The role of poly(aspartic acid) in the precipitation of calcium phosphate in confinement

Abstract

Many questions remain regarding the formation of ultrathin hydroxapatite (HAP) crystals within the confines of collagen fibrils of bones. These structures form through the interplay of the collagen matrix and non-collagenous proteins, and in vitro mineralization studies employing poly(aspartic acid) (PAsp) as a mimic of the non-collagenous proteins have generated mineralized fibrils with structures comparable to their biogenic counterparts. In this article, we employ the nanoscale cylindrical pores perforating track-etch filtration membranes to investigate the role of PAsp in controlling the infiltration and crystallization of calcium phosphate (CaP) within confined volumes. Oriented polycrystalline HAP and non-oriented octacalcium phosphate (OCP) rods precipitated within the membrane pores via an amorphous calcium phosphate (ACP) precursor, where PAsp increased the proportion of OCP rods. Further, ACP crystallized faster within the membranes than in bulk solution when PAsp was present, suggesting that PAsp inhibits crystallization in solution, but promotes it when bound to a substrate. Finally, in contrast to the collagen system, PAsp reduced the yield of intra-membrane mineral and failed to enhance infiltration. This suggests that a specific interaction between the collagen matrix and ACP/PAsp precursor particles drives effective infiltration. Thus, while orientation of HAP crystals can be achieved by confinement alone, the chemistry of the collagen matrix is necessary for efficient mineralisation with CaP.

Confinement generates single-crystal aragonite rods at room temperature

“…We have previously shown that TE membranes provide a versatile means of studying the effects of confinement on the polymorph, orientation, and single-crystal/polycrystalline structure of inorganic crystals (21)(22)(23)(24)(25)(26). Briefly reviewing these studies, our early work employed pores as small as 200 nm and used low temperatures and 100 mM CaCl 2 and Na 2 CO 3 solutions to generate single crystals of calcite via an amorphous calcium carbonate (ACC) precursor phase (22,23).…”

Confinement Increases the Lifetimes of Hydroxyapatite Precursors

“…Confinement plays a key role in the formation of bone, which begins with the growth of ultrathin platelets of nonstoichiometric carbonated hydroxyapatite (HAP) within gaps in the collagen fibrils. 20 These 2-6 nm thick platelets are the smallest known biogenic crystal 21,22 and are organized so membranes to show that confinement alone can direct the orientation of HAP, 42,43 and demonstrate that both octacalcium phosphate (OCP) and amorphous calcium phosphate (ACP) can be effectively stabilized in confinement. Consideration of the mechanism by which confinement increases the lifetime of these phases suggests that is distinct from that found with CaCO 3 and CaSO 4 , and emphasizes the need to study multiple systems to fully understand the effects of confinement on crystallization processes, and therefore to employ confinement as a route to controlling crystallization.…”

Tunability of collagen matrix mechanical properties via multiple modes of mineralization

“…This is different from PAsp-induced mineralization, in which PAsp infiltrates the collagen and preferentially binds in the gap zones, promoting intrafibrillar mineralization. Although there is evidence that PAsp may act as an inhibitor when not bound to collagen [60]. Prior studies examining the mineralization of dense collagen scaffolds with fetuin have demonstrated similar results: mineralization occurs preferentially within the matrix, often concentrated near the surface, likely due to diffusion limitations [20,53,61].…”