Towards the Development of Artificial Bone Grafts: Combining Synthetic Biomineralisation with 3D Printing.

A synthetic technique inspired by the biomineralisation process in nacre has been previously reported to be effective in replicating the nanostructural elements of nacre in 2D chitosan hydrogel films. Here we evaluate the applicability of this synthetic biomineralisation technique, herein called the McGrath method, in replicating the flat tabular morphology of calcium carbonate and other nanostructural elements obtained when 2D chitosan hydrogel films were used, on a 3D porous chitosan hydrogel-based scaffold, hence developing 3D chitosan-calcium carbonate composites. Nozzle extrusion-based 3D printing technology was used to develop 3D porous scaffolds using chitosan hydrogel as the printing ink in a custom-designed 3D printer. The rheology of the printing ink and print parameters were optimised in order to fabricate 3D cylindrical structures with a cubic lattice-based internal structure. The effects of various dehydration techniques, including air-drying, critical point-drying and freeze-drying, on the structural integrity of the as-printed scaffolds from the nano to macroscale, were evaluated. The final 3D composite materials were characterised using scanning electron microscopy, X-ray diffraction and energy dispersive X-ray spectroscopy. The study has shown that McGrath method can be used to develop chitosan-calcium carbonate composites wherein the mineral and matrix are in intimate association with each other at the nanoscale. This process can be successfully integrated with 3D printing technology to develop 3D compartmentalised polymer-mineral composites.

Molecular Dynamics Simulation of ß-Lactoglobulin at Different Oil/Water Interfaces.

Controlling and manipulating protein behavior at an interface is of immense relevance to a broad range of physicochemical and biological phenomena and technological processes. Although many experimental studies have contributed to rapid progress in the fundamental knowledge of protein behavior at interfaces, detailed molecular-level understanding of the mechanism of protein adsorption at an interface is still remarkably lacking. In this study, atomistic molecular dynamics simulations were used to characterize the adsorption of ß-lactoglobulin at two different oil/water (O/W) interfaces, where the oil was either the marginally hydrophilic octanol or the more hydrophilic triolein, and the results were compared to those of a previous study utilizing the hydrophobic oil decane. Both the approach to the surface and the mechanism of adsorption depend upon the hydrophilicity of the oil and the interfacial tension of the O/W interface, with the nature of the adsorption, the accompanying structural changes, and the energetic driving force differing markedly between the different oils. Intriguingly, the behavior of the protein resembles that predicted for a soft spherical particle at an O/W interface. The results are also in agreement with key experimental findings, particularly the observation that proteins undergo more conformational change upon adsorption to hydrophobic surfaces, flattening out to expose hydrophobic interior residues to the surface, and that a thicker layer of native-like adsorbed protein forms at hydrophilic surfaces, and reveal structural and mechanistic detail behind each mechanism of adsorption.

Deciphering ß-Lactoglobulin Interactions at an Oil-Water Interface: A Molecular Dynamics Study.

Protein adsorption at liquid-liquid interfaces is of immense relevance to many biological processes and dairy-based functional foods. Due to experimental limitations, however, there is still a remarkable lack of understanding of the adsorption mechanism, particularly at a molecular level. In this study, atomistic molecular dynamics simulations were used to elucidate the approach and adsorption mechanism of ß-lactoglobulin (ß-LG) at a decane-water interface. Through multiple independent simulations starting from three representative initial orientations of ß-LG relative to the decane surface the rate at which ß-LG approaches the oil/water interface is found to be independent of its initial orientation, and largely stochastic in nature. While the residues that first make contact with the decane and the final orientation of ß-LG upon adsorption are similar in all cases, the adsorption process is driven predominantly by structural rearrangements that preserve the secondary structure but expose hydrophobic residues to the decane surface. This detailed characterization of the adsorption of ß-LG at an oil/water interface should inform the design and development of novel encapsulation and delivery systems in the food and pharmaceutical sciences.

