Giuseppe Falini

Magnesium influence on hydroxyapatite crystallization

Abstract X-ray diffraction, infrared absorption, and chemical investigations have been carried out on hydroxyapatite synthesized in the presence of different magnesium concentrations in solution. Magnesium inhibits the crystallization of hydroxyapatite through a reduction of Ca/P molar ratio and crystal sizes of apatite. The reduction of the crystal sizes is also very great for very low magnesium content and increases on increasing magnesium concentration in solution up to 35 Mg atom percent with respect to the total metal ions. The samples are completely amorphous between 35 and 50 Mg atom percent. For higher magnesium concentration different crystalline phases are formed. The results of the x-ray powder pattern fitting indicate that the HA crystal structure at most hosts magnesium amounts of about seven percent. Magnesium substitutes only the calcium atoms which form the channels containing the hydroxy ions. Since magnesium content is much smaller than that found in the solid phase, the greatest amount of magnesium must not be lattice bound. The extent of hydroxyapatite conversion into magnesium substituted β-tricalcium phosphate on heat treatment appears strongly related to magnesium content of the apatitic solid phase. On the basis of these results, the key role of magnesium on the crystallization, crystal growth, and thermal stability of hydroxyapatite has been used to explain the relevant properties of biological apatites.

Biologically inspired growth of hydroxyapatite nanocrystals inside self-assembled collagen fibers

Abstract Bone defects are generally filled using autologous implants because artificial bone materials have low bioaffinity. However, natural bone can induce infections and antigenic reaction, therefore, the preparation of artificial material with composition, structure and biological feature comparable to those of bone is a goal to be pursued. The aim of this work was to follow a biologically inspired approach performing a direct nucleation of hydroxyapatite (HA) on self-assembled collagen fibers to set up a collagen–hydroxyapatite nanocrystals composite as a new particularly attractive material for bone repair and reconstruction. X-ray diffractometric technique, thermogravimetric (TG–DTG), spectroscopic (FT-IR, ICP), microscopic (SEM, TEM) analyses have been used to highlight the likeness of the artificial biomimetic HA/Col composite with natural bone tissue.

Oriented Crystallization of Vaterite in Collagenous Matrices

The influence of high super- saturation on kinetic control and the importance of the polypeptide structure in the crystallization of calcium carbo- nate polymorphs were studied in cross- linked gelatin films containing high concentrations of the polypeptides poly-l-aspartate and poly-l-glutamate. Oriented crystallization of vaterite oc- curs in uniaxially deformed gelatin films containing poly-l-aspartate at concenta- tions greater than 100 mg per gram of gelatin. The fact that no orientation of the mineral phase was observed with entrapped poly-l-glutamate at the same concentrations suggests that the orient- ed crystallization is controlled by the b sheet structure assumed by poly-l-as- partate in the presence of calcium ions. These results indicate that local super- saturation in the microenvironment in which nucleation and growth occur plays an important role in controlling the deposition of vaterite in cross-linked gelatin films. However, collagen bundles and the ordered and oriented polypep- tide chains of poly-l-aspartate can con- tribute to the control of polymorphism by inducing the formation of a specific phase by epitaxial crystallization, as suggested by the preferentially oriented deposition of vaterite and aragonite. This is of potential significance in bio- mineralization processes and in materi- als science.

Rietveld structure refinements of calcium hydroxylapatite containing magnesium

The crystal structures of four hydroxylapatite (HA) samples prepared from solutions in the presence of 10, 15, 25 and 30 Mg-atom-% have been investigated by X-ray powder pattern fitting. The total magnesium content of the solid samples, as determined by chemical analysis, was 4.9, 14.1, 20.4 and 30.6 Mg-atom-%, respectively. Rietveld analysis was performed using the computer program PREFIN implemented with routines which allow the refinements of the average crystallite sizes. Different refinement procedures were carried out in order to evaluate the effect of the amorphous and background profiles on the occupancy factor data. For comparison, magnesium-free hydroxylapatite was refined with the same strategies. The results of the different approaches indicate that the degree of magnesium substitution for calcium in the HA structure can be at most ~ 10 atom-%. Magnesium substitutes calcium preferentially at the 6(h) site. The broadening of the diffraction peaks increases on increasing the total magnesium content in the solid phase, which is always significantly higher than the amount incorporated into the HA structure. The excess is probably located in the amorphous phase and/or on the crystallite surface.

