Formulation, in vitro physico-chemical and biological assessment of calcium pyrophosphate dihydrate cement for bone tissue engineering.

Calcium pyrophosphate dihydrate (CPPD: Ca₂P₂O₇·2H₂O) crystals are known for their inflammatory potential as pathological calcifications in osteoarthritis while several studies showed the promising potential of calcium pyrophosphate-based materials as bioactive bone substitutes. This study presents for the first time the feasibility to formulate a m-CPPDc cement consisting of pure agglomerated monoclinic CPPD (m-CPPD) crystals with a lower inflammatory potential than a biomimetic carbonated apatite cement (Apc): m-CPPDc-induced interleukin-1β production was 2 times lower than Apc and 2.5 times lower than isolated m-CPPD crystals. In addition, the possibility to reach a biphasic cement composition (m-CPPD associated with the monoclinic calcium pyrophosphate tetrahydrate (m-CPPT-β) metastable phase) was exemplified by varying a formulation parameter which could be of interest to modulate and controlled the material resorbability and inflammatory response. Through an original methodology combining ortho- and pyrophosphate ions titration, in vitro biological and acellular cement physico-chemical evolution tests, we can correlate the enhanced hydrolysis of pyrophosphate ions released from this cement to the action of ALP enzyme (about 3 times more orthophosphate ions at day 2 and 6 whereas no pyrophosphate was detected) and/or hMSC cells (about 37 % less pyroP at day 4). Interestingly m-CPPDc cement combines a high stability during its evolution in different aqueous media (SBF, TRIS buffer, TRIS buffer including ALP enzymes) at 37 °C while releasing higher calcium (3 times more) and orthophosphate ions concentration than a biomimetic apatite cement. Overall, these results illustrate that the cement formulation strategy implemented in this study opens perspectives to develop a new family of phosphocalcic cements fully composed of hydrated calcium pyrophosphate(s) and intrinsically biologically responsive in vitro. STATEMENT OF SIGNIFICANCE: We developed a formulation strategy demonstrating for the first time the feasibility to obtain a pure calcium pyrophosphate dihydrate (CPPD) cement but also the possibility to reach biphasic cement compositions by playing with hydrated calcium pyrophosphate phases. This material consisting of agglomerated CPPD crystals combines low inflammatory potential, cytocompatibility and a high stability in different aqueous media at 37 °C while releasing higher calcium and orthophosphate ions than a biomimetic apatite cement. Both in vitro cell test including an original pyrophosphate follow-up method and physico-chemical testing revealed a correlation between the hydrolysis of pyrophosphate released from cement to the action of enzymes and/or hMSC cells paving the way to a new family of biologically responsive phosphocalcic bone cements.