Biominerals and Biomimetics

Biominerals are organic-inorganic composites formed by biological organisms. There are more than 70 different types of biominerals found in organisms. These bioceramic hybrid materials are found in the hard tissues of many organisms and serve a variety of functions, ranging from structural support in skeletons to magnetoreception in bacteria. Materials scientists have long been fascinated by the elaborate microstructures that are created by the cellular processes in organisms, particularly since physiological conditions provide a rather limited supply of materials to work with, mainly mineral salt precipitates.

Because of these limitations, organisms have evolutionarily adapted a means to fabricate hierarchically structured composites which can enhance the properties of the biominerals. This includes, for example, the ‘optical fibers’ in the siliceous glass skeleton of some sea sponges, the protective exoskeletons of marine invertebrates, or the strength and toughness of bones and teeth in vertebrates, all of which are made from ceramic materials that are normally very brittle. The organic component in these bioceramics, which includes biopolymers such as proteins, polysaccharides, and phospholipids, is often only a minor constituent of the biocomposite, yet it plays a decisive role in both the properties and the fabrication of the biomineral. Such biopolymers act as crystal growth modifiers that are able to regulate crystal size, shape, phase, orientation, composition, texture, and location of the mineral crystals.

Biomimetic engineers seek to understand and develop novel microstructural processing techniques that will lead to the high degree of control exerted by biomineralizing organisms. A primary goal of our group is to develop a molecular took-kit that can be used to modulate the growth of inorganic crystals and build novel hybrid composite structures. We are inspired by both the beauty and utility of the materials hidden in the microstructures of the world around us.

Calcium Phosphate Biominerals in Bone

Classic examples of hierarchically-structured biominerals are found in the bones and teeth of vertebrates. For example, dental enamel, which needs to be hard and tough, is made out of ‘rods’ of extremely long prismatic crystals of hydroxyapatite (HAp), which are then organized into species specific microstructures, such as the ‘woven fibers’ of rat enamel. In contrast, bone consists of extremely small nanocrystals of hydroxyapatite that are embedded within an organic matrix. In both cases, the mineral needs to be strong and tough to provide structural support, yet hydroxyapatite (HAp) is a brittle ceramic. Nature has found a way to get around this limitation by intercalating the nanocrystals throughout an organic matrix to enhance the fracture toughness. Collagen, the primary constituent of this organic matrix in bone, is an amazing macromolecule that self assembles into triple helices that assemble further into fibrils . These fibrils provide a scaffold for the deposition of the mineral phase in a highly organized structure. Extremely small nanocrystals of hydroxyapatite (HAp) are formed within and throughout the interstices of collagen fibrils, such that the intercalated crystals become protected by the ductile collagen, providing bone with both high strength and toughness.

The interpenetrating nanostructure of bone is created by ‘intrafibrillar’ mineralization , which our group has successfully been able to reproduce in the beaker. Our trick to achieving intrafibrillar mineralization relates to the use of polypeptide additives that mimic the acidic proteins found to be closely associated with bone and other biominerals. These proteins are unusual in that they contain a large number of amino acids with carboxylate or phosphate side groups, and this creates a protein far more negatively charged than any other proteins found in the body. In our in vitro model system of bone formation, we find that similar negatively charged polypeptides sequester mineral ions to form a highly hydrated amorphous mineral precursor. nanodroplets of this “polymer-induced liquid-precursor” (PILP) phase are formed, and we hypothesize that these droplets can then infiltrate the collagen fibrils by capillary action to form an interpenetrating nanostructured architecture that closely mimics that of bone.

We are currently working toward fabricating the next level of hierarchy in bone, the lamellar microstructure found in osteonal bone, which may allow us to prepare artificial bone-like composites with mechanical properties that match bone. In addition, the close similarity between the composition and structure of our biomimetic bone and that of native bone may provide a biomaterial that osteoclasts and osteoblasts (bone resorbing and bone forming cells, respectively) recognize as looking like natural bone, allowing them remodel the biomaterial using the same cellular processes involved in natural bone remodeling and repair. The ultimate goal is to fabricate biomimetic bone-like materials for applications in tissue engineering and load-bearing bioresorbable bone graft substitutes. In addition, the ability to tailor the properties, such as by controlling the assembly of the collagen scaffold and/or degree of mineralization, provides a model system that can contribute to investigation of bone’s unique mechanical properties.

