Norberto Roveri

Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking

The mechanical, thermal, swelling and release properties of glutaraldehyde (GTA) crosslinked gelatin films have been investigated in order to verify the influence of GTA concentration on the stability of the films. Air-dried films were submitted to treatment with GTA solutions at concentrations ranging from 0.05 to 2.5 wt%. At the smallest GTA concentration, the crosslinking degree, determined by trinitrobenzensulfonic acid assay, amounts to about 60% and increases up to values near 100%, obtained with GTA concentrations > or = 1 wt%. Simultaneously, the deformability of the films decreases, whereas the stress at break, sigmab, and the Youngs modulus, E, increase. A crosslinking degree of about 85%, obtained using 0.25% GTA, is enough to prevent gelatin release in buffer solution and to provoke a significant reduction of the swelling in physiological solution. Furthermore, crosslinking greatly affects the thermal stability of the samples, as indicated by the results of differential scanning calorimetry (d.s.c.) investigation carried out on wet and air-dried films. The data suggest that the use of GTA at low concentration, which is desiderable to prevent toxicity, allows to modulate the physico-chemical properties of gelatin films, in order to obtain stable materials with a wide range of possible biomedical applications.

Stabilization of gelatin films by crosslinking with genipin

The possibility to stabilize gelatin films by crosslinking with genipin was investigated through a mechanical, chemical and thermal characterization of samples treated with genipin solutions at different concentrations. The extent of crosslinking, evaluated as difference between the number of free epsilon -amino groups before and after crosslinking, increases as a function of genipin concentration up to about 85%. Simultaneously, the deformability of the films decreases whereas the Youngs modulus E, increases. Furthermore, crosslinking provokes a significant reduction of the swelling in physiological solution, and enhances the thermal stability of the samples, as indicated by the results of the d.s.c. investigation. The data obtained from the films treated with genipin at concentrations higher than 0.67% are quite similar, and indicative of a good stabilizing effect of genipin. In spite of the small gelatin release (2%) observed after 1 month of storage in buffer solution, the mechanical, thermal and swelling properties of the films are very close to those previously obtained for glutaraldehyde crosslinked gelatin, and suggest that genipin, which is by far less cytotoxic, can be considered a valid alternative for crosslinking gelatin biomaterials.

Chemical and structural characterization of the mineral phase from cortical and trabecular bone

X-ray diffraction, infrared spectroscopy and chemical investigations have been carried out on the inorganic phases from rat cortical and trabecular bone. Although both inorganic phases consist of poorly crystalline B carbonated apatite, several significant differences have been observed. In particular, trabecular bone apatite displays reduced crystallite sizes, Ca/P molar ratio, and carbonate content, and exhibits a greater extent of thermal conversion into beta-tricalcium phosphate than cortical bone apatite. These differences can be related to the different extents of collagen posttranslational modifications exhibited by the two types of bone, in agreement with their different biological functions.

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.

The role of magnesium on the structure of biological apatites

SummaryX-ray diffraction, infrared absorption spectroscopy, and chemical investigation have been carried out on deproteinated samples of turkey leg tendon at different degrees of calcification. The inorganic phase consists of poorly crystalline B carbonated apatite. On increasing calcification, the apatite crystal size, as well as its thermal stability, increase while the relative magnesium content is reduced. On the other hand, synchrotron X-ray diffraction data clearly indicate that apatite lattice parameters do not change as the crystals get larger. At the last stage of calcification the crystal size, chemical composition, and thermal conversion of the apatite crystallites approximate those of bone samples, which have been examined for comparison. The results provide a quantitative relationship between relative magnesium content and extent of apatite conversion into B-tricalcium phosphate by heat treatment. Furthermore, they suggest that the smaller crystallites laid down inside the gap region of the collagen fibrils are richer in magnesium than the longer ones that fill the space between collagen fibrils.

