Enamel Mineralization Induced by Pyrophosphate-Stabilized Calcium Phosphate Solutions

Inorganic pyrophosphate (PP_i) is a potent inhibitor of biological mineralization. It has been suggested that specific enzymes control bone and cartilage mineralization by regulating PP_i formation and degradation within supersaturated fluids. The potential use of a similar approach for mineralized tissue repair, however, has not been explored. Objective: This study was conducted to determine if PP_i-stabilized, highly supersaturated calcium phosphate mineralization solutions could be used to induce the mineralization of human enamel in vitro in the absence and presence of an enzyme we previously showed to degrade PP_i and reverse its inhibitory activity. Methods: Acid-etched sections (n=3-4) of human enamel were suspended in 100 mL of PP_i-stabilized mineralization solutions (1.3 mM CaCl₂, 1.5 mM KH₂PO₄, 50 mM NaCl, pH 7.4, and 5-10 µM PP_i.) and maintained at 37^oC with stirring. Experiments were carried out in the presence and absence of alkaline phosphatase (3.2 U/ml) from calf, AP-C (Worthington). The pH was monitored continuously and SEM images were used to assess crystal growth. Results: In the absence of AP-C, little to no change in the enamel rod structure was observed following 4 to 24 hr exposures to the PP_i-stabilized mineralization solutions. In contrast, with added AP-C, large amounts of mineral formed in 4 hr that were specifically associated with the enamel surface, with no mineralization occurring in the bulk solution. Randomly-oriented, plate-like structures were apparent and crystal growth appeared to have commenced as a series of waves resulting in layers of mineral deposition, covering variable portions of the enamel surface. Conclusions: The rate and extent of enamel-associated mineralization can be greatly enhanced through the enzymatic degradation of PP_i, using stabilized, highly supersaturated calcium phosphate solutions. These results suggest that such solutions could potentially be used to induce mineralization of damaged or diseased enamel tissues. Supported by NIDCR grant DE-15163.