Calcium Carbonate Biominerals in Shells and Exoskeletons

Another classical example of hierarchically structured biominerals is found in mollusk shells. The shells are organized into multilayered composites with each layer exhibiting different properties because they are composed of different phases of calcium carbonate. For example, calcite and aragonite, two phases of CaCO3, are found in the prismatic and nacreous layers of these shells, respectively. Interestingly, these layers are formed under similar reaction conditions, yet the crystal phase is regulated as well as the morphologies of the crystals. The crystals are then assembled into the two layers with differing microstructures, where the hard outer shell consists of prismatic layer comprised of columns of calcite while the inner nacreous layer (known for itspearlescent appearance) is composed of a ‘brick-n-mortar’ architecture, with ‘bricks’ of oriented aragonite tablets ‘glued’ together with a chitin organic matrix. In both cases, the crystals have unique morphologies that bear little resemblance to crystals ofcalcite and aragonite grown in the beaker. Overall, mollusk shells demonstrate that the processes involved in biomineralization provide the ability to control crystal size, shape, orientation, phase, texture, composition, and location.

This is accomplished primarily through interactions of the growing crystals with the surrounding organic matrix, which can template crystal nucleation and/or modulate crystal growth, as well as regulate aggregation or assembly. For example, an insoluble organic matrix can provide a template to control the location of the nucleation event, or can be assembled into a compartment within which the biomineral crystal is formed to regulate its growth and subsequent shape. Notably, this “molding” of crystals occurs under physiological conditions, where clearly the mineral cannot be melted and formed. A classic example of a ‘molded’ crystal is found in the spine of a sea urchin, which has a fenestrated microporous architecture.

We have put forth the hypothesis that such non-equilibrium crystal morphologies might be generated by a polymer-induced liquid–precursor (PILP) process. This hypothesis was prompted by the discovery of strange helical structures and mineral films in a calcium carbonate reaction containing poly(aspartic acid) additive. It became apparent that this with this non-classical crystallization system, a fluidic amorphous precursor to the mineral might provide a means for “molding” crystals such that an endless array of crystal morphologies might be feasible.

The hallmark of biomineralization is the elaborate morphologies that are formed. With the PILP system, we have been able to reproduce, in the beaker, many of the nano- and micro-structural features found in biominerals. For example, single-crystalline aragonite tablets that mimic mollusk nacre can be prepared; or mineral films can be patterned on organic templates; crystals can molded using a compartment from a replica of the sea urchin spine; nanofibers emulating the ‘fibers’ found in urchin teeth can be grown using a solution-precursor-solid (SPS) mechanism; crystals can be doped to high levels due to entrapment of the impurities within the solidifying precursor phase; fibrous crystals of non-biological minerals such as BaCO3 and SrCO3; and as described above, intrafibrillar crystals with orientation and nanostructure mimicking bone nanostructure. Crystals formed by the PILP process also exhibit a nanocolloidal and nanolaminated texture that emulates that observed by atomic force microscopy (AFM) in many biominerals. We believe such features may point to mineralogical signatures of the mechanisms by which the biomineral crystals are formed. Calcium carbonate system has a few commercial applications (e.g. filler particles and biodegradable core-shell microcapsules for controlled-release pharmaceutical applications), but it also provides a convenient model system for exploring the influence of macromolecules on crystal growth, which ultimately may help us to unravel Mother Nature’s secrets in the formation of biological hard tissues.

Calcium Oxalate and Phosphate Biominerals in Kidney Stones

Biominerals can also be found in pathological mineral deposits, such as in kidney stones, biomaterials encrustation, and atherosclerotic plaque. In the case of kidney stones, our group has developed an in vitro model system of uro/nephrolithiasis, where we have been able to reproduce several of the unusual crystallographic features found in stones, again using peptide additives that mimic the acidic proteins found in urine.

In kidney stones, the biominerals are typically composed of a combination of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals, which grow into aggregates that are attached to, or get lodged within, the nephrons of the kidney. Urine is a complex medium where many proteins and lipids are present, but we hypothesize that some of the acidic proteins (similar to those found in bone formation) may be inadvertently generating a PILP type process that could cause aggregation of crystals, or initiate the amorphous calcium phosphate found in Randall’s plaque, or create large spherulites of Ca0x that overgrows this CaP nidus to form a stone. Several of these features have been mimicked in our in vitro model system, such as globules of ACP, CaOx ‘dumbbells’, and concentrically laminated spherules. The value of an in vitro model system is that it provides a less complex environment for determining the crystallization mechanisms involved in the formation of these features. Such features can then provide mineralogical ‘signatures’ that help to decipher the mechanisms involved in stone formation, so that therapeutic approaches can be developed to reduce or eliminate the painful and sometimes debilitating condition of stone formation.

Oxide based Biominerals

One also finds oxide based ceramics, such as silica and iron oxides, in invertebrate biominerals, and these are areas that our group hopes to explore in the future. Magnetite is found in the ‘teeth’ of chiton, which enhances the hardness of the teeth, enabling the mollusk to scrape algae off of rocks. On the other hand, magnetite is used for its magnetic properties in magnetotactic bacteria. A small chain of magnetite nanoparticles provides a ‘compass’ that directs the swimming of the microorganism into the favorable microaerobic sediment. From a materials perspective, one finds that the nanoparticles are highly regulated in terms of size and uniformity, which enables them to have single domain magnetic properties. In some cases, the nanoparticles are fabricated into elongated bullet-shaped particles.

Generally, it is difficult to achieve such an anisotropic shape in magnetite due to the high symmetry of its lattice. Elongated magnetite crystals are also found in the teeth of the chiton, which are oriented perpendicular to the impact zone to enhance the hardness of the teeth. A similar arrangement is found in dental enamel of vertebrates and outer layer of urchin teeth; yet calcium phosphate and calcium carbonate are mineral of choice for their biominerals.

Biosilica is found in two very different marine organisms, diatoms and sponges. Both have beautiful and elaborate structures. These systems also demonstrate the hierarchical structuring found in biominerals, starting with a colloidal texture at the nanoscale, and continuing with concentric laminations in the case of sponges (similar to the textures observed in PILP formed crystals), or exacting symmetry in the porosity of the diatom frustules. Even mechanically overlapped glass ‘joints and hinges’ are found in the biosilicifying organisms. Interestingly, these organisms also used highly charged macromolecules to regulate the biomineralization process, but in this case, the amine containing macromolecules are positively charged. This is an area we would like to explore due to the similarities yet differences between the roles of the biopolymers found in silica versus calcium based biominerals.

Molecular Recognition in Organic-Inorganic Hybrid Materials

Regulation of biomineralization entails both specific and non-specific interactions between the organic and inorganic phases. The PILP process is considered non-specific because it does not require molecular recognition between the mineral lattice and the soluble polypeptide because the polymer simply sequesters ions to generate an amorphous precursor. However, there are many properties other than crystals morphology that are regulated in biominerals, as mentioned above. Some of these presumably require more specific interactions between the organic template and the biomineral. Biological systems are famous for their capabilities of molecular recognition, but this has generally been considered from the perspective of ‘lock-and-key’ interactions in bioreceptors, enzymes, and antibody binding to antigens, etc.. The role of molecular recognition between organics and inorganics is less well understood. It seems likely that specific interactions based on these principles might enable control over crystal phase and orientation from an organic matrix template. Therefore, we have recently expanded our studies to include more specialized peptides that have specific binding affinity to inorganic surfaces. Using phage display techniques, we are ‘biopanning’ for inorganic binding peptides that bind to electronic materials. Using a combinatorial approach, phage (virus) clones can be used to isolate inorganic binding peptides that serve as linkers between a microelectronics device and a bioreceptor. Our goal is to develop molecular construction kits for the fabrication of highly specialized biosensor devices.