Biomineralization mechanisms: a new paradigm for crystal nucleation in organic matrices

Abstract

There is substantial practical interest in the mechanism by which the carbonated apatite of bone mineral can be initiated specifically in a matrix. The current literature is replete with studies aimed at mimicking the properties of vertebrate bone, teeth, and other hard tissues by creating organic matrices that can be mineralized in vitro and either functionally substitute for bone on a permanent basis or serve as a temporary structure that can be replaced by normal remodeling processes. A key element in this is mineralization of an implant with the matrix and mineral arranged in the proper orientations and relationships. This review examines the pathway to crystallization from a supersaturated calcium phosphate solution in vitro, focusing on the basic mechanistic questions concerning mineral nucleation and growth. Since bone and dentin mineral forms within collagenous matrices, we consider how the in vitro crystallization mechanisms might or might not be applicable to understanding the in vivo processes of biomineralization in bone and dentin. We propose that the pathway to crystallization from the calcium phosphate-supersaturated tissue fluids involves the formation of a dense liquid phase of first-layer bound-water hydrated calcium and phosphate ions in which the crystallization is nucleated. SIBLING proteins and their in vitro analogs, such as polyaspartic acids, have similar dense liquid first-layer bound-water surfaces which interact with the dense liquid calcium phosphate nucleation clusters and modulate the rate of crystallization within the bone and dentin collagen fibril matrix.

Figure 1

The generalized Gibbs formulation of crystal nucleation, from Equation 1. The upper blue curve is the unfavorable gain in free energy because of creation of the new surface interface, the lower blue curve is the favorable decrease in free energy because the stability of the crystal relative to the initial solution free energy. G* is the critical free energy at cluster size n*, the cluster size at which the probability of release of particles from the surface of the cluster is balanced with the probability cluster growth. When n > n* the cluster will have a greater probability of further growth. From Vekilov [26] with permission.

Figure 2

Schematic illustration of the two-step mechanism of nucleation of crystals, modified from work presented by Vekilov [25,26]. In this two step mechanism a dense liquid cluster forms, and a crystal nucleus may form inside the cluster. (a) Microscopic viewpoint shows crystal formation in the plane of two order parameters (Concentration and Structure) inspired by the work of ten Wolde and Frenkel [23, 24]; (b) macroscopic viewpoint of events along the energy pathways (solid lines) depicted in (c). (i) The supersaturated solution. (ii) The formation of the dense liquid depicted in most of (a). (iii) The formation of the critical cluster for the crystal at n₂* within the dense liquid, also shown in (a). (iv) The creation of the nucleation cluster and subsequent growth of the crystal. (v) The fully formed crystal. (c) The pathway for the change in free-energy ΔG, along two possible versions of the two-step nucleation mechanism. If dense liquid is unstable and ΔG⁰ _DL > G_SS(ΔG⁰_DL standard free energy of formation of dense liquid phase), the upper curve applies; if dense liquid is stabilized with the introduction of a foreign interface, ΔG⁰ _DL < 0, the lower curve applies. ΔG₁* is the barrier for formation of a cluster of dense liquid, ΔG₂* is the barriegr for a formation of a crystalline nucleus inside the dense liquid.

Figure 3

Structured water around a Ca²⁺ ion. The central Ca ion is indicated in yellow, the 6 oxygen atoms of the first water shell, coordinated directly to the Ca are in red. The oxygen atoms of the 12 second shell waters are in green. The polarity of the water molecules is quite well fixed and reduces the effective charge on the central Ca ion in so far as long range electrostatic interactions between ions is considered. Pavlov et al. [53], with permission.

Figure 4

Structured water around a PO₄³⁻ ion. (a) The central P atom is indicated in gold, 3 waters of the first shell are shown coordinated directly to the phosphate molecule. (b) The central P atom is indicated in gold with the oxygen atoms on the phosphate indicated in yellow. The 12 waters of the first shell are illustrated. (a) From Pribil et al. [55] with permission. (b) Is our construction of the complete first shell, as suggested [55], with the understanding that the orientations of the non-axial waters will be affected by the waters of the second shell.