Amorphous calcium pyrophosphate bone cement sets without crystallization: a physico-chemical and <i>in vitro</i> biological study

Calcium phosphate cements have been developed since the 1980s for bone reconstruction. Generally, mixing the solid phase with the liquid phase results in the formation of agglomerated apatite or brushite crystals. This study aimed at formulating the first amorphous calcium phosphate cement constituted of pure amorphous calcium pyrophosphate (a-CPPc: Ca₂P₂O₇·<i>n</i>H₂O) after setting and hardening at 37 °C in view of developing a bioactive material for bone applications. The amorphous phases involved at each step of the cement preparation were thoroughly characterized using complementary techniques to investigate on the chemical setting reaction. The key role of the structural water content (<i>n</i>) and the presence of some orthophosphate ions on the stability of these amorphous phases were evidenced, preventing crystallization but not cement setting and hardening. The acellular <i>in vitro</i> study in SBF or TRIS buffer medium including or not alkaline phosphatase enzymes (ALP) showed the bioactivity of this amorphous cement, which remained amorphous after two weeks in SBF solution. In TRIS it transforms into a more stable calcium pyrophosphate crystalline phase and its orthophosphate and calcium ions release is enhanced in presence of ALP. The hydrolysis by ALP of pyrophosphate ions released was demonstrated by phosphate ions titration. The cement cytotoxicity assessment combined with chemical titration allowed optimizing potassium pyrophosphate to be incorporated into the liquid phase (2.5% w/w). Overall, this first set of physico-chemical and biological results on this original biologically responsive a-CPPc cement formulation paves the way to widen the family of bioactive phosphocalcic bone cements with this amorphous hydrated calcium pyrophosphate cement.