Fabio Nudelman

The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors

Bone is a composite material in which collagen fibrils form a scaffold for a highly organized arrangement of uniaxially oriented apatite crystals. In the periodic 67 nm cross-striated pattern of the collagen fibril, the less dense 40-nm-long gap zone has been implicated as the place where apatite crystals nucleate from an amorphous phase, and subsequently grow. This process is believed to be directed by highly acidic non-collagenous proteins; however, the role of the collagen matrix during bone apatite mineralization remains unknown. Here, combining nanometre-scale resolution cryogenic transmission electron microscopy and cryogenic electron tomography with molecular modelling, we show that collagen functions in synergy with inhibitors of hydroxyapatite nucleation to actively control mineralization. The positive net charge close to the C-terminal end of the collagen molecules promotes the infiltration of the fibrils with amorphous calcium phosphate (ACP). Furthermore, the clusters of charged amino acids, both in gap and overlap regions, form nucleation sites controlling the conversion of ACP into a parallel array of oriented apatite crystals. We developed a model describing the mechanisms through which the structure, supramolecular assembly and charge distribution of collagen can control mineralization in the presence of inhibitors of hydroxyapatite nucleation.

Biomineralization as an Inspiration for materials chemistry

Living organisms are well known for building a wide range of specially designed organic-inorganic hybrid materials such as bone, teeth, and shells, which are highly sophisticated in terms of their adaptation to function. This has inspired physicists, chemists, and materials scientists to mimic such structures and their properties. In this Review we describe how strategies used by nature to build and tune the properties of biominerals have been applied to the synthesis of materials for biomedical, industrial, and technological purposes. Bio-inspired approaches such as molecular templating, supramolecular templating, organized surfaces, and phage display as well as methods to replicate the structure and function of biominerals are discussed. We also show that the application of in situ techniques to study and visualize the bio-inspired materials is of paramount importance to understand, control, and optimize their preparation. Biominerals are synthesized in aqueous media under ambient conditions, and these approaches can lead to materials with a reduced ecological footprint than can traditional methods.

In vitro models of collagen biomineralization

Over the last several years, significant progress has been made toward understanding the mechanisms involved in the mineralization of hard collagenous tissues, such as bone and dentin. Particularly notable are the identification of transient mineral phases that are precursors to carbonated hydroxyapatite, the identification and characterization of non-collagenous proteins that are involved in controlling mineralization, and significant improvements in our understanding of the structure of collagen. These advances not only represent a paradigm shift in the way collagen mineralization is viewed and understood, but have also brought new challenges to light. In this review, we discuss how recent in vitro models have addressed critical questions regarding the role of the non-collagenous proteins in controlling mineralization, the nature of the interactions between amorphous calcium phosphate and collagen during the early stages of mineralization, and the role of collagen in the mineralization process. We discuss the significance of these findings in expanding our understanding of collagen biomineralization, while addressing some of the limitations that are inherent to in vitro systems.

Temperature-Responsive Nanospheres with Bicontinuous Internal Structures from a Semicrystalline Amphiphilic Block Copolymer

Internally structured self-assembled nanospheres, cubosomes, are formed from a semicrystalline block copolymer, poly(ethylene oxide)-block-poly(octadecyl methacrylate) (PEO(39)-b-PODMA(17)), in aqueous dispersion. The PODMA block provides them with a temperature-responsive structure and morphology. Using cryo-electron tomography, we show that at room temperature these internally bicontinuous aggregates undergo an unprecedented order-disorder transition of the microphase-separated domains that is accompanied by a change in the overall aggregate morphology. This allows switching between spheres with ordered bicontinuous internal structures at temperatures below the transition temperature and more planar oblate spheroids with a disordered microphase-separated state above the transition temperature. The bicontinuous structures offer a number of possibilities for application as templates, e.g., for biomimetic mineralization or polymerization. Furthermore, the unique nature of the thermal transition observed for this system offers up considerable possibilities for their application as temperature-controlled release vessels.

Uniting Polypeptides with Sequence-Designed Peptides: Synthesis and Assembly of Poly(γ-benzyl l-glutamate)-b-Coiled-Coil Peptide Copolymers

A new class of peptide has been created, polypeptide-b-designed peptides, which unites the useful qualities of the two constituent peptide types. We demonstrate the synthesis and self-assembly possibilities of this class of peptide chimera with a series of amphiphilic polypeptide-b-designed peptides in which the hydrophobic block is poly(gamma-benzyl l-glutamate) (PBLG) and the hydrophilic block is a coiled-coil forming peptide (denoted E). The synthetic approach was to synthesize the coiled-coil forming peptide on the solid phase, followed by the ring-opening polymerization of gamma-benzyl l-glutamate N-carboxyanhydride, initiated from the N-terminal amine of the peptide E on the solid support. The polypeptide-b-peptide was then cleaved from the resin, requiring no further purification. Peptide E contains 22 amino acids, while the average length of the PBLG block ranged from 36 to 250 residues. This new class of peptide was applied to create a modular system, which relied on juxtaposing the properties of the component peptide types, namely the broad size range and structure-inducing characteristics of the polypeptide PBLG blocks, and the complex functionality of the sequence-designed peptide. Specifically, the different PBLG block lengths could be connected noncovalently with various hydrophilic blocks via the specific coiled-coil folding of E with K or K-poly(ethylene glycol), where K is a peptide of complementary amino acid sequence to E. In this way, nanostructures could be formed in water at neutral pH over the entire compositional range, which has not been demonstrated previously with such large PBLG blocks. It was found that the size, morphology (polymersomes or bicelles), and surface functionality could be specified by combining the appropriate modular building blocks. The self-assembled structures were characterized by dynamic light scattering, circular dichroism, scanning electron microscopy, cryogenic-transmission electron microscopy, fluorescence spectroscopy, and zeta-potential measurements. Finally, as the structures are able to encapsulate water-soluble compounds, and the surfaces are easily functionalized via the coiled-coil binding, it is expected that these peptide-based nanocapsules will be able to act as delivery vehicles to specific targets in the body.

