Controlled crystallisation of calcium phosphate and calcium carbonate via bio-inspired approaches: additives and confinement

This thesis describes the investigation of the two bio-inspired approaches, confinement and additives, to manipulate the crystallization of calcium carbonate and calcium phosphate. The first experimental chapter deals with the investigation of calcium phosphate rods grown in confined environments in the absence and presence of polyaspartic acid. Although similar results were obtained in the absence and presence of the additive, growing calcium phosphate in confinement allowed formation of polycrystalline rods with an orientation comparable to bone. This demonstrated that confinement may play a more significant role in bone formation than previously anticipated. The second chapter deals with the effect of positively charged additives on the crystallisation of CaCO3. Although neglected before in literature, this chapter demonstrates that positively charged additives have a profound effect on the crystallisation of CaCO3 changing the morphology to films and fibers. This morphology change was linked to a phase separation process, forming hydrated amorphous droplets of calcium carbonate by a carbonate-polymer interaction, which had the tendency to coalesce and form films. Fiber formation was attributed to oriented attachment of anisotropic particles due to unequal distribution of charge. In the third chapter, based on bone, the mineralisation of collagen by CaCO3 was investigated. By formation of a highly hydrated liquid-like amorphous phase of CaCO3, it was possible to infiltrate the nanoscale gaps of collagen. After crystallisation, nanocrystals of calcite and vaterite were formed, 5 nm thick, but randomly oriented, demonstrating collagen templates the shape but not the orientation of the crystals. In a final chapter hollow rods of CaCO3 were formed by templating them inside membrane pores. The influence of time, pore size, additives and surface chemistry was investigated. Most hollow rods were formed at early timescales which filled up at later times. By changing the surface chemistry, the amount of hollow rods increased significantly in the 200 nm pore

Formation and structure of calcium carbonate thin films and nanofibers precipitated in the presence of poly(allylamine hydrochloride) and magnesium ions

That the cationic polyelectrolyte poly(allylamine hydrochloride) (PAH) exerts a significant influence on CaCO₃ precipitation challenges the idea that only anionic additives have this effect. Here, we show that in common with anionic polyelectrolytes such as poly(aspartic acid), PAH supports the growth of calcite thin films and abundant nanofibers. While investigating the formation of these structures, we also perform the first detailed structural analysis of the nanofibers by transmission electron microscopy (TEM) and selected area electron diffraction. The nanofibers are shown to be principally single crystal, with isolated domains of polycrystallinity, and the single crystal structure is even preserved in regions where the nanofibers dramatically change direction. The formation mechanism of the fibers, which are often hundreds of micrometers long, has been the subject of intense speculation. Our results suggest that they form by aggregation of amorphous particles, which are incorporated into the fibers uniquely at their tips, before crystallizing. Extrusion of polymer during crystallization may inhibit particle addition at the fiber walls and result in local variations in the fiber nanostructure. Finally, we investigate the influence of Mg²+ on CaCO₃ precipitation in the presence of PAH, which gives thinner and smoother films, together with fibers with more polycrystalline, granular structures

Characterization of soluble TLR2 and CD14 levels during acute dengue virus infection

Dengue virus infection results in a broad spectrum of diseases ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Hitherto, there is no consensus biomarker for the prediction of severe dengue disease in patients. Yet, early identification of patients who progress to severe dengue is pivotal for better clinical management. We have recently reported that an increased frequency of classical (CD14 ++CD16 -) monocytes with sustained high TLR2 expression in acutely infected dengue patients correlates with severe dengue development. Here, we hypothesized that the relatively lower TLR2 and CD14 expression in mild dengue patients is due to the shedding of their soluble forms (sTLR2 and sCD14) and that these could be used as indicators of disease progression. Therefore, using commercial sandwich ELISAs, we evaluated the release of sTLR2 and sCD14 by peripheral blood mononuclear cells (PBMCs) in response to in vitro dengue virus (DENV) infection and assessed their levels in acute-phase plasma of 109 dengue patients. We show that while both sTLR2 and sCD14 are released by PBMCs in response to DENV infection in vitro, their co-circulation in an acute phase of the disease is not always apparent. In fact, sTLR2 was found only in 20% of patients irrespective of disease status. In contrast, sCD14 levels were detected in all patients and were significantly elevated in DF patients when compared to DHF patients and age-matched healthy donors. Altogether, our results suggest that sCD14 may help in identifying patients at risk of severe dengue at hospital admittance. </p

Stable Formation of Gold Nanoparticles onto Redox-Active Solid Biosubstrates Made of Squid Suckerin Proteins

The use of biomolecules to synthesize inorganic nanomaterials, including metallic nanoparticles, offers the ability to induce controlled growth under mild environmental conditions. Here, recently discovered silk-like “suckerin” proteins are used to induce the formation of gold nanoparticles (AuNPs). Advantage is taken of the distinctive biological and physico-chemical characteristics of suckerins, namely their facile recombinant expression, their solubility in aqueous solutions, and their modular primary structure with high molar content of redox-active tyrosine (Tyr) residues to induce the formation of AuNPs not only in solution, but also from nanostructured solid substrates fabricated from suckerins.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore

Bioinspired pH and magnetic responsive catechol-functionalized chitosan hydrogels with tunable elastic properties

We have developed pH- and magnetic-responsive hydrogels that are stabilized by both covalent bonding and catechol/Fe3+ ligands. The viscoelastic properties of the gels are regulated by the complexation valence and can be used to tune drug release profiles. The stable incorporation of magnetic nanoparticles further expands control over the mechanical response and drug release, in addition to providing magnetic stimuli-responsivity to the gels.Published versio

Formation and Structure of Calcium Carbonate Thin Films and Nanofibers Precipitated in the Presence of Poly(Allylamine Hydrochloride) and Magnesium Ions

That the cationic polyelectrolyte poly­(allylamine hydrochloride) (PAH) exerts a significant influence on CaCO₃ precipitation challenges the idea that only anionic additives have this effect. Here, we show that in common with anionic polyelectrolytes such as poly­(aspartic acid), PAH supports the growth of calcite thin films and abundant nanofibers. While investigating the formation of these structures, we also perform the first detailed structural analysis of the nanofibers by transmission electron microscopy (TEM) and selected area electron diffraction. The nanofibers are shown to be principally single crystal, with isolated domains of polycrystallinity, and the single crystal structure is even preserved in regions where the nanofibers dramatically change direction. The formation mechanism of the fibers, which are often hundreds of micrometers long, has been the subject of intense speculation. Our results suggest that they form by aggregation of amorphous particles, which are incorporated into the fibers uniquely at their tips, before crystallizing. Extrusion of polymer during crystallization may inhibit particle addition at the fiber walls and result in local variations in the fiber nanostructure. Finally, we investigate the influence of Mg²⁺ on CaCO₃ precipitation in the presence of PAH, which gives thinner and smoother films, together with fibers with more polycrystalline, granular structures

Systematic Study of the Effects of Polyamines on Calcium Carbonate Precipitation

While negatively charged organic additives are widely used as an effective means to control CaCO₃ precipitation, positively charged additives are generally considered to be much less active. Nevertheless, the cationic polyelectrolyte poly­(allylamine hydrochloride) has recently been shown to exert significant control over CaCO₃ precipitation, driving the formation of thin films and fibers, and other examples suggest that many positively charged additives promote vaterite formation. This article aims to bring together these sometimes conflicting views of the activity of positively charged additives. The effect of a series of polyamines on CaCO₃ precipitation was studied, where the polyamines were selected such that the amine group type, the p<i>K</i>_a value (of the corresponding conjugated acid), the molecular weight, and the side chain length of the polymers could be evaluated. The results unambiguously demonstrate that polyamines carrying primary amine groups are capable of exerting a significant effect and that the activity of this class of polyamines is strongly dependent on the length of the side chain. In contrast, polyamines comprising with quaternary amines have negligible effect, despite carrying a permanent positive charge. The activity of the most active polyamines therefore depends on their ability to complex with carbonate ions present in solution, and electrostatic attraction alone is not sufficient

On Duality of Set-Valued Optimization (Nonlinear Analysis and Convex Analysis)

Inspired by the observation that crystalline calcium carbonate and calcium phosphate biominerals frequently form via amorphous precursors, a wide range of studies have been performed which demonstrate that many inorganic crystals can precipitate from solution via amorphous phases. This article considers the crystallization mechanism of calcium oxalate, which is a significant biomineral in many plants and the primary constituent of kidney stones in vertebrates, and shows that this can also precipitate via an amorphous precursor phase from aqueous solution. A range of approaches were employed to study calcium oxalate formation, including precipitation in bulk solution in the presence and absence of additives and in the spatially confined volumes offered by track etched membranes and a crossed cylinders apparatus. A freeze concentration method was also used to generate sufficient quantities of amorphous calcium oxalate (ACO) for analysis. The results show that amorphous calcium oxalate crystallizes rapidly in bulk solution but can be significantly stabilized through the concerted activity of additives and confinement. We also demonstrate that ACO has a composition of ≈CaC₂O₄:H₂O. These data suggest that calcium oxalate biominerals, in common with their carbonate and phosphate counterparts, may also precipitate via amorphous phases

TLR2 on blood monocytes senses dengue virus infection and its expression correlates with disease pathogenesis

Vascular permeability and plasma leakage are immune-pathologies of severe dengue virus (DENV) infection, but the mechanisms underlying the exacerbated inflammation during DENV pathogenesis are unclear. Here, we demonstrate that TLR2, together with its co-receptors CD14 and TLR6, is an innate sensor of DENV particles inducing inflammatory cytokine expression and impairing vascular integrity in vitro. Blocking TLR2 prior to DENV infection in vitro abrogates NF-κB activation while CD14 and TLR6 block has a moderate effect. Moreover, TLR2 block prior to DENV infection of peripheral blood mononuclear cells prevents activation of human vascular endothelium, suggesting a potential role of the TLR2-responses in vascular integrity. TLR2 expression on CD14 + + classical monocytes isolated in an acute phase from DENV-infected pediatric patients correlates with severe disease development. Altogether, these data identify a role for TLR2 in DENV infection and provide insights into the complex interaction between the virus and innate receptors that may underlie disease pathogenesis

Systematic study of the effects of polyamines on calcium carbonate precipitation

While negatively charged organic additives are widely used as an effective means to control CaCO3 precipitation, positively charged additives are generally considered to be much less active. Nevertheless, the cationic polyelectrolyte poly(allylamine hydrochloride) has recently been shown to exert significant control over CaCO3 precipitation, driving the formation of thin films and fibers, and other examples suggest that many positively charged additives promote vaterite formation. This article aims to bring together these sometimes conflicting views of the activity of positively charged additives. The effect of a series of polyamines on CaCO3 precipitation was studied, where the polyamines were selected such that the amine group type, the pKa value (of the corresponding conjugated acid), the molecular weight, and the side chain length of the polymers could be evaluated. The results unambiguously demonstrate that polyamines carrying primary amine groups are capable of exerting a significant effect and that the activity of this class of polyamines is strongly dependent on the length of the side chain. In contrast, polyamines comprising with quaternary amines have negligible effect, despite carrying a permanent positive charge. The activity of the most active polyamines therefore depends on their ability to complex with carbonate ions present in solution, and electrostatic attraction alone is not sufficient