How does oil type determine emulsion characteristics in concentrated Na-caseinate emulsions?

Macroscopic properties and ensemble average diffusion of concentrated (dispersed phase 50-60 wt%) Na-caseinate-stabilised emulsions for three different oils (soybean oil, palm olein and tetradecane) were explored. On a volume fraction basis, pulsed gradient stimulated echo (PGSTE)-NMR data show that droplet dynamics for all three systems are similar within a region of the emulsion morphology diagram. The exact limits of the emulsion space depend however on which oil is considered. The reduced solubility of tetradecane in water, and Na-caseinate in tetradecane, result in the stabilisation of flocs during formulation. Floc formation is not observed when soybean oil or palm olein is used under identical emulsion formulation conditions. Linear rheology experiments provide indirect evidence that the local structure and the properties of the thin film interfacial domain of tetradecane emulsions vary from those of soybean oil and palm olein emulsions. Collectively these data indicate that protein/oil interactions within a system dominate over specific oil droplet structure and size distribution, which are similar in the three systems.

How important is polyelectrolyte complex formation in biomimetic mineralisation? Manipulation via alcohol addition.

Understanding the formation of biominerals like nacre can lead to the fabrication of more advanced biomimetic materials. Several factors are known to influence the final form of both native nacre and biomimetic synthetic variants. Two important components in calcium carbonate biominerals such as nacre are the organic scaffold and the acidic proteins. Interactions between these two components may also influence final composite characteristics. In this investigation chitosan hydrogels were prepared from acidic aqueous solution using four alcohols as cosolvents. The addition of alcohol enables direct modification of the network of the chitosan hydrogel (and thereby the nanometre and micrometre length-scale structure of the hydrogel). Both alcohol-modified chitosan and subsequently reacetylated chitin scaffolds were then mineralised with a combined soaking mineralisation method in the presence of poly(acrylic acid), the latter of which mimics the role of the acidic proteins in the native system. The effects of these structural variations of the hydrogel, induced by the presence of alcohol during fabrication, on (1) the formation of a polyelectrolyte complex between the chitosan or chitin and the poly(acrylic acid) and (2) the subsequent polymorph and morphology of calcium carbonate crystals mineralised within the hydrogel scaffold were investigated. Increasing the amount of the alcohols 1,2-propanediol or 1,3-propanediol led to increased disruption of the hydrogen bonding of the hydrogel scaffold and significant changes to, or reduced formation of, the polyelectrolyte complex formed between the scaffold carbohydrate and the poly(acrylic acid). The disruption of the polyelectrolyte complex in turn led to a loss of control over which polymorph of calcium carbonate is nucleated. These results show that the physical form of the polymer scaffold in these organic/inorganic composites, and the formation of the polyelectrolyte complex play a crucial role in determining the final composite structure and the calcium carbonate polymorphs and morphologies.

In situ continuous growth formation of synthetic biominerals.

Continuous self-assembled growth of both the organic and inorganic components of materials with nacre-like structure is achieved upon mineralisation of chitin and chitosan scaffolds using a combined soaking method and the inclusion of poly(acrylic acid) and chitosan oligomers as additives.

Size and charge characterisation of a submicrometre oil-in-water emulsion using resistive pulse sensing with tunable pores.

Resistive pulse sensing (RPS) with tunable pores (TPs) has been used to investigate an oil-in-water emulsion stabilised with ß-lactoglobulin (BLG). The mode of the droplet size distribution steadily increased over four months, from less than 150 nm to more than 200 nm. Results suggest that the dominant growth mechanism was migration of oil to relatively large droplets, as in Ostwald ripening. In contrast, the growth dynamics for salt-induced aggregation suggest flocculation and coalescence of droplets coming into contact. The charge measurement method recently developed by Vogel et al. was also applied to the emulsion. The two data analysis methods used yielded average droplet ζ-potentials of -18.9 mV and -21.8 mV, compared with -27.6 mV obtained using light scattering. Methods for measuring emulsion droplet deformation and the charge on individual droplets are under development. Tunable pores are a useful tool for improved characterisation of submicrometre emulsions, as well as other synthetic and biological particles, as they provide better precision than light scattering for particle number distributions.

Na-caseinate/oil/water systems: emulsion morphology diagrams.

The concentrated (dispersed phase 50-70 wt%) composition space of Na-caseinate, a family of milk proteins, stabilised emulsions was investigated for three different oils: soybean oil, palm olein and tetradecane with pH 6.8 phosphate buffer continuous phase. The variation of emulsion stability and microstructure were explored using static light scattering, diffusion nuclear magnetic resonance, cryo-scanning electron microscopy, rheology and the time varying macroscopic phase separation of the emulsions. For soybean oil and palm olein a rich diversity of emulsion microstructures and stabilities are realised. Five emulsion domains, each having a different microstructure and macroscopic stability have been identified within the composition space probed. For the lowest concentrations of emulsifier bridging flocculation is evident and emulsions are of low stability. Increasing Na-caseinate concentration leads to an increased stability and the existence of distinct individual oil droplets, visualised using cryo-scanning electron microscopy. Further increases in Na-caseinate concentration reduce emulsion stability due to depletion flocculation. Na-caseinate self-assembly is then initiated. At sufficiently high Na-caseinate and/or oil concentrations the continuous phase of the emulsion is a three-dimensional protein network and emulsion stability is again enhanced. At the limits of the emulsion composition space a gel-like paste is formed. The diversity of emulsion microstructure is reduced when tetradecane is the discrete phase. Na-caseinate self-assembly is limited and there is no evidence for formation of a protein network.

Biomimetic approach to forming chitin/aragonite composites.

Biomimetic materials which display the complexity of biominerals like nacre are synthetically difficult to prepare. The formation of chitin/calcium carbonate composites, where CaCO(3) is present as aragonite, was achieved via reacetylation of preformed chitosan scaffolds followed by the combination of presoaking of chitin templates with mineral solutions in the presence of poly(acrylic acid). The as-synthesised composites are comprised of well-ordered ribbons of aragonite crystals held within an organic matrix, mimicking the structure of nacre.

Biomimetic mineralisation of polymeric scaffolds using a combined soaking and Kitano approach.

Chitosan hydrogels are of considerable interest in synthetic biomimetic mineralisation strategies due to their favourable characteristics such as the presentation of a large surface area for crystal nucleation within a structured yet responsive scaffold. Chitosan hydrogels were prepared and subsequently calcium carbonate mineralisation was initiated using a method which combines alternate soaking of the films with precursor solutions followed by treatment with Kitano solution. This combined approach allows for increased extent of mineralisation, inducement of mineralisation uniformly throughout the hydrogel rather than only at the peripheral surface and ready scalability and shape manipulation. The base synthetic system is readily modified through the introduction of additives that manipulate the nucleation and growth of the calcium carbonate. Addition of poly(acrylic acid) inhibits nucleation and induces tangential crystal growth along the internal and external interfaces of the hydrogel. The resulting composite is comprised of stacked overlapping plates of calcium carbonate intercalated with carbohydrate. The method is applicable in combination with a variety of hydrogels including macroporous chitosan, chitosan-alginate bilayers and pure alginate hydrogels. The composite materials were analysed by SEM, XRD, microRaman spectroscopy and mechanical strength testing.

Biomimetic mineralisation of polymeric scaffolds using a combined soaking approach: adaptation with various mineral salts.

