We have used the pH-induced self-assembly of a peptide-amphiphile to make a nanostructured fibrous scaffold reminiscent of extracellular matrix. The design of this peptide-amphiphile allows the nanofibers to be reversibly cross-linked to enhance or decrease their structural integrity. After cross-linking, the fibers are able to direct mineralization of hydroxyapatite to form a composite material in which the crystallographic c axes of hydroxyapatite are aligned with the long axes of the fibers. This alignment is the same as that observed between collagen fibrils and hydroxyapatite crystals in bone.

http://science.sciencemag.org/content/sci/294/5547/1684.full.pdf

Self-assembly and mineralization of peptide-amphiphile nanofibers

Natural materials such as bone, tooth, and nacre are nanocomposites of proteins and minerals with superior strength. Why is the nanometer scale so important to such materials? Can we learn from this to produce superior nanomaterials in the laboratory? These questions motivate the present study where we show that the nanocomposites in nature exhibit a generic mechanical structure in which the nanometer size of mineral particles is selected to ensure optimum strength and maximum tolerance of flaws (robustness). We further show that the widely used engineering concept of stress concentration at flaws is no longer valid for nanomaterial design.

https://www.pnas.org/content/pnas/100/10/5597.full.pdf

Materials become insensitive to flaws at nanoscale: lessons from nature.

1.1. Biomineralization
The study of biomineralization is not only important to gain an understanding of how mineral-rich tissues are created in vivo but also because it is a great source of inspiration for the design of advanced materials.1-7 Mineralized tissues have remarkable hierarchical structures that have evolved over time to achieve great functions in a large variety of organisms. Organic phases play a key role in templating the structure of mineralized tissues; therefore, their matrices are often hybrid in composition, varying widely in the relative content of organic and inorganic substances. Understanding the complex integration of hard and soft phases that biology achieves in mineralized matrices across scales and its link to properties is knowledge of great value to materials chemistry. At the same time, the synthetic mechanisms used by biology to create mineralized matrices could also offer some useful strategies to create synthetic hybrid materials. Often, the amount of organic material utilized by Nature to modify mechanical properties of mineralized structures is vanishingly small. One example is the role of occluded proteins in the toughness of biogenic calcite.8 The study of mammalian bone and teeth in the biomineralization and biomimetic context is particularly interesting since the information derived could contribute a significant biomedical impact on therapies and strategies to repair or regenerate human mineralized tissues. This is an important area given the continually rising average life span of humans. The materials of interest could be highly sophisticated bioactive scaffolds to regenerate bone and possibly dental tissues as well. This review focuses on the formation of hydroxyapatite (HA) in synthetic systems designed primarily in the biomimetic context of bone or enamel mineralization for therapeutic approaches in repair of human tissues. Bone and enamel share the same mineral composition, HA, but have different morphologies and organic content. Enamel is almost entirely inorganic in composition, and bone has a relatively high organic composition. Knowledge acquired in this field may inspire the chemical synthesis of novel hybrid materials, including apatite-based structures for the regeneration of human bone and dental tissues.

Biomimetic Systems for Hydroxyapatite Mineralization Inspired By Bone and Enamel

http://www.biofilling.com/article/7_1Mineral.pdf

Mineral trioxide aggregate: a comprehensive literature review--part II: leakage and biocompatibility investigations.