Cryo-electron tomography: 3-dimensional imaging of soft matter

The advent of cryogenic-transmission electron microscopy (cryoTEM) signified a breakthrough in the in situ imaging of hydrated specimens of biological and synthetic origin allowing their study in a state of preservation that is close to native. An inherent limitation to cryoTEM, however, is that images are 2-dimensional projections of the 3-dimensional objects, resulting in the overlapping of multiple features that cannot be discerned. Cryo-electron tomography (cryoET) is essential to overcome this limitation. In this technique images of the specimen are acquired at different tilt angles and then reconstructed into the 3-dimensional object, revealing detailed information on the structure, morphology or 3-dimensional spatial organization of (bio)macromolecules and (macro)molecular assemblies. This information then can be coupled to processes happening in the 3-dimensional space, making cryoET an invaluable tool to bridge between the structural organization in space and the function or activity of macromolecular complexes at the nanometre scale.

Stabilization of amorphous calcium carbonate by controlling its particle size

Amorphous calcium carbonate (ACC) nanoparticles of different size are prepared using a flow system. Post-synthesis stabilization with a layer of poly[(α,β)-dl-aspartic acid] leads to stabilization of the ACC, but only for particles <100 nm. Larger and uncoated particles readily convert into the crystalline forms of CaCO₃. This shows that ACC is intrinsically stable below 100 nm.

Controlling Internal Pore Sizes in Bicontinuous Polymeric Nanospheres

Complex polymeric nanospheres were formed in water from comb-like amphiphilic block copolymers. Their internal morphology was determined by three-dimensional cryo-electron tomographic analysis. Varying the polymer molecular weight (MW) and the hydrophilic block weight content allowed for fine control over the internal structure. Construction of a partial phase diagram allowed us to determine the criteria for the formation of bicontinuous polymer nanosphere (BPN), namely for copolymers with MW of up to 17 kDa and hydrophilic weight fractions of ≤0.25; and varying the organic solvent to water ratio used in their preparation allowed for control over nanosphere diameters from 70 to 460 nm. Significantly, altering the block copolymer hydrophilic–hydrophobic balance enabled control of the internal pore diameter of the BPNs from 10 to 19 nm.

The role of the amorphous phase on the biomimetic mineralization of collagen.

Bone is a hierarchically structured composite material whose basic building block is the mineralized collagen fibril, where the collagen is the scaffold into which the hydroxyapatite (HA) crystals nucleate and grow. Understanding the mechanisms of hydroxyapatite formation inside the collagen is key to unravelling osteogenesis. In this work, we employed a biomimetic in vitro mineralization system to investigate the role of the amorphous precursor calcium phosphate phase in the mineralization of collagen. We observed that the rate of collagen mineralization is highly dependent on the concentration of polyaspartic acid, an inhibitor of hydroxyapatite nucleation and inducer of intrafibrillar mineralization. The lower the concentration of the polymer, the faster the mineralization and crystallization. Addition of the non-collagenous protein C-DMP1, a nucleator of hydroxyapatite, substantially accelerates mineral infiltration as well as HA nucleation. We have also demonstrated that Cu ions interfere with the mineralization process first by inhibiting the entry of the calcium phosphate into the collagen, and secondly by stabilizing the ACP, such that it does not convert into HA. Interestingly, under these conditions mineralization happens preferentially in the overlap regions of the collagen fibril. Our results show that the interactions between the amorphous precursor phase and the collagen fibril play an important role in the control over mineralization.

Nacre biomineralisation: A review on the mechanisms of crystal nucleation

The wide diversity of biogenic minerals that is found in nature, each with its own morphology, mechanical properties and composition, is remarkable. In order to produce minerals that are optimally adapted for their function, biomineralisation usually occurs under strict cellular control. This control is exerted by specialised proteins and polysaccharides that assemble into a 3-dimensional organic matrix framework, forming a microenvironment where mineral deposition takes place. Molluscs are unique in that they use a striking variety of structural motifs to build their shells, each made of crystals with different morphologies and different calcium carbonate polymorphs. Much of want is known about mollusc shell formation comes from studies on the nacreous layer, or mother-of-pearl. In this review, we discuss two existing models on the nucleation of aragonite crystals during nacre formation: heteroepitaxial nucleation and mineral bridges. The heteroepitaxial nucleation model is based on the identification of chemical functional groups and aragonite-nucleating proteins at the centre of crystal imprints. It proposes that during nacre formation, each aragonite tablet nucleates independently on a nucleation site that is formed by acidic proteins and/or glycoproteins adsorbed on the chitin scaffold. The mineral bridges model is based on the identification of physical connections between the crystals in a stack, which results in a large number of crystals across several layers sharing the same crystallographic orientation. These observations suggest that there is one nucleation event per stack of tablets. Once the first crystal nucleates and reaches the top interlamellar matrix, it continues growing through pores, giving rise to the next layer of nacre, subsequently propagating into a stack. We compare both models and propose that they work in concert to control crystal nucleation in nacre. De novo crystal nucleation has to occur at least once per stack of aligned crystals, and is induced by nucleation sites. We suggest that further growth is controlled both by mineral bridges and nucleation sites. Finally, we discuss the role of amorphous calcium carbonate precursor in nacre formation.