Fabrication of nitrogen-doped carbon dots for screening the purine metabolic disorder in human fluids.

Fabrication of nitrogen-doped carbon dots (N-CDs) electrode for the screening of purine metabolic disorder was described in this paper. Peroxynitrite is a short-lived oxidant species that is a potent inducer of cell death. Uric acid (UA) can scavenge the peroxynitrite to avoid the formation of nitrotyrosine, which is formed from the reaction between peroxynitrite and tyrosine (Try). Scavenging the peroxynitrite avoids the inactivation of cellular enzymes and modification of the cytoskeleton. Reduced level of UA decreases the ability of the body from preventing the peroxynitrite toxicity. On the other hand, the abnormal level of UA leads to gout and hyperuricemia. Allopurinol (AP) is administered in UA lowering therapy. Thus, the simultaneous determination of UA, Try and AP using N-CDs modified glassy carbon (GC) electrode was demonstrated for the first time. Initially, N-CDs were prepared from L-asparagine by pyrolysis and characterized by different spectroscopic and microscopic techniques. The HR-TEM image shows that the average size of the prepared N-CDs was 1.8±0.03nm. Further, the N-CDs were directly attached on GC electrode by simple immersion, follows Micheal's nucleophilic addition. XPS of N-CDs shows a peak at 285.3eV corresponds to the formation of C-N bond. The GC/N-CDs electrode shows higher electrocatalytic activity towards UA, Tyr and AP by not only shifting their oxidation potentials toward less positive potential but also enhanced their oxidation currents in contrast to bare GC electrode. The GC/N-CDs electrode shows the limit of detection of 13×10-10M (S/N=3) and the sensitivity of 924µAmM-1cm-2 towards the determination of UA. Finally, the N-CDs modified electrode was utilized for the determination of UA, Tyr and AP in human blood serum and urine samples.

Chemico-physical characterization of gelatin films modified with oxidized alginate.

The possibility of using low concentrations of dialdehyde alginate (ADA) to crosslink and stabilize gelatin films was investigated. The films were prepared from gelatin solutions at different concentrations (5, 10 and 15wt.%) containing different amounts of oxidized alginate (0, 1 and 3wt.% with respect to the weight of gelatin). The extent of crosslinking increases as a function of ADA concentration, up to about 23%. The presence of oxidized alginate provokes a significant reduction in the degree of swelling and of gelatin release in phosphate-buffered saline solution, enhancing the effect of gelatin concentration. Furthermore, the values of the Young's modulus, E, and of the stress at break, sigma(b), increase with increasing ADA concentration. The observed small, but appreciable, increase in thermal stability found by differential scanning calorimetric investigation is supported by X-ray diffraction results.

Effect of strontium and gelatin on the reactivity of alpha-tricalcium phosphate.

The hydrolysis reaction of alpha-tricalcium phosphate (alpha-TCP) is of great interest because of its widespread use in the preparation of biomaterials for hard tissue repair. The aim of this study was to investigate how this reaction is influenced by the presence of a bioactive ion, Sr(2+), and of a biopolymer, gelatin, which were previously reported to affect the setting reaction of alpha-TCP-based cements. Hydrolysis experiments were carried out at different Sr(2+) concentrations (0, 5, 10, 20 at.%) in solutions at different gelatin concentrations (0, 0.1, 0.5, 1.0 wt.%). The results indicate that Sr(2+) delays the conversion of alpha-TCP into calcium-deficient hydroxyapatite (CDHA). The structural and morphological modifications of CDHA obtained from solutions at increasing Sr(2+) concentrations indicate that during hydrolysis strontium enters the structure of CDHA, where it partially substitutes for calcium. On the contrary, alpha-TCP hydrolysis rate increases on increasing gelatin concentration. Gelatin promotes conversion of alpha-TCP into octacalcium phosphate, and strongly interacts with the nucleating and growing crystals.

Porous composite scaffolds based on gelatin and partially hydrolyzed alpha-tricalcium phosphate.

Porous composite scaffolds of varying compositions were prepared by freeze-drying gelatin foams containing increasing amounts of alpha-tricalcium phosphate (alpha-TCP), up to about 40 wt.%. Due to the presence of gelatin, a partial hydrolysis of alpha-TCP into octacalcium phosphate (OCP) occurs during foaming. As a consequence, the scaffolds contain both alpha-TCP and OCP, in relative amounts of about 74% and 26%, respectively, independent of the initial composition. In physiological conditions the inorganic component of the scaffolds undergoes a further hydrolysis as shown by the finding that after immersion in phosphate-buffered saline at 37 degrees C for 1 week the scaffolds contain poorly crystalline hydroxyapatite together with OCP. The scaffolds display a porous interconnected microstructure. The mean dimensions of the pores decrease from about 350 to about 170 microm as the inorganic phase content increases. Simultaneously, the mean values of the compression strength and Young's modulus increase. Stabilization of the scaffolds was obtained by using a natural, non-toxic, crosslinking agent, genipin, which significantly improves their mechanical properties.

Nanocrystalline hydroxyapatite-polyaspartate composites.

The direct synthesis of hydroxyapatite-poly-L-aspartic acid (HA-PASP) nanocrystals has been carried out in presence of increasing amounts of PASP in solution up to 56 mmol/l. WAXS, TEM, TGA, IR and chemical analyses were used to characterize the structure, morphology and composition of the products. PASP is quantitatively incorporated into HA crystals, provoking a reduction of the coherent length of the crystalline domains. Furthermore, composite crystals display a greater length/width ratio with respect to the control HA crystals, and show a remarkable trend towards aggregation. 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 comparison between the morphological and structural data allows to suggest a specific interaction between PASP and HA structure.

In vitro mineralization of gelatin-polyacrylic acid complex matrices.

Gelatin-polyacrylic acid (gel-PAA) matrices were obtained by slow diffusion of polyacrylic acid into gelatin gels. The matrices were submitted to uniaxial stretching, which induces a preferential orientation of the collagen molecules, and used as biomimetic substrates for the nucleation of hydroxyapatite from simulated body fluid (SBF). The relative amount of hydroxyapatite deposited from 1.5SBF increases as a function of polyelectrolyte content in the matrices, up to about 30 wt%. In the absence of PAA, the inorganic phase is laid down on the surface of the gelatin matrices as hemispherical aggregates. At variance, hydroxyapatite deposition in the gel-PAA composite matrices at relatively low PAA content occurs preferentially in the spaces between the layers on the surface of the matrices and displays a tablet-like morphology. At high polyelectrolyte concentration, an almost uniform layer of hydroxyapatite covers the whole surface of the matrices. The preferential orientation of the (002) hydroxyapatite reflection indicates a close relationship between the inorganic crystals and the collagen molecules.

Relationship between triple-helix content and mechanical properties of gelatin films.

This paper reports a study on the influence of the renaturation level of gelatin on the mechanical and swelling properties of gelatin films. Films at different renaturation level were obtained from gelatin samples with different Bloom index. It was verified that the triple-helix content, calculated from the values of the enthalpy of denaturation associated to the endothermal transition at about 41 degrees C of gelatin, increases with the Bloom index. The d.s.c. data are further supported by the results of the X-ray diffraction investigation carried out on the same samples. The increase of triple-helix content provokes a significant reduction in the degree of swelling, and a remarkable improvement of the mechanical properties of the films. The elastic Young's modulus, E, increases linearly with the renaturation level, from 3.6 to 12.0 MPa. Crosslinking with GTA 1% remarkably reduces the degree of swelling of all the samples, and induces a further increase of the Young's modulus, which reaches values up to 27 MPa.

Alpha-Tricalcium phosphate-gelatin composite cements.

This study investigates gelatin influence on calcium phosphate cement (CPC) setting properties. Cements of different compositions were prepared using |Alpha -tricalcium phosphate (|Alpha -TCP) enriched with a small amount of dicalcium phosphate dihydrate (DCPD), and different gelatin amounts up to 20 wt%. The cements, prepared with a liquid/powder ratio of 0.3 ml/g, were soaked in simulated body fluid (SBF) for different times inverted exclamation markU21 days. The setting reaction of the control cement prepared without gelatin occurred in 7 days, whereas the transformation of the cements at high gelatin content into apatite was almost complete in 2-day aging in SBF. Gelatin presence reduced the total porosity of the cements, and significantly modified their microstructure. The fractured surface of the aged control cement was covered with entangled plate-like apatite crystals, whereas the gelatin cements displayed more compact surfaces, most likely due to the inhibiting effect of gelatin on apatite crystal growth. The microstructural modifications were in agreement with the mprovement of the mechanical properties in the aged cements, the compressive strength of which increased linearly as a function of gelatin content, from 2.0 inverted exclamation markA 0.8 to 14 inverted exclamation markA 1 MPa.

Interaction of acidic poly-amino acids with octacalcium phosphate.

Octacalcium phosphate (OCP) hydrolysis into hydroxyapatite (HA) has been investigated in aqueous solutions at different concentrations of poly-L-aspartate (PASP) and poly-L-glutamate (PGLU). In the absence of the polyelectrolytes, the transformation of OCP into HA is complete in 48 h. Both poly-L-aspartate and poly-L-glutamate inhibit OCP hydrolysis. However, PGLU displays a greater inhibiting effect, as a result of the different extent of phase transformation obtained at the same polyelectrolyte concentration. The inhibition takes place through polyelectrolyte adsorption on the (100) face of OCP crystals, which prevents the splitting of OCP crystals along their c-axis and the transformation into the final very long, needle-like, apatitic crystals.

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 Young's 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.

Alpha-tricalcium phosphate hydrolysis to octacalcium phosphate: effect of sodium polyacrylate.

Alpha-Tricalcium phosphate (alpha-TCP) hydrolysis into octacalcium phosphate (OCP) has been investigated in phosphoric acid solution at different concentrations of sodium polyacrylate (NaPA). The hydrolysis process has been followed by powder X-ray diffraction, infrared absorption and scanning electron microscopy analyses. In the absence of the polyelectrolyte, alpha-TCP undergoes a complete transformation into OCP in 24 h. The presence of polyacrylate in solution inhibits the hydrolysis so that a NaPA concentration of 0.5 microm is sufficient to lengthen the time required to complete the hydrolysis to 4 days. The variation of Ca2+ concentration in the soaking solution suggests that the transformation occurs through alpha-TCP dissolution followed by OCP precipitation. The delayed OCP nucleation and growth in the presence of polyacrylate implies a preferential adsorption of the polyelectrolyte on the growing OCP crystals, which induces an anisotropic reduction of the coherence lengths of the perfect crystalline domains.

Bonelike apatite growth on hydroxyapatite-gelatin sponges from simulated body fluid.

In vitro bioactivity of gelatin sponges and hydroxyapatite-enriched gelatin sponges was tested through evaluation of the variations in their composition and morphology after soaking in simulated body fluid (1.5) for periods up to 21 days at 37 degrees C. The presence of hydroxyapatite inside the sponges promotes the deposition of bonelike apatite crystals. The deposits are laid down as spherical aggregates, with mean diameters increasing from about 1-2 microm, after 4 days of soaking in simulated body fluid solution, up to about 3.5 microm in the samples soaked for 21 days. Simultaneously, the relative amount of inorganic phase increases up to about 56% wt, leading to a composite material with a composition quite close to that of bone tissue. The inorganic phase is a poor crystalline carbonated apatite similar to trabecular bone apatite.

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 Young's 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.

Structural aspects of the calcification process of lower vertebrate collagen.

In order to investigate the structural relationship between inorganic phase and collagen fibrils in the calcified tissues of lower vertebrates we have carried out a wide and small angle X-ray diffraction investigation on carp scales and bone samples. The small angle patterns from decalcified bone and scales, as well as uncalcified tendon samples from carp are very similar to that of type I collagen from higher vertebrates. The D-axial period, 67 nm, is the same as that of higher vertebrate type I collagen, while the most significant difference is the relatively low intensity of the first order reflection, which is, however, the most intense. The relative intensity distributions of the meridional reflections recorded from fish bone and scales are in agreement with an electron density distribution according to a step function. The calculated step length is very close to the values previously reported for calcified tissues from higher vertebrates. The small angle reflections from calcified, as well as decalcified, scales display different directions of orientation, which could be in agreement with a plywood arrangement of collagen fibrils in successive sheets parallel to the plane of the scale.