Jens-Petter Andreassen

Alginate-controlled formation of nanoscale calcium carbonate and hydroxyapatite mineral phase within hydrogel networks.

A one-step method was used to make nanostructured composites from alginate and calcium carbonate or calcium phosphate. Nanometer-scale mineral phase was successfully formed within the gel network of alginate gel beads, and the composites were characterized. It was found that calcite was the dominating polymorph in the calcium carbonate mineralized beads, while stoichiometric hydroxyapatite was formed in the calcium phosphate mineralized beads. A combination of electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis and powder X-ray diffraction showed that alginate played an active role in controlling mineral size, morphology and polymorphy. For the calcium phosphate mineralized beads, alginate was shown to modulate stoichiometric hydroxyapatite with low crystallinity at room temperature, which may have important applications in tissue engineering. The results presented in this work demonstrate important aspects of alginate-controlled crystallization, which contributes to the understanding of composite material design.

Biocomposites prepared by alkaline phosphatase mediated mineralization of alginate microbeads

This study set out to develop a biomimetic scaffold by incorporating osteoinductive hydroxyapatite (HA) particles into a porous alginate gel matrix via a cell-friendly pathway. Two types of alginate/calcium phosphate (Alg/CP) composites were prepared through alkaline phosphatase (ALP) mediated mineralization and counter-diffusion precipitation. Structural characteristics were analyzed by scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM). Thermal stability and mineral content were studied by means of thermogravimetric (TG) analysis. X-ray diffraction (XRD) and Rietveld refinement showed the presence of bone-like hydroxyapatite. Our studies suggested that the gradual nature of enzymatic process together with alginate matrices provide regulation of nanocrystalline hydroxyapatite formation. The ALP-mediated mineralization process has great advantages over counter-diffusion precipitation, providing homogenous mineral distributions, smaller crystal sizes, and increased apparent Youngs moduli, which creates a better structure for bone defect repair scaffolds.

Biomimetic type morphologies of calcium carbonate grown in absence of additives

This report demonstrates how typical particle morphologies documented in biomimetic mineralization studies of calcium carbonate will precipitate also from solutions without adding modulating additives, at supersaturation levels below the solubility level of amorphous calcium carbonate (ACC). In the literature, hexagonal plates and flower structures of vaterite, as well as dumbbell structures of aragonite are explained by non-classical aggregation mechanisms from precursor crystals, assisted and stabilized by biomolecules, ions and templates, or by transformation from ACC. By performing experiments at both depleting and constant low supersaturation ratios for a range of temperatures, we show that the vaterite morphology changes from hexagonal monocrystalline plates expressing the basal (001) faces, to dendritic flower shapes and finally to spherulites, as a function of increasing supersaturation. Aragonite goes through a similar transition from monocrystalline elongated structures to polycrystalline dumbbells and spherical structures. We conclude that the key to understand the shape development is quantification of the activity based supersaturation and the realization that calcium carbonate forms along classical crystallization pathways. The higher number of crystals required for aggregation based growth is not favoured at these low supersaturation values. Dislocation and surface nucleation driven crystal growth is responsible for faceted morphologies at moderate supersaturation, whereas dendritic and spherulitic growth patterns appear due to interface instability at higher driving forces.

Controlled mineralisation and recrystallisation of brushite within alginate hydrogels.

Due to high solubility and fast resorption behaviour under physiological conditions, brushite (CaHPO4⋅2H2O, calcium monohydrogen phosphate dihydrate, dicalcium phosphate dihydrate) has great potential in bone regeneration applications, both in combination with scaffolds or as a component of calcium phosphate cements. The use of brushite in combination with hydrogels opens up possibilities for new cell-based tissue engineering applications of this promising material. However, published preparation methods of brushite composites, in which the mineral phase is precipitated within the hydrogel network, fail to offer the necessary degree of control over the mineral phase, content and distribution within the hydrogel matrix. The main focus of this study is to address these shortcomings by determining the precise fabrication parameters needed to prepare composites with controlled composition and properties. Composite alginate microbeads were prepared using a counter-diffusion technique, which allows for the simultaneous crosslinking of the hydrogel and precipitation of an inorganic mineral phase. Reliable nucleation of a desired mineral phase within the alginate network proved more challenging than simple aqueous precipitation. This was largely due to ion transport within the hydrogel producing concentration gradients that modified levels of supersaturation and favoured the nucleation of other phases such as hydroxyapatite and octacalcium phosphate, which would otherwise not form. To overcome this, the incorporation of brushite seed crystals resulted in good control during the mineral phase, and by adjusting the number of seeds and amount of precursor concentration, the amount of mineral could be tuned. The material was characterised with a range of physical techniques, including scanning electron microscopy, powder x-ray diffraction and Rietveld refinement, Fourier transform infrared spectroscopy, and thermogravimetric analysis, in order to assess the mineral morphology, phase and amount within the organic matrix. The mineral content of the composite material converted from brushite into hydroxyapatite when submerged in simulated body fluid, indicating possible bioactivity. Additionally, initial cell culture studies revealed that both the material and the synthesis procedure are compatible with cells relevant to bone tissue engineering.

Gelling kinetics and in situ mineralization of alginate hydrogels: A correlative spatiotemporal characterization toolbox

UNLABELLED Due to their large water content and structural similarities to the extracellular matrix, hydrogels are an attractive class of material in the tissue engineering field. Polymers capable of ionotropic gelation are of special interest due to their ability to form gels at mild conditions. In this study we have developed an experimental toolbox to measure the gelling kinetics of alginate upon crosslinking with calcium ions. A reaction-diffusion model for gelation has been used to describe the diffusion of calcium within the hydrogel and was shown to match experimental observations well. In particular, a single set of parameters was able to predict gelation kinetics over a wide range of gelling ion concentrations. The developed model was used to predict the gelling time for a number of geometries, including microspheres typically used for cell encapsulation. We also demonstrate that this toolbox can be used to spatiotemporally investigate the formation and evolution of mineral within the hydrogel network via correlative Raman microspectroscopy, confocal laser scanning microscopy and electron microscopy. STATEMENT OF SIGNIFICANCE Hydrogels show great promise in cell-based tissue engineering, however new fabrication and modification methods are needed to realize the full potential of hydrogel based materials. The inclusion of an inorganic phase is one such approach and is known to affect both cell-material interactions and mechanical properties. This article describes the development of a correlative experimental approach where gel formation and mineralization has been investigated with spatial and temporal resolution by applying Raman microspectroscopy, optical and electron microscopy and a reaction-diffusion modeling scheme. Modeling allows us to predict gelling kinetics for other geometries and sizes than those investigated experimentally. Our experimental system enables non-destructive study of composite hydrogel systems relevant for, but not limited to, applications within bone tissue engineering.

Classical and Nonclassical Theories of Crystal Growth

In this chapter, we discuss classical and nonclassical concepts of crystal growth that coexist in the literature as explanations for the formation of both mono- and polycrystalline particles, often of the same substances. Crystalline particles with intraparticle nanosized subunits, nanoparticulate surface features, and complex morphologies have led to the development of new nonclassical theories of crystal growth based on the aggregation of nanocrystals in solution. At the same time, similar morphologies are explained by monomer incorporation at conditions of stress incorporation, which results in nucleation at the growth front and accompanying branching at the nanoscale. The two mechanisms are differently affected by important process variables like supersaturation, temperature, or additives and are analyzed with respect to their capability of predicting crystal growth rates. A quantitative description of the formation kinetics of the solid phases is essential for the design and operation of industrial precipitation and crystallization processes and for the understanding of fundamental principles in material design and biomineralization processes. In this chapter, we emphasize the importance of supersaturation in order to account for the extensive nanoparticle formation required to build micron-sized particles by nano-aggregative growth, as well as the accompanying change in the population density.

A correlative spatiotemporal microscale study of calcium phosphate formation and transformation within an alginate hydrogel matrix

UNLABELLED The modification of soft hydrogels with hard inorganic components is a method used to form composite materials with application in non-load-bearing bone tissue engineering. The inclusion of an inorganic component may provide mechanical enhancement, introduce osteoconductive or osteoinductive properties, or change other aspects of interactions between native or implanted cells and the material. A thorough understanding of the interactions between such components is needed to improve the rational design of such biomaterials. To achieve this goal, model systems which could allow study of the formation and transformation of mineral phases within a hydrogel network with a range of experimental methods and high spatial and time resolution are needed. Here, we report a detailed investigation of the formation and transformation process of calcium phosphate mineral within an alginate hydrogel matrix. A combination of optical microscopy, confocal Raman microspectroscopy and electron microscopy was used to investigate the spatial distribution, morphology and crystal phase of the calcium phosphate mineral, as well as to study transformation of the mineral phases during the hydrogel mineralization process and upon incubation in a simulated body fluid. It was found, that under the conditions used in this work, mineral initially formed as a metastable amorphous calcium phosphate phase (ACP). The ACP particles had a distinctive spherical morphology and transformed within minutes into brushite in the presence of brushite seed crystals or into octacalcium phosphate, when no seeds were present in the hydrogel matrix. Incubation of brushite-alginate composites in simulated body fluid resulted in formation of hydroxyapatite. The characterization strategy presented here allows for non-destructive, in situ observation of mineralization processes in optically transparent hydrogels with little to no sample preparation. STATEMENT OF SIGNIFICANCE The precipitation and transformations of calcium phosphates (CaP) is a complex process, where both formation kinetics and the stability of different mineral phases control the outcome. This situation is even more complex if CaP is precipitated in a hydrogel matrix, where one can expect the organic matrix to modulate crystallization by introducing supersaturation gradients or changing the nucleation and growth kinetics of crystals. In this study we apply a range of characterization techniques to study the mineral formation and transformations of CaP within an alginate matrix with spatiotemporal resolution. It demonstrates how a detailed investigation of the mineral precipitation and transformations can aid in the future rational design of hydrogel-based materials for bone tissue engineering and studies of biomineralization processes.

Analysis of Filtration Characteristics for Compressible Polycrystalline Particles by Partial Least Squares Regression

Crystal size and morphology have been varied by changing the initial supersaturation ratio and the temperature in reactive crystallization experiments. The influence of the chord length distribution, average cake porosity, and filtration pressure difference on the average cake resistance of polycrystalline particles of an industrially produced aromatic amine has been investigated by means of partial least squares (PLS) regression and sensitivity analysis. Analysis of the results has disclosed that wider chord length distributions as well as lower values of the measured average porosity lead to higher values for the average cake resistance. However, PLS regression and sensitivity analysis have identified the applied pressure difference itself as the most significant parameter influencing the magnitude of the cake resistance. This unexpected behavior is accounted for by compression of the filter cake occurring predominantly in small layers above the filter cloth characteristic for highly compressible cakes.

Filtration Properties of Ferric Hydroxide Precipitate in Nickel Production

Ferric iron removal from the nickel electrolyte at Glencore Nikkelverk AS in Kristiansand, Norway, is achieved by precipitation of iron hydroxide followed by filtration, and it is desirable to improve filtration properties of the precipitated iron hydroxide in order to decrease the filtration time. The effect of increasing the residence time from the current 45 min to 75 min and temperature from 65 °C to 90 °C on the filtration properties of the precipitated iron hydroxide (akaganeite) was studied by simulating the industrial process conditions in a continuous reactor setup operating at steady state, using ferrous-containing process solution as feed material. It was possible to decrease the filter cake resistance by both increasing residence time and temperature, but temperature gave the most pronounced effect and reduced the filter cake resistance by one order of magnitude.

Growing gold nanostructures for shape-selective cellular uptake

With development in the synthesis of shape- and size-dependent gold (Au) nanostructures (NSs) and their applications in nanomedicine, one of the biggest challenges is to understand the interaction of these shapes with cancer cells. Herein, we study the interaction of Au NSs of five different shapes with glioblastoma-astrocytoma cells. Three different shapes (nanorods, tetrahexahedra, and bipyramids), possessing tunable optical properties, have been synthesized by a single-step seed-mediated growth approach employing binary surfactant mixtures of CTAB and a secondary surfactant. By the use of two-step seed-mediated approach, we obtained new NSs, named nanomakura (Makura is a Japanese word used for pillow) which is reported for the first time here. Spherical Au nanoparticles were prepared by the Turkevich method. To study NS-cell interactions, we functionalized the NSs using thiolated PEG followed by 11-Mercaptoundecanoic acid. The influence of shape and concentration of NSs on the cytotoxicity were assessed with a LIVE/DEAD assay in glioblastoma-astrocytoma cells. Furthermore, the time-dependent uptake of nanomakura was studied with TEM. Our results indicate that unlike the other shapes studied here, the nanomakura were taken up both via receptor-mediated endocytosis and macropinocytosis. Thus, from our library of different NSs with similar surface functionality, the shape is found to be an important parameter for cellular uptake.