The effect of polyaspartate chain length on in vitro remineralization of dental tissues

Abstract

Event Abstract Back to Event The effect of polyaspartate chain length on in vitro remineralization of dental tissues Bryan Quan1 and Eli D. Sone1, 2, 3 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 University of Toronto, Faculty of Dentistry, Canada 3 University of Toronto, Department of Materials Science & Engineering, Canada Introduction: Most mineralized mammalian tissues are composites of hydroxyapatite (HAP) and collagen fibrils, with between 10 % and 80 % of bone and dentin HAP estimated to be intrafibrillar. Though intrafibrillar HAP is essential to the mechanical properties of bony tissues, the mechanism of intrafibrillar mineralization is not well known. However, it is thought that mineralizing proteins are involved in directing the process. These proteins are typically enriched in aspartate or glutamate residues[1], which is important for their mineralization-regulating properties[2]. Recently polyaspartate (pAsp) and polyacrylate, used as analogs of mineralizing proteins, have been shown to mediate intrafibrillar HAP formation in spontaneously precipitating calcium-phosphate (CaP) solutions[3]. Currently, the roles of these polycarboxylate additives and the parameters which govern their effectiveness are not well described. Here, we investigate the effect of pAsp chain length on remineralization of dental tissues and characterize the remineralizing solutions to gain insight into the mechanisms of intrafibrillar mineralization. These studies will lead to improved materials for fixation, replacement, and repair of bony tissues. Materials and Methods: We remineralize ultrathin sections of demineralized mouse dental tissues directly on TEM grids in spontaneously precipitating solutions of calcium and phosphate ions[4], using either no pAsp as a control, or low polydispersity pAsp additives with nominal degrees of polymerization (DP) of 6, 10, and 100 to mediate intrafibrillar remineralization. Remineralization outcomes are characterized by conventional transmission electron microscopy (TEM), while the remineralizing solutions are characterized by a combination of Ca2+-ion selective electrode (ISE) measurements, dynamic light scattering (DLS), and TEM. Results and Discussion: Remineralization experiments (Figure 1) show that with no pAsp, or with pAsp hexapeptide, superficial HAP forms quickly and without selectivity, while selected area electron diffraction (SAED) indicates no preferential HAP orientation. Conversely, pAsp with DP 10 or 100 induces selective remineralization of only the natively mineralizing tissues dentin and cementum, and SAED of shows that the c-axes of HAP crystals orient with the long axes of collagen fibrils. This demonstrates a role for both pAsp and tissue-specific mineralization factors in determining mineralization selectivity. Though the affinity of pAsp for Ca2+ is relatively insensitive to DP, ISE measurements show that higher DP pAsp better inhibits crystallization, while DLS suggests that a higher DP pAsp better inhibits the formation of amorphous CaP precursors (not shown). These results suggest that pAsp inhibits CaP precipitation in the solution compartment, thereby maintaining a high supersaturation and supporting intrafibrillar mineralization. Conclusion: We show that while higher DP pAsp is an effective mediator of intrafibrillar mineralization, pAsp with DP 6 is not. However, pAsp with DP as low as 10 does mediate intrafibrillar mineralization. This demonstrates a lower size limit for the effective inhibition of amorphous CaP formation and CaP crystallization in solution by aspartate repeats in mineralizing proteins. Figure 1 - TEM micrographs and SAED of sections remineralized in solution control (a) or with 25μg/mL pAsp6 (b), pAsp10 (c), or pAsp100 (d).