Calcium orthophosphates are the main mineral constituents of bones and teeth, and there is great interest in understanding the physical mechanisms that underlie their growth, dissolution, and phase stability. By definition, all calcium orthophosphates consist of three major chemical elements: calcium (oxidation state +2), phosphorus (oxidation state +5), and oxygen (oxidation state −2).1 The orthophosphate group (PO43−) is structurally different from meta (PO3−), pyro (P2O74−), and poly (PO3)nn−. In this review, only calcium orthophosphates will be discussed. The chemical composition of many calcium orthophosphates includes hydrogen, either as an acidic orthophosphate anion such as HPO42− or H2PO4−, and/or incorporated water as in dicalcium phosphate dihydrate (CaHPO4 · 2H2O).1 Most calcium orthophosphates are sparingly soluble in water, but all dissolve in acids; the calcium to phosphate molar ratios (Ca/P) and the solubilities are important parameters to distinguish between the phases (Table 1) with crystallographic data summarized in Table 2. In general, the lower the Ca/P ratio, the more acidic and soluble the calcium phosphate phase.2 It is now generally recognized that the crystallization of many calcium phosphates involves the formation of metastable precursor phases that subsequently dissolve as the precipitation reactions proceed. Thus, complex intermediate phases can participate in the crystallization process. Moreover, the in vivo presence of small peptides, proteins, and inorganic additives other than calcium and phosphate has a considerable influence on crystallization, making it difficult to predict the possible phases that may form.3 Studies of apatite mineral formation are complicated by the possibility of forming several calcium phosphate phases. The least soluble, hydroxyapatite (HAP), is preferentially formed under neutral or basic conditions. In more acidic solutions, phases such as brushite (DCPD) and octacalcium phosphate (OCP) are often encountered. Even under ideal HAP precipitation conditions, the precipitates are generally nonstoichiometric, suggesting the formation of calcium-deficient apatites. Both DCPD and OCP have been implicated as possible precursors to the formation of apatite. This may occur by the initial precipitation of DCPD and/or OCP followed by transformation to a more apatitic phase. Although DCPD and OCP are often detected during in vitro crystallization, in vivo studies of bone formation rarely show the presence of these acidic calcium phosphate phases. In the latter case, the situation is more complicated, since a large number of ions and molecules are present that can be incorporated into the crystal lattice or adsorbed at the crystallite surfaces. In biological apatite, DCPD and OCP are usually detected only during pathological calcification, where the pH is often relatively low. In normal in vivo calcifications, these phases have not been found, suggesting the involvement of other precursors or the formation of an initial amorphous calcium phosphate phase (ACP) followed by transformation to apatite.



Table 1

Ca/P Molar Ratios, Chemical Formulas, and Solubilitiesa of Some Calcium Orthophosphate Minerals1,3,4





Table 2

Crystallographic Data of Calcium Orthophosphates1,4,5



In this review, we will discuss some important parameters related to crystal nucleation and growth/dissolution including the supersaturation/undersaturation, pH, ionic strength and the ratio of calcium to phosphate activities (Table 3). We then focus on the dynamics of crystallization/dissolution in the presence of additive molecules pertinent to biogenic calcium phosphate minerals.



Table 3

Crystal Growth Controls and Their Effect on the Bulk Solution and the Crystal Surfaces6




2. Biologically Related Calcium Phosphate Phases
2.1. Structure, Composition, and Phase Stability


2.1.1. Amorphous Calcium Phosphate (ACP) 
During the synthesis of HAP crystals through the interaction of calcium and phosphate ions in neutral to basic solution, a precursor amorphous phase is formed that is structurally and chemically distinct from HAP.7 However, calculations have shown that the phase consisted of individual or groups of HAP unit cells.8 Chemical analysis of the precursor phase indicated this noncrystalline phase to be a hydrated calcium phosphate (Ca3(PO4)2 · xH2O) with a Ca/P ratio 1.50,8 consisting of roughly spherical Ca9(PO4)6 “Posner’s clusters” (PC) close-packed to form larger spherical particles with water in the interstices.9 PCs appeared to be energetically favored in comparison to alternative candidates including Ca3(PO4)2 and Ca6(PO4)4 clusters.10 The structure of PCs in isolated form is notably different from that in a HAP environment.11 In particular, the chirality feature of PCs found in the HAP environment is suggested to disappear in an isolated form and in aqueous solution. The reconsideration of PCs as possible components in the actual structural model of ACP resulted from the cluster growth model of the HAP crystal.12 Ab initio calculations confirmed that stable isomers exist on the [Ca3(PO4)2]3 potential energy surface (PES).12,13 These isomers correspond to compact arrangements, i.e., arrangements in which the Ca and PO4 are disposed closely together. Their geometries are compatible with the terms “roughly spherical” used in Posner’s hypothesis. The calculations performed on the monomer and dimer PES revealed that the relative energies of the different isomers are governed by a specific bonding pattern in which a calcium atom interacts with two PO43− groups, forming four CaO bonds.12,13 The compact isomers on the trimer PES are energetically favored in comparison to monomer or dimer isomers. This is rationalized by the appearance of a specific bonding pattern for the trimer case in which a calcium forms six CaO bonds with six different PO4 groups. This type of bonding in encountered in HAP.13