Nanocrystals of magnesium and fluoride substituted hydroxyapatite

Hydroxyapatite nanocrystals synthetized in the presence of different concentrations of magnesium and fluoride ions in solutions--1, 5 and 10 at.% have been submitted to a structural and chemical characterization. The syntheses were carried out in the presence of low molecular weight polyacrylic acid, which has been verified to inhibit hydroxyapatite crystallization. The polyelectrolyte is adsorbed into the crystals during the synthesis and provokes a reduction of the mean crystal sizes. The reduction is greater along the direction orthogonal to the c-axis, suggesting a preferential adsorption of the polyelectrolyte on the crystalline faces parallel to the c-axis. Both magnesium and fluoride can be incorporated into the hydroxyapatite structure. On the basis of the values of the lattice constants and of the magnesium relative content of the solid phase, it can be suggested that probably just a part of magnesium is substituted for calcium, the remainder being adsorbed on the crystal surface. However, magnesium destabilizes the apatitic structure favouring its thermal conversion into beta-tricalcium phosphate, and displays an inhibiting effect on the crystallization of hydroxyapatite. This last effect is enhanced by the simultaneous presence of polyacrylic acid. Fluoride substitution for hydroxyl ions into hydroxyapatite structure induces a slight increase of the crystal sizes along the c-axis direction. The data indicate that the experimental approach can be successfully used to prepare nanoapatite with crystallinity, crystal dimensions, composition, structure and stability very close to those characteristics of biological apatites.

Inhibiting effect of zinc on hydroxylapatite crystallization

Abstract X-Ray diffraction and spectrophotometric analysis have been used to investigate the role of zinc on hydroxylapatite (HA) crystallization. The presence of zinc in solution strongly inhibits the crystallization of hydroxylapatite, which can be synthesized as a unique crystalline phase only up to zinc concentration of about 25 atom %. This phase exhibits a reduction of Ca/P molar ratio and crystal sizes with increasing zinc concentration. Although the Ca/Zn ratio in the solid phase is almost equivalent to that in solution, the values of the cell parameters of the apatitic phase indicate that zinc cannot appreciably substitute for calcium in HA structure. Therefore, zinc must be assumed to be adsorbed on the surface of apatite crystallites and/or in the amorphous phase. The extent of thermal conversion of HA into s-tricalcium phosphate (s-TCP) increases with increasing zinc concentration in the solid phase, either when it is obtained by means of synthesis in solution or after cyclic pH fluctuation. The decrease of the lattice constants of s-tricalcium phosphate with increasing zinc concentration in the solid phase indicates that zinc partially replaces calcium in this structure. The inhibiting effect of zinc on HA crystallization and its preference for s-TCP structure closely resembles the behavior previously observed for magnesium.

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.

Drawn gelatin films with improved mechanical properties

Chain anisotropic distribution in gelatin films has been obtained by uniaxial stretching at constant relative humidity, followed by air drying and successive cross-linking with glutaraldehyde. The drawn samples have been characterized by mechanical tests, differential scanning calorimetry and scanning electron microscopy. The Youngs modulus, E, and the stress at break, sigma(b), increase linearly with the draw ratio and reach values which are about five times those characteristic of undrawn samples. Furthermore, on stretching the alignment of the gelatin strands along the direction of deformation increases while the thickness of the layers decreases significantly. The renaturation level, that is the fraction of gelatin in a collagen-like structure, has been calculated as the ratio between the melting enthalpy of gelatin samples and that of tendon collagen. The results indicate that the improvement of mechanical properties achieved by drawn gelatin is closely related to the renaturation level. The experimental approach utilized to induce segmental orientation in gelatin films, allows to obtain anisotropic materials with improved mechanical properties in the direction of deformation, and can be usefully applied in the preparation of biomaterials.

HYDROXYAPATITE GELATIN FILMS: A STRUCTURAL AND MECHANICAL CHARACTERIZATION

Composite films of gelatin and hydroxyapatite were prepared and characterized by mechanical tests, scanning electron microscopy and X-ray diffraction investigation. The mechanical properties of the films are greatly affected by the presence of hydroxyapatite and change as a function of inorganic phase content. On stretching, the long axis of the collagen molecular portions align parallel to the direction of deformation and the gelatin coarse layered structure becomes more evident and ordered. Furthermore, under deformation the inorganic crystals, which are embedded in the gelatin layers, seem to squeeze out in the interlayer spaces and assume a preferential orientation parallel to the force trajectories. Thus, as the inorganic phase stiffens the gelatin films, the macromolecular matrix distributes the stress promoting the preferential orientation of the apatitic crystals. The results indicate that this experimental approach can be used to prepare composites with anisotropic properties, which can be modulated through variation in composition and mechanical deformation in order to get biomaterials suitable to fulfill specific mechanical functions.