Many mineralizing organisms selectively form either calcite or aragonite, two polymorphs of calcium carbonate with very similar crystalline structures. Understanding how these organisms achieve this control has represented a major challenge in the field of biomineralization. Macromolecules extracted from the aragonitic shell layers of some mollusks induced aragonite formation in vitro when preadsorbed on a substrate of β-chitin and silk fibroin. Macromolecules from calcitic shell layers induced mainly calcite formation under the same conditions. The results suggest that these macromolecules are responsible for the precipitation of either aragonite or calcite in vivo.

https://science.sciencemag.org/content/sci/271/5245/67.full.pdf

Control of Aragonite or Calcite Polymorphism by Mollusk Shell Macromolecules

Bone is a hierarchically structured composite material which, in addition to its obvious biological value, has been well studied by the materials engineering community because of its unique structure and mechanical properties. This article will review the existing bone literature, with emphasis on the prevailing theories regarding bone formation and structure, which lay the groundwork for proposing a new model to explain how intrafibrillar mineralization of collagen can be achieved during bone formation. Intrafibrillar refers to the fact that growth of the mineral phase is somehow directed by the collagen matrix, which leads to a nanostructured architecture consisting of uniaxially oriented nanocrystals of hydroxyapatite embedded within and roughly [0 0 1] aligned parallel to the long collagen fibril axes. Secondary (osteonal) bone, the focus of this review, is a laminated organic–inorganic composite composed primarily of collagen, hydroxyapatite, and water; but minor constituents, such as non-collagenous proteins (NCPs), are also present and are thought to play an important role in bone formation. To date, there has been no clear understanding of the role of these NCPs, although it has been generally assumed that the NCPs regulate solution crystal growth via some type of ‘epitaxial’ relationship between specific crystallographic faces and specific protein conformers. Indeed, ‘epitaxial’ relationships have been calculated; but in practice, it has not been demonstrated that intrafibrillar mineralization can be accomplished via this route. Because of the difficulty in examining biomineralization processes in vivo , the authors of this article have turned to using in vitro model systems to investigate the possible physicochemical mechanisms that may be involved in biomineralization. In the case of bone biomineral, we have now been able to duplicate the most fundamental level of bone structure, the interpenetrating nanostructured architecture, using relatively simple anionic polypeptides that mimic the polyanionic character of the NCPs. We propose that the charged polymer acts as a process-directing agent, by which the conventional solution crystallization is converted into a precursor process. This polymer-induced liquid-precursor (PILP) process generates an amorphous liquid-phase mineral precursor to hydroxyapatite which facilitates intrafibrillar mineralization of type-I collagen because the fluidic character of the amorphous precursor phase enables it to be drawn into the nanoscopic gaps and grooves of collagen fibrils by capillary action. The precursor then solidifies and crystallizes upon loss of hydration waters into the more thermodynamically stable phase, leaving the collagen fibrils embedded with nanoscopic hydroxyapatite (HA) crystals. Electron diffraction patterns of the highly mineralized collagen fibrils are nearly identical to those of natural bone, indicating that the HA crystallites are preferentially aligned with [0 0 1] orientation along the collagen fibril axes. In addition, studies of etched samples of natural bone and our mineralized collagen suggest that the long accepted “deck of cards” model of bone's nanostructured architecture is not entirely accurate. Most importantly, this in vitro model demonstrates that a highly specific, epitaxial-type interaction with NCPs is not needed to stimulate crystal nucleation and regulate crystal orientation, as has long been assumed. Instead, we propose that collagen is the primary template for crystal organization, but with the important caveat that this templating occurs only for crystals formed from an infiltrated amorphous precursor. These results suggest that the 25-year-old debate regarding bone formation via an amorphous precursor phase needs to be revisited. From a biomedical perspective, in addition to providing possible insight into the role of NCPs in bone formation, this in vitro system may pave the way toward the ultimate goal of fabricating a synthetic bone substitute that not only has a composition similar to bone, but has comparable mechanical properties and bioresorptive potential as natural bone. From a materials chemistry perspective, the non-specificity of the PILP process and capillary infiltration mechanism suggests that non-biological materials could also be fabricated into nanostructured composites using this “biomimetic” strategy.

Bone structure and formation: A new perspective

Organisms are capable of forming a diverse array of minerals, some of which cannot be formed inorganically in the biosphere. The initial precipitates may differ from the form in which they are finally stabilized, or during development of the organism one mineral may substitute for another. Biogenic minerals commonly have attributes which distinguish them from their inorganic counterparts. They fulfill important biological functions. They have been formed in ever-increasing amounts during the last 600 million years and have radically altered the character of the biosphere.

