In vertebrates and invertebrates biomineralization is controlled by the cell and the proteins they produce. A large number of these proteins are intrinsically disordered, gaining some secondary structure when they interact with their binding partners. These partners include the component ions of the mineral being deposited, the crystals themselves, the template on which the initial crystals form, and other intrinsically disordered proteins and peptides. This review speculates why intrinsically disordered proteins are so important for biomineralization, providing illustrations from the SIBLING (small integrin binding N-glycosylated) proteins and their peptides. It is concluded that the flexible structure, and the ability of the intrinsically disordered proteins to bind to a multitude of surfaces is crucial, but details on the precise-interactions, energetics and kinetics of binding remain to be determined.

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Bone is a highly organized tissue in which each structural level influences the macroscopic and microscopic mechanical behavior. In particular, the quantity, quality, and distribution of the different bone components, i.e. collagen matrix and hydroxyapatite crystals, are associated with bone strength or fragility. Common spectroscopic techniques used to assess bone composition have resolutions limited to the micrometer range. In this study, our aims were two-fold: i) to develop and validate the AFM-IR methodology for skeletal tissues and ii) to apply the methodology to sheep cancellous bone with the objective to obtain novel findings on the composition and structure of trabecular packets.To develop the methodology, we assessed spatial and temporal reproducibility using a known homogeneous material (polymethylmethacrylate, PMMA). We verified that the major peak positions were similar and not shifted when compared to traditional Fourier Transform Infrared imaging (FTIRI). When AFM-IR was applied to sheep cancellous bone, the mineral-to-matrix ratio increased and the acid phosphate substitution ratio decreased as a function of tissue maturity. The resolution of the technique enabled visualization of different stages of the bone maturation process, particularly newly-formed osteoid prior to mineralization. We also observed alternating patterns of IR parameters in line and imaging measurements, suggesting the apposition of layers of alternating structure and / or composition that were not visible with traditional spectroscopic methods. In conclusion, nanoscale IR spectroscopy demonstrates novel compositional and structural changes within trabecular packets in cancellous bone. Based on these results, AFM-IR is a valuable tool to investigate cancellous bone at the nanoscale and, more generally, to analyze small dynamic areas that are invisible to traditional spectroscopic methods.

Dentin phosphoprotein (DPP) is a protein expressed mainly in dentin and to a lesser extent in bone. DPP has a disordered structure, rich in glutamic acid, aspartic acid and phosphorylated serine/threonine residues. It has a high capacity for binding to calcium ions and to hydroxyapatite (HA) crystal surfaces. We used molecular dynamics (MD) simulations as a method for virtually screening interactions between DPP motifs and HA. The goal was to determine which motifs are absorbed to HA surfaces. For these simulations, we considered five peptides from the human DPP sequence. All-atom MD simulations were performed using GROMACS, the peptides were oriented parallel to the {100} HA crystal surface, the distance between the HA and the peptide was 3 nm. The system was simulated for 20 ns. Preliminary results show that for the unphosphorylated peptides, the acidic amino acids present an electrostatic attraction where their side chains are oriented towards HA. This attraction, however, is slow to facilitate bulk transport to the crystal surface. On the other hand, the phosphorylated (PP) peptides are rapidly absorbed on the surface of the HA with their centers of mass closer to the HA surface. More importantly, the root mean square fluctuation (RMSF) indicates that the average structures of the phosphorylated peptides are very inflexible and elongate, while that of the unphosphorylated peptides are flexible. Radius of gyration (Rg) analysis showed the compactness of un-phosphorylated peptides is lower than phosphorylated peptides. Phosphorylation of the DPP peptides is necessary for binding to HA surfaces.

Dentin phosphoprotein (DPP) is the most acidic protein in vertebrates and structurally is classified as an intrinsically disordered protein. Functionally, DPP is related with dentin and with bone formation, however the specifics of such association remain unknown. Here, we used atomistic molecular dynamics simulations to screen selected binding domains of DPP onto hydroxyapatite (HA), which is one of its important interacting partners. From these results, we selected a functionally relevant peptide, Ace-SSDSSDSSDSSDSSD-NH2 (named P5) and its phosphorylated form (named P5P), for experimental characterization. SAXS experiments indicated that in solution P5 was disordered, possibly in an extended conformation while P5P displayed more compact globular conformations. Circular dichroism and FTIR confirmed that, either in the presence or absence of Ca2+/HA, P5 adopts a random coil structure, whereas its phosphorylated counterpart, P5P, has a more compact arrangement associated with conformations that display β-sheet and α-helix motifs when bound to HA. In solution, P5 inhibited HA crystal growth, whereas at similar concentrations, P5P stimulated it. These findings suggest that phosphorylation controls the transient formation of secondary and tertiary structure of DPP peptides, and, most likely of DPP itself, which in turn controls HA growth in solution and possibly HA growth in mineralized tissues.

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Composite materials are used in bone tissue engineering because they mimic the structure of the extracellular matrix of bone. In this work, polylactic acid (PLA) fiber scaffolds prepared by air-jet spinning technique, were doped with different concentrations of SBA-15 ceramic (0.05, 0.1, and 0.15 wt%). The SBA-15 ceramic powder was made by the Sol-Gel process. Physicochemical characterization of PLA, SBA-15, and composite fiber scaffold was done by XRD, SEM, BET, FTIR, TGA, mechanical test, and biocompatibility assay, which were performed in a cell culture model with osteoblast cells. Our results showed a random nanofibers composite scaffold with an improvement in the physicochemical properties. The PLA fiber diameter increases as increases the content of SBA-15, and the mechanical properties were dose-dependent. SBA-15 shows the well-ordered mesoporous hexagonal structure with a pore size of 5.8 ± 0.2 nm and a specific surface area with a value of 1042 ± 89 m 2 /g. PLA fibers and composites have thermal stability up to 300°C, and thermal decomposition in the range 316-367°C. In vitro biocompatibility results showed that PLA/SBA-15 composite scaffold had no cytotoxicity effect in terms of cell adhesion and viability of osteoblast cells. Furthermore, the doped SBA-15 with 0.05% wt onto the polymer matrix could be useful in biomedical applications for bone tissue engineering.