Biomimetic strategies which utilise hydrogels have been targeted due to favourable hydrogel characteristics such as the presentation of a large surface area for crystal nucleation within a structured yet responsive scaffold. Chitosan hydrogels were prepared and mineralised using a combined method which involves alternate soaking of the films with precursor solutions, followed by treatment with saturated mineral solution. This method has been shown to be effective for the synthesis of calcium carbonate-chitosan composite materials with tensile strength comparable to nacre. The ratio of organic to inorganic is readily controlled through the presoaking solution concentrations. The ubiquity of this method is shown here with respect to switching out both the anion (CaHPO(4)) and the cation (BaSO(4)). Cation doping is also readily achieved allowing formation of Mg-rich CaCO(3). Poly(acrylic acid) added to (Mg,Ca)CO(3)-chitosan systems induces the formation of two polymorphs (vaterite and calcite) which coexist within the composite material. The mineralised scaffolds were analysed by SEM and powder XRD. The successful mineralisation of chitosan templates with various inorganic compounds shows that this combined approach is widely applicable as a biomimetic approach.

Nonequilibrium 2-hydroxyoctadecanoic acid monolayers: effect of electrolytes.

2-Hydroxyacids display complex monolayer phase behavior due to the additional hydrogen bonding afforded by the presence of the second hydroxy group. The placement of this group at the position α to the carboxylic acid functionality also introduces the possibility of chelation, a utility important in crystallization including biomineralization. Biomineralization, like many biological processes, is inherently a nonequilibrium process. The nonequilibrium monolayer phase behavior of 2-hydroxyoctadecanoic acid was investigated on each of pure water, calcium chloride, sodium bicarbonate and calcium carbonate crystallizing subphases as a precursor study to a model calcium carbonate biomineralizing system, each at a pH of ∼6. The role of the bicarbonate co-ion in manipulating the monolayer structure was determined by comparison with monolayer phase behavior on a sodium chloride subphase. Monolayer phase behavior was probed using surface pressure/area isotherms, surface potential, Brewster angle microscopy, and synchrotron-based grazing incidence X-ray diffraction and X-ray reflectivity. Complex phase behavior was observed for all but the sodium chloride subphase with hydrogen bonding, electrostatic and steric effects defining the symmetry of the monolayer. On a pure water subphase hydrogen bonding dominates with three phases coexisting at low pressures. Introduction of calcium ions into the aqueous subphase ensures strong cation binding to the surfactant head groups through chelation. The monolayer becomes very unstable in the presence of bicarbonate ions within the subphase due to short-range hydrogen bonding interactions between the monolayer and bicarbonate ions facilitated by the sodium cation enhancing surfactant solubility. The combined effects of electrostatics and hydrogen bonding are observed on the calcium carbonate crystallizing subphase.

Assembly of two- and three-dimensionally patterned silicate materials using responsive soft templates.

We report a soft and straightforward method for synthesizing two- and three-dimensionally patterned silicate materials by phase separation using nonionic emulsion templates. Our liquid-state method involves, under controlled atmosphere, the mixing of a condensed silica solution with an oil-in-water emulsion in the presence of a solution of a nonionic emulsifier, Triton X-100. The preparation is stabilized using an organic solvent. The morphology of the silicate materials is significantly modified by changing the reaction conditions or the concentration of the reagents. Three-dimensionally macro and nanoporous continuous films and nanoporous individual spherical particles, both made of amorphous silica, are obtained. The structure of the films and particles is defined by the emulsion template. Films were on average 20 microm thick with a volume-based porosity of approximately 7 x 10(-2) cm(3) g(-1), with pore size correlating well with the size of the oil droplets in the templating emulsion. The siliceous films are bicontinuous leading to large surface areas and openly accessible pores. Individual spheres ranged in size from approximately 1 to 6 microm in diameter with nanoporous openings of 300 nm in diameter. The porosity and integrity of all materials are maintained upon calcination.

The microstructural and rheological properties of Na-caseinate dispersions.