http://science.sciencemag.org/content/sci/211/4487/1126.full.pdf

Minerals formed by organisms

Mother-of-pearl (nacre) is a platelet-reinforced composite, highly filled with calcium carbonate (aragonite). The Young modulus, determined from beams of a span-to-depth ratio of no less than 15 (a necessary precaution), is of the order of 70 GPa (dry) and 60 GPa (wet), much higher than previously recorded values. These values can be derived from ‘shear-lag’ models developed for platey composites, suggesting that nacre is a near-ideal material. The tensile strength of nacre is of the order of 170 MPa (dry) and 140 MPa (wet), values which are best modelled assuming that pull-out of the platelets is the main mode of failure. In three-point bending, depending on the span-to-depth ratio and degree of hydration, the work to fracture across the platelets varies from 350 to 1240 J m -2 . In general, the effect of water is to increase the ductility of nacre and increase the toughness almost tenfold by the associated introduction of plastic work. The pull-out model is sufficient to account for the toughness of dry nacre, but accounts for only a third of the toughness of wet nacre. The additional contribution probably comes from debonding within the thin layer of matrix material. Electron microscopy reveals that the ductility of wet nacre is caused by cohesive fracture along platelet lamellae at right angles to the main crack. The matrix appears to be well bonded to the lamellae, enabling the matrix to be stretched across the delamination cracks without breaking, thereby sustaining a force across a wider crack. Such a mechanism also explains why toughness is dependent on the span-to-depth ratio of the test piece. With this last observation as a possible exception, nacre does not employ any really novel mechanisms to achieve its mechanical properties. It is simply ‘well made’. The importance of nacre to the mollusc depends both on the material and the size of the shell. Catastrophic failure will be very likely in whole, undamaged shells which behave like unnotched beams at large span-to-depth ratios. This tendency is increased by the fact that predators act as ‘soft’ machines and store strain energy which can be fed into the material very quickly once the fracture stress has been reached. It may therefore be advantageous to have a shell made of an intrinsically less tough material which is better at stopping cracks (e. g. crossed lamellar). However, nacre may still be preferred for the short, thick shells of young molluscs, as these have a low span-to-depth ratio and can make better use of ductility mechanisms.

The Mechanical Design of Nacre

Organisms have been producing mineralized skeletons for the past
550
million years. They have evolved many different strategies for
improving
these materials at almost all hierarchical levels from
Angstroms to
millimetres. Key components of biological materials are the
macromolecules, which are intimately involved in controlling
nucleation,
growth, shaping and adapting mechanical properties of the mineral
phase to
function. One interesting tendency that we have noted is that
organisms
have developed several strategies to produce materials that have
more
isotropic properties. Much can still be learned from studying the
principles of structure–function relations of biological
materials.
Some of this information may also provide new ideas for improved
design of
synthetic materials.

Design strategies in mineralized biological materials

The purpose of this study was to investigate the mineral induction capacity in vitro of polyanionic proteins covalently bound to a surface. Rat dentin gamma-carboxyglutamate-containing protein of the osteocalcin type (Gla-protein), proteoglycan (PG), and phosphoprotein (PP-H), as well as phosvitin (PhV) and bovine serum albumin (BSA), were covalently linked to agarose beads. There were incubated at 37 degrees C in solutions with a Ca/P molar ratio of 1.67, [Ca][P] molar products in the range 1.0-1.8 mM2, and an ionic strength of 0.165. The incubations were performed at constant pH and composition conditions; no spontaneous precipitation occurred under these conditions. Mineral formation, as monitored by scanning electron microscopy (SEM), was induced by all immobilized polyanions, including enzymatically dephosphorylated PP-H and PhV. No mineral was induced by BSA. The mineral inductive capacity of immobilized polyanionic proteins, as judged by the SEM after identical incubations, was found to differ between the different ligands. The mineral induced by PP-H and PG was shown by X-ray diffraction to be apatitic. It was concluded that, although polyanionic proteins in solution may inhibit mineral induction and growth, very minute quantities of such molecules, when immobilized on a surface, induce mineral at physiological concentrations of calcium and phosphate ions. The data presented may be taken to suggest that PP-H and PG, and perhaps other polyanions, may possibly be responsible for mineral nucleation in dentin and bone. The results, however, also point to the rather limited specificity in this type of reaction.

Mineral induction by immobilized polyanionic proteins.

Mineral induction by immobilized phosphoproteins.

Decalcification at the mantle-shell interface in Mercenaria tnercenaria was studied through the changes in the chemical composition of the extrapallial fluid, and by the measurement of Ca45-deposition and solution. Measurements of O2-tension demonstrated that the clam was anaerobic soon after the valves were closed. Measurements of calcium, carbon dioxide, and hydrogen ion concentration showed that all of these components of the extrapallial fluid increase with increasing time of closure. These measurements, and measurements of calcium and succinic acid in the tissues and fluids of the clam, indicated thatsuccinic acid produced by the anaerobic metabolism of the clam was neutralized by the dissolution of previously deposited shell.

/pdf/decalcification-at-the-mantle-shell-interface-in-molluscs-4nub7wyki1.pdf

Decalcification at the Mantle-Shell Interface in Molluscs

The hydrophilic, sulfated fraction of the organic matrix found in molluscan shells appears to be involved in crystal nucleation. It is located primarily at the sites of initial nucleation. The hydrophilic fraction favors in vitro formation of calcified deposits, when it is fixed in place on the hydrophobic fraction. Calcium is bound by the hydrophillic fraction with high affinity and selectivity. Enzymatic desulfation reduces the calcium binding. However, the binding stoichiometry of one calcium for every two ester sulfates is not altered. The calcium binding induces local anion binding, which induces secondary calcium binding. This coordinated ion binding is known as ionotropy. The resultant local high concentration of ions is thought to bring about nucleation.

/pdf/ionotropic-nucleation-of-calcium-carbonate-by-molluscan-1c4krtgb8x.pdf

Miles A. Crenshaw

Papers

Mineral induction by immobilized polyanionic proteins.

Mineral induction by immobilized phosphoproteins.

Decalcification at the Mantle-Shell Interface in Molluscs

Ionotropic Nucleation of Calcium Carbonate by Molluscan Matrix

Induction and inhibition of hydroxyapatite formation by rat dentine phosphoprotein in vitro.