Anabela C. Fernandes

Apatite deposition on titanium surfaces — the role of albumin adsorption

Titanium implant surfaces are known to spontaneously nucleate apatite layers when in contact with simulated body fluids. However, adsorption of proteins may influence the process of apatite layer formation. In this study the role of bovine serum albumin (BSA) adsorption in the process of apatite deposition on titanium substrates is investigated. Deposition of calcium phosphate was induced by immersing titanium substrates in a Hanks balanced salt solution (HBSS) for times ranging from 1 to 23 days. The resulting substrates were studied by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), wettability measurements and electrochemical impedance determinations. All these methods indicate the presence of a calcium phosphate layer. The same procedure was repeated substituting HBSS with a solution of BSA in HBSS. Although SEM, EDS and electrochemical impedance spectra do not reveal the presence of an apatite layer, XPS analysis strongly indicates that the inhibition of apatite formation by BSA is only partial. The competition between BSA adsorption and apatite deposition seems to lead to a mixed film where the protein co-exists with calcium phosphate. Wettability studies suggest that this surface film is heterogeneous and porous, similar to the thicker films formed in albumin-free HBSS.

Mineralisation of two phosphate ceramics in HBSS: role of albumin

The role of albumin in the mineralisation process of commercial hydroxyapatite (HAp) and synthesised biphasic (HAp-tricalcium phosphate) ceramics in a bufferless simulated inorganic plasma (HBSS) was investigated by conventional in vitro tests and static and dynamic wettability measurements. Albumin was either pre-adsorbed or solubilised in HBSS. It was found that calcium complexation by albumin plays a key role in early mineralisation kinetics, so that mineralisation is favoured when albumin is pre-adsorbed and hindered when it is dissolved in HBSS. In the biphasic ceramic this picture is complicated by the fact that albumin, in solution, seems to promote the dissolution of tricalcium phosphate, and simultaneously compete for calcium with the ceramic. It also appears that albumin has a stabilising effect of octacalcium phosphate present in deposits on commercial HAp. The same effect may be present in the case of the biphasic ceramic, at earlier mineralisation times, when octacalcium phosphate appears as a precursor of HAp. Octacalcium phosphate formation on commercial apatite is accompanied by carbonate substitution in phosphate positions.

Thermotropic mesomorphism of a model system for the plant epicuticular wax layer.

As a model for the epicuticular wax layer of plant cuticular membranes, we have studied the phase behavior of 1-tetradecanol and 1-octadecanol and their binary mixtures between 5 and 70 degrees C, using differential scanning calorimetry and Fourier transform infrared spectroscopy (FTIR). Both pure compounds show two exothermic phase transitions corresponding to a transformation from a liquid phase to a hexagonally packed solid phase (S(HEX)), which at lower temperatures transforms to an orthorhombically-packed solid phase (S(ORT)). On heating the S(ORT) solid a single endothermic transition with a transition enthalpy corresponding to the sum of the exothermic transition enthalpies is obtained. These transitions were also followed using FTIR spectroscopy in the CH(2)-stretching (symmetric and asymmetric) and CH(2)-rocking vibration modes. The FTIR spectra of the pure compounds in the liquid, S(HEX), and S(ORT) phases were used to simulate experimental spectra in the phase transition regions. The simulations allowed us to estimate the molar fractions of each phase in the transition regions of the pure compounds. A phase diagram for the binary mixture of 1-tetradecanol and 1-octadecanol was obtained using differential scanning calorimetry and FTIR. FTIR studies on binary mixtures prepared from one perdeuterated component and the other nondeuterated permitted studying the thermotropic behavior of each component in the mixture independently. The mixture shows an eutectic behavior with an eutectic point between a molar fraction of octadecanol (X(18)) of 0.12 and 0.18 and a temperature of approximately 32 degrees C. Below 32 degrees C, a binary mixture of solid phases, one an S(ORT) phase and the other an S(HEX) phase, coexist up to approximately 25 degrees C, below which both solid phases are S(ORT) phases. We discuss the possible relevance of this complex phase behavior in a simple binary mixture of two long-chain alkanols in the context of the far more complex phase behavior expected for the plant epicuticular wax layer.

The dipolar relaxation behaviour of a liquid-crystalline side-chain polymer as studied by thermally stimulated discharge currents

Abstract The various dipolar relaxation mechanisms of a liquid-crystalline side-chain polymer have been studied in detail by the technique of thermally stimulated currents. The α relaxation, or glass transition relaxation, showed a compensation behaviour, i.e. a correlation between the activation enthalpy and the activation entropy of its components. Two sub-glass relaxations were observed. The lower-temperature one shows a small activation enthalpy (5–6 kcal mol−1), and is attributed to slightly hindered internal rotations in the side group. The higher-temperature one, on the other hand, shows a higher activation enthalpy (23 kcal mol−1) and a low positive activation entropy, and is attributed to restricted internal rotations of the side groups around bonds near the main chain, eventually in cooperation with the movements of the main-chain segments. An upper glass relaxation was also observed. This relaxation shows a Williams-Landel-Ferry or Vogel behaviour and high values of its mean activation enthalpy and entropy. The attribution of this discharge at the molecular level is discussed.

THE INFLUENCE OF PROTEINS ON CALCIUM PHOSPHATE DEPOSITION OVER TITANIUM IMPLANTS STUDIED BY DYNAMIC CONTACT ANGLE ANALYSIS AND XPS

Abstract The spontaneous formation of a calcium phosphate (apatite-like) layer on the surface of titanium implants in contact with biological model fluids is well known, but the effect of the presence of proteins in real biological fluids is not yet well understood. In this work, the process of calcium phosphate deposition on titanimn surfaces immersed in Hanks balanced salt solution (HBSS) containing dissolved bovine serum albumin (BSA) was studied by dynamic contact angle analysis (DCA) and X-ray photoelectron spectroscopy (XPS). Previous studies using electrochemical and wettability techniques as well as scanning electron microscopy (SEM) and XPS showed that although the deposition of calcium and phosphate ions still occurs in the presence of albumin, the growth of a calcium phosphate layer is inhibited. The present study suggests that when the Ti sample contacts a solution where both protein molecules and calcium and phosphate ions are present, the protein adsorbs first followed by the deposition of the ions. The same behaviour was observed for Ti substrates pre-covered by an apatite-like layer. Ti samples previously coated with a protein film, obtained by incubation in an isotonic saline solution of albumