2.1. Preparation of Cement Mixtures and Cement Samples

The used preparation procedure allowed us to synthesize the composite biocements with the addition of up to 4 wt.% of amino acids in one step. The selected amino acid mixture in cement contained three basic collagen components (GLY, PRO and HYP) and also ARG or LYS. Tetracalcium phosphate (Ca₄(PO₄)₂O, TTCP) was prepared by annealing of an equimolar mixture of calcium carbonate (CaCO₃, analytical grade, Sigma-Aldrich, Saint Louis, MO, USA) and dicalcium phosphate anhydrous (DCPA) (CaHPO₄ (Ph.Eur.), Fluka), at 1450 °C for 5 h. Following this, the TTCP was milled in a planetary ball mill (Fritsch, 730 rpm, agate balls and vessel, d50 = 7 µm) for 2 h. The tetracalcium/monetite powder cement mixture, which was free of amino acids (C cement), was synthesized by an in situ reaction of TTCP and orthophosphoric acid (86% analytical grade, Merck, Darmstadt, Germany) in 80 v/v % ethanol (reaction solution) using a planetary ball mill with agate balls and vessel for 30 min [37]. The orthophosphoric acid was added in such an amount to be the Ca/P mole ratio in cements close to 1.67. Two different amino acid/cement powder mixtures—glycine:hydroxyproline:proline:arginine (CAK, mass ratio = 4:2:2:1) and glycine:hydroxyproline:proline:lysine (CAL, mass ratio = 4:2:2:1)—were tested. The amino acids were dissolved in reaction solution with the formation of a saturated solution of phosphate–amino acids. To maintain sterile conditions for in vivo testing, amino acid solutions were filtered through a 0.2 μm membrane filter (Millipore, PVDF, Darmstadt, Germany) in a laminar box (ESCO, class II, Esco Micro Pte Ltd, Singapore, Singapore); the sterile solution was then added to TTCP (sterilized at 160 °C/2 h) and the suspension was milled under the same conditions, as in the case of C cement, and dried at 100 °C under sterile conditions. Resulting CAK and CAL cement powder mixtures contained 4 wt.% of amino acids (glycine, L-proline, 4-hydroxy-L-proline, L-lysine or L-arginine (all Sigma)). The cements were prepared by the mixing of powder mixtures with 2% NaH₂PO₄ (as hardening liquid, sterile solution) at P/L ratio = 2.

The amount of released calcium and phosphorus, as a composition of hardened cements (Ca/P ratio), was determined by ICP (Horiba Activa, HORIBA Jobin Yvon Inc., Park Avenue, Edison, NJ, USA) after their dissolution in HNO₃ (20%, analytical grade, Merck, Darmstadt, Germany).