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The extracellular matrix of hard connective tissues is composed primarily of mineralized collagen fibrils. Acidic noncollagenous proteins play important roles in mediating mineralization of collagen. Polyaspartate, a homopolymer substitute for such proteins, has been used extensively in in vitro models to produce biomimetic mineralized collagen. Polyglutamate behaves differently in mineralization models, despite its chemical similarity. We show that polyaspartate is a 350 times more effective inhibitor of solution precipitation of hydroxyapatite than polyglutamate. Supersaturated CaP solutions stabilized with polyaspartic acid produce collagen with aligned intrafibrillar mineral, while solutions containing polyglutamate lead to the formation of unaligned mineral clusters on the fibril surface. Molecular analysis showed that the commercial polyaspartic acid contains substantial isomerization, unlike polyglutamic acid. Hence, the secondary structure of polyaspartic acid is more disordered than that of polyglutamic acid. The increased flexibility of the polyaspartic acid chain may explain its potency as an inhibitor of solution crystallization and a mediator of intrafibrillar collagen mineralization.

Formation of hydroxyapatite (HAP) within collagen fibrils, as found in bone, dentine and cementum, is thought to be mediated by proteins rich in aspartate (Asp) and glutamate such as osteopontin and bone sialoprotein, respectively. Indeed polyaspartate (pAsp), a homopolymer analogue of such proteins, has been shown to induce intrafibrillar mineralization of collagen from solutions of calcium and phosphate that are supersaturated with respect to HAP. To elucidate the role of pAsp in mineralization of collagen, we explored the effect of pAsp chain length on in vitroHAP deposition in demineralized mouse periodontal tissue sections. Through characterization of both tissue sections and mineralizing solution, we show that chain length contributes to the effectiveness of pAsp in mediating intrafibrillar mineralization. This function appears to be associated with inhibition of otherwise kinetically favoured crystallization in the bulk solution, which allows for intrafibrillar crystallization, though this does not preclude the possibility of a more active role for pAsp in addition. Inhibition of crystallization in solution by pAsp occurs by slowing the growth of amorphous calcium phosphate and stabilization of this phase, rather than by sequestration of Ca2+ions. These results suggest that the length of Asp-rich sequences of mineralizing proteins may be essential to their function, and could also be useful in optimization of mineralized tissue replacement synthesis.