The self-diffusion coefficients of water and casein aggregates in aqueous Na-caseinate dispersions were measured using a pulsed gradient stimulated echo nuclear magnetic resonance technique (PGSTE-NMR). The results for water self-diffusion are discussed in terms of the cell model. The influence of Na-caseinate concentration on water self-diffusion can be explained by obstruction and tortuosity effects arising due to the presence of casein aggregates. Unrestricted diffusion of casein aggregates was measured for all concentrations at short observation times ( approximately 20ms). Despite a near uniform size distribution on increasing Na-caseinate concentration the measured unrestricted diffusion coefficient was a decreasing function of Na-caseinate concentration. Enhanced packing interaction between the aggregates and the slight augmentation of the large tail in the size distribution is the main reason for this observed decrease. On increasing the experimental observation times restricted diffusion of the aggregates is measured. The rheological response of the dispersions was investigated. The stress evolution of the sample with time exhibits three distinct behaviours, irrespective of Na-caseinate concentration and applied shear rate: a spontaneous increase of stress at very short times (<1s), followed by a plateau with fine structure superposed and lastly a sharp increase in stress attributed to irreversible gelation. The latter occurs at significantly reduced times on increasing Na-caseinate concentration. Linear rheology measurements indicate that the dispersions are predominantly locally liquid-like while being macroscopically in a gelled state.

The role of subphase chemistry in controlling monolayer behaviour.

Intermolecular interactions lie at the root of many fundamental processes in nature. For example, they define membrane formation and crystallisation processes. Here, using Langmuir monolayers, we have investigated the role of intermolecular interactions in defining non-equilibrium monolayer structure and response, specific to a nucleating subphase chemistry. In particular, the interactions in mixed alcohol/carboxylic acid systems have been explored on four different subphases: ultrapure water, calcium chloride, sodium bicarbonate and a calcium carbonate crystallising subphase. Area per molecule, surface pressure and surface potential were analysed as a function of mol% ratio of acid to alcohol on each of the subphases. Comparison of the data across the subphases showed that three main interactions dictate the behaviour of the monolayer: (1) formation of a hydrogen-bonding network (predominantly dominating intramonolayer structure), (2) electrostatic interactions between the charged monolayer and strongly binding counterions and (3) the formation of a hydrogen-bonded soap network induced by the presence of bicarbonate coions. The latter provides a potential mechanism for defining directionality of interfacial interactions, and therefore face-selective nucleation.

Cell membrane structures during exocytosis.

Exocytosis is a key biological process that controls the neurotransmission and release of hormones from cells. In endocrine cells, hormones are packed into secretory vesicles and released into the extracellular environment via openings in the plasma membrane, a few hundred nanometers wide, which form as a result of fusion of the membranes of the granule and cell. The complex processes and dynamics that result in the formation of the fusion pore, as well as its structure, remain scantly understood. A number of different exocytosis mechanisms have been postulated. Furthermore, the possibility exists that several mechanisms occur simultaneously. We present here an investigation of the cell membrane dynamics during exocytosis in anterior pituitary cells, especially gonadotropes, which secrete LH, a hormone central to ovulation. Gonadotrope enrichment was achieved using immunolabeled magnetic nanobeads. Three complementary imaging techniques were used to realize a fine structure study of the dynamics of the exocytosis-like sites occurring during secretion. Living pituitary and gonadotrope-enriched cells were imaged with atomic force microscopy, as well as cells that had been fixed to obtain better resolution. Atomic force microscopy, along with scanning and transmission electron microscopy, studies of these cells revealed that there are at least two different site configurations: simple single fusion pores and a complex association of pores consisting of a simple primary site combined with secondary attachments.

Evolution of a lamellar domain structure for an equilibrating lyotropic liquid crystal.