It is now generally agreed that, both in vitro and in vivo, precipitation reactions at sufficiently high supersaturation and pH result in the initial formation of an amorphous calcium phosphate with a molar calcium/phosphate ratio of about 1.18–2.50. The chemical composition of ACP is strongly dependent on the solution pH: ACP phases with Ca/P ratios in the range of 1.18:1 precipitated at pH 6.6 to 1.53:1 at pH 11.7 and even as high as 2.5:1.4 Two amorphous calcium phosphates, ACP1 and ACP2, have been reported with the same composition, but differing in morphology and solubility.14,15 The formation of ACP precipitate with little long-range order tends to consist of aggregates of primary nuclei (roughly spherical clusters) with composition Ca9(PO4)65 dependent on the conditions of formation. It hydrolyzes almost instantaneously to more stable phases. These amorphous clusters served as seeds during HAP crystallization via a stepwise assembly process12 and were presumed to pack randomly with respect to each other,16 forming large 300–800 A spheres. Recent experimental studies found that ACP has definite local atomic microcrystalline order rather than a random network structure. NMR of thoroughly dried ACP suggests that the tightly held water resides in the interstices between clusters,17 but these are probably not of intrinsic importance in the structure of ACP. It is well-known that ACP contains 10–20% by weight of tightly bound water, which is removed by vacuum drying at elevated temperature.9 However, drying does not alter the calcium and phosphorus atomic arrangement. The side band intensities of dried ACP suggest that its chemical shift anisotropy is similar to or identical with that of normal ACP.17 ACP has an apatitic short-range structure, but with a crystal size so small that it appears to be amorphous by X-ray analysis. This is supported by extended X-ray absorption fine structure (EXAFS) on biogenic and synthetic ACP samples.18–20

The CaP amorphous phase transforms to HAP microcrystalline in the presence of water. The lifetime of the metastable amorphous precursor in aqueous solution was reported to be a function of the presence of additive molecules and ions, pH, ionic strength, and temperature.21 The transformation kinetics from ACP to HAP, which can be described by a ”first-order” rate law, is a function only of the pH of the mediating solution at constant temperature. The solution-mediated transformation depends upon the conditions which regulate both the dissolution of ACP and the formation of the early HAP nuclei.22 Tropp et al. used 31P NMR to demonstrate that the strength of ACP side bands is due to a characteristic structural distortion of unprotonated phosphate and not to a mixture of protonated and unprotonated phosphates,17 suggesting that ACP could contain substantial amounts of protonated phosphate not in the form of any known phase of calcium phosphate crystals. Yin and Stott suggested that, in the transformation from ACP to HAP, ACP need only dissociate into clusters rather than undergo complete ionic solvation. The cluster with C1 symmetry is the most stable isomer in vacuum. The interaction of Posner’s cluster with sodium ions and especially with protons leads to a considerable stability increase, and surprisingly, the cluster with six protons and six OH− recovers the C3 symmetry and similar atomic arrangement that it has as a structural unit in the HAP crystal. This may be a key factor in the transformation from ACP to HAP crystal.23

In general, ACP is a highly unstable phase that hydrolyzes almost instantaneously to more stable phases. In the presence of other ions and macromolecules or under in vivo conditions, ACP may persist for appreciable periods3 and retain the amporphous state under some specific experimental conditions.24

/pdf/calcium-orthophosphates-crystallization-and-dissolution-1c1riyx5dd.pdf

Calcium orthophosphates: crystallization and dissolution.

Many proteins found in mineralized tissues have been proposed to function as regulators of the mineralization process, either as nucleators or inhibitors of hydroxyapatite (HA) formation. We have studied the HA-nucleating and HA-inhibiting properties of proteins from bone [osteocalcin (OC), osteopontin (OPN), osteonectin (ON) and bone sialoprotein (BSP)], dentine [phosphophoryn (DPP)] and calcified cartilage [chondrocalcin (CC)] over a wide range of concentrations. Nucleation of HA was studied with a steady-state agarose gel system at sub-threshold [Ca] x [PO4] product. BSP and DPP exhibited nucleation activity at minimum concentrations of 0.3 microgram/ml (9 nM) and 10 micrograms/ml (67 nM) respectively. OC, OPN, ON and CC all lacked nucleation activity at concentrations up to 100 micrograms/ml. Inhibition of HA formation de novo was studied with calcium phosphate solutions buffered by autotitration. OPN was found to be a potent inhibitor of HA formation [IC50 = 0.32 microgram/ml (0.01 microM)] whereas OC was of lower potency [IC50 = 6.1 micrograms/ml (1.1 microM)]; BSP, ON and CC all lacked inhibitory activity at concentrations up to 10 micrograms/ml. The effect of OPN on HA formation de novo is mainly to inhibit crystal growth, whereas OC delays nucleation. These findings are consistent with the view that BSP and DPP may play roles in the initiation of mineralization in bone and dentine respectively. OPN seems to be the mineralized tissue protein most likely to function in the inhibition of HA formation, possibly by preventing phase separation in tissue fluids of high supersaturation.

A R Poole

Papers

Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins.