaCl₂·2H₂O; #C3306 Sigma, Taufkirchen; München, Germany) were added dropwise to the polyP solution. After stirring for 12 h the particles formed were collected by filtration through Nalgene Filter Units (pore size 0.45 μm; Cole-Parmer, Kehl/Rhein; Wertheim-Mondfel, Germany). Then the particles were washed twice with ethanol to remove the unbound Ca²⁺. Finally, the amorphous microparticles, “Ca-polyP-MP”, were dried at 60 °C overnight.
The particles formed from sodium orthophosphate (tri-sodium phosphate; Sigma #342483) were prepared in the same way. Two g of tri-sodium phosphate were dissolved in 100 mL of water; the pH value was adjusted to pH 10; then 60 mL of the CaCl₂ solution (containing 5 g CaCl₂) were added. The resulting crystalline particles, Ca-phosphate-NP, were processed as described above.