Aerosol OT/water exhibits a lamellar phase over a wide range of concentrations. We show, by magnetic resonance (NMR) and scanning electron microscopy (SEM), that the morphology of the lamellar phase varies significantly across that range and that the rate of equilibration depends strongly on concentration (25, 33, and 50 wt %) with, paradoxically, the faster equilibration at higher surfactant concentrations. We find that the 25 wt % sample exhibits a defect-rich local structure, characteristic of a superposed L(3) character. Further into the lamellar region, at 33 wt %, this defect-rich structure persists heterogeneously, while, at 50 wt %, the lamellar phase domains are highly ordered. The NMR methods used here included (2)H spectroscopy and the two-dimensional NMR method, diffusion-diffusion exchange spectroscopy (DEXSY). The latter was used to obtain quantitative information on the domain sizes and defects within the polydomain lamellar mesophase. Comparison of the NMR with the SEM results suggests that, at 25 wt % AOT, bilayer defects play an important role in influencing the (2)H NMR and DEXSY NMR results.

Orientational anisotropy in the polydomain lamellar phase of a lyotropic liquid crystal.

Diffusion-diffusion correlation measurements by NMR are used to investigate the degree of orientational order in the lamellar phase of Aerosol OT (bis(2-ethylhexyl) sodium sulfosuccinate) and water at a range of surfactant concentrations (25, 33, and 50 wt %). We show that true isotropy of the domains is found at the lowest concentration but that at higher concentrations deviations from isotropy can be found, as evidenced by asymmetry on the 2D correlation distributions. We further discuss the significance of asymmetry in diffusion-diffusion exchange experiments, 2D distributions that should always be symmetric in steady state.

Aging of oil-in-water emulsions: the role of the oil.

Controlling stability and aging of emulsions is important from commercial and scientific perspectives. Achieving such control comes through gaining an understanding of the relationship between emulsion constituents and microstructure and how these influence the kinetics and mechanism of destabilisation. We present here an investigation determining the rate of destabilisation as a function of time for a series of water/n-alkane/Triton X-100 oil-in-water emulsions. The time dependence of the emulsions was investigated using static light scattering, PFG-NMR and measurement of gross phase separation. By changing the chain length of the oil from hexane to tetradecane, an almost five orders of magnitude variation in emulsion lifetime could be achieved, while maintaining most of the other chemical and physical characteristics of the emulsions. Further, we show that while Ostwald ripening is the dominant destabilisation mechanism, two distinct regimes are evident. Initially, we observed an enhanced Ostwald ripening regime due to the presence of oil-swollen micelles in the aqueous continuum, that is a depletion flocculation mechanism is followed. The presence of oil-swollen micelles was confirmed using PFG-NMR. The micelles aid the gross oil transport between the discrete oil domains. Upon phase separation the oil-swollen micelles are predominantly removed from the emulsion along with the excess water resulting in a concomitant reduction in the ripening rate, producing the more general Ostwald ripening cubic dependence of droplet radius as a function of time for the lower molecular weight oils. The oils with higher molecular weight (decane and above), however, were observed to switch over to destabilisation via creaming. PFG-NMR was shown to be a powerful technique to fully probe emulsion microstructure as a function of time with droplet size and spacing being directly obtained from the data.

A study of anisotropic water self-diffusion and defects in the lamellar mesophase.

The correlation of molecular diffusion coefficients obtained via a novel two-dimensional pulsed gradient spin-echo (PGSE) NMR method has been shown to reveal detailed structural information on the mesophases of lyotropic liquid crystals. A four-component system containing both nonionic (pentaethylene glycol monododecyl ether) and ionic (sodium dodecyl sulfate) surfactants, water, and decane was prepared and left to equilibrate. In the temperature region around 309 K, a lamellar mesophase forms. A two-dimensional Laplace inverse transformation was performed on the (gammadeltag)2(delta - delta/3) domain data to separate any multiexponential behavior that resulted from local anisotropy. The results of the double PGSE experiment with contiguous gradient pulse pairs, applied both collinearly and orthogonally, clearly show the presence of local anisotropic self-diffusion of the water molecules and suggest a preferred orientation of the lamellae. Information about defects/domain size was obtained by the insertion of a mixing time (t(m)') between the successive gradient pulse pairs. This work highlights the value of this new NMR correlation method in the study of surfactant systems.