Regulation of Inorganic Pyrophosphate Inhibition of Hydroxyapatite Crystal Growth

Inorganic pyrophosphate (PP_i) is a potent inhibitor of biological mineralization. It has been suggested that bone and cartilage mineralization may be controlled by specific enzymes that regulate PP_i formation and degradation within supersaturated fluids. However, studies have not examined the potential of such an approach for mineralized tissue repair. Objective: As a first step, this study was conducted to determine if PP_i stabilization of a highly supersaturated calcium phosphate solution could be reversed by the presence of a mineral phase and/or enzymatically. Methods: Varying concentrations ( 0.1 to 20 µM) of PP_i were studied to determine its effect on spontaneous and seeded precipitation of an unstable supersaturated solution (1.3 mM CaCl₂, 1.5 mM KH₂PO₄, 50 mM NaCl, pH 7.4) in the presence and absence of hydroxyapatite (HA, NIST, 18.3 m²/g). Experiments were conducted at 37^oC with stirring. The pH was monitored continuously and aliquots were removed at selected times for calcium and phosphate analysis. The effect of alkaline phosphatase (AP, chicken intestine) was examined. Results: We found that as little as 0.1 µM PP_i could completely inhibit spontaneous precipitation for up to 24 hr. This inhibition could not be reversed with AP (2mg/mL). However, in the presence 0.1 µM PP_i, immediate crystal growth was observed upon adding HA (15 mg). At higher PP_i concentrations, crystal growth was reduced and nearly completely inhibited at concentrations of 10 µM and greater in the presence of HA. However, reversal of crystal growth inhibition (at 10 µM PP_i) was not apparent upon the addition of AP (up to 50 mg/mL). Conclusions: Although further studies are needed to verify the feasibility of using an enzyme to regulate the kinetics of this process, our results suggest that PP_i-stabilized CaP solutions can potentially induce mineralization of damage mineralized tissues (e.g., carious lesions). Supported by NIDCR grant DE-15163.