Chitin-Silk Fibroin Interactions: Relevance to Calcium Carbonate Formation in Invertebrates

In mineralized tissues chitin is almost always associated with proteins, many of which are known to have chitin recognition consensus sequences. It has been observed in some mollusk shells that there is a well-defined spatial relation between the crystallographic axes of the crystals and the chitin fibrils. This implies that the chitin functions directly or indirectly as a template for nucleation of the mineral phase. It is thus of much interest to understand the exact nature of the interface between the chitin and the proteins at the molecular level in mineralized tissues. Chitin/silk fibroin interactions were studied in vitro at the molecular level using homogenous films composed of the two macromolecules. The results show that the silk fibroin intercalates between the molecular planes of the chitin, and that the interactions are mainly through the chitin acetyl groups. Published X-ray diffraction patterns and infrared spectra of mineralized tissue organic matrices, as well as infrared spectra reported here of the squid pen and lobster cuticle, all show that in vivo the chitin and protein are not intimately mixed, but exist as two phases. We deduce that there is an interfacial plane between them in which the interactions are through the amide groups.

Crystallization of calcium carbonate in presence of magnesium and polyelectrolytes

Abstract The results of X-ray diffraction and morphological investigations carried out on calcium carbonate crystals grown from solutions containing different Mg 2+ /Ca 2+ molar ratios and polyelectrolytes are reported. Polyelectrolytes containing charged groups important in biomineralization processes are used. Polymers containing sulphate (carrageenans K and J) and carboxylate (polyacrylate, poly-L-glutamate and poly-L-aspartate) charged groups do not influence the magnesium substitution for calcium in calcite. When the Mg 2+ /Ca 2+ molar ratio is high, the control of polymorph type seems to be dictated almost exclusively by magnesium ions independent of the presence of poly-L-glutamate, polyacrylate or carrageenans. In these conditions only aragonite crystallizes. The behaviour of poly-L-aspartate, which assumes a β-sheet structure for long stretches of chain in the presence of calcium ions, is remarkably different. Since, in the presence of this polymer, calcite crystallizes together with aragonite also when Mg 2+ /Ca 2+ molar ratio is high, it can be assumed that the β-sheet structure of poly-L-aspartate plays an important role in determining the calcite crystallization in the presence of magnesium ions.

Protein crystallization on polymeric film surfaces

Abstract Polymeric films containing ionizable groups, such as sulfonated polystyrene, cross-linked gelatin films with adsorbed poly- l -lysine or entrapped poly- l -aspartate and silk fibroin with entrapped poly- l -lysine or poly- l -aspartate, have been tested as heterogeneous nucleant surfaces for proteins. Concanavalin A from jack bean and chicken egg-white lysozyme were used as models. It was found that the crystallization of concanavalin A by the vapor diffusion technique, is strongly influenced by the presence of ionizable groups on the film surface. Both the induction time and protein concentration necessary for the crystal nucleation decrease whereas the nucleation density increases on going from the reference siliconized cover slip to the uncharged polymeric surfaces and even more to the charged ones. Non-specific attractive and local interactions between the protein and the film surface might promote molecular collisions and the clustering with the due symmetry for the formation of the crystal nuclei. The results suggest that the studied polymeric film surfaces could be particularly useful for the crystallization of proteins from solutions at low starting concentration, thus using small quantities of protein, and for proteins with very long crystallization time.

Hydroxyapatite/polyacrylic acid nanocrystals

Hydroxyapatite nanocrystals have been synthesized in the presence of polyacrylic acid in solution. The method leads to apatite crystals with a polyelectrolyte relative content which increases as a function of its concentration in solution and reaches a limit value of about 15% wt. Polyacrylic acid affects the degree of crystallinity, the coherent length of the perfect crystalline domains, as well as the dimensions and the morphology of the crystals. TEM images indicate that on increasing polyelectrolyte content, the dimensions of the crystals are reduced, while their morphology changes from plate-like to acicular, and, at high polyacrylic acid content, the crystals tend to aggregate in clusters. In agreement, the broadening of the X-ray diffraction reflections indicate a reduction of the coherent length along the long dimension (002) and the cross section (310) of the apatite crystals. The reduction is greater along the direction orthogonal to the c-axis, which can be rationalized by a preferential interaction of the carboxylate groups of the polyelectrolyte with the phosphate sites on the crystalline faces parallel to the c-axis.

Magnesium calcite crystallizatin from water–alcohol mixtures

Magnesium calcite with unusual magnesium contents up to 14 mol% crystallizes from water–alcohol mixtures. Morphological changes result from interactions of solvent and Mg2+ with specific planes of the growing crystals.

Protein crystallisation on chemically modified mica surfaces

Chemically modified mica sheets have been tested as heterogeneous nucleant surfaces for lysozyme, concanavalin A and thaumatin. Smooth mica surfaces with reduced hydrophilic properties and different density of ionisable groups have been prepared by a silanisation reaction using mixtures of n-propyltriethoxysilane and 3-aminopropyltriethoxysilane in different percentages starting from 0 to 100% of aminosilane. The crystallisation experiments were carried out with the hanging drop vapour diffusion technique. The results suggest that these mica surfaces act as heterogeneous nucleant agents, whose effectiveness is due to non-specific attractive and local interactions between charged residues of the protein and the ionisable groups on the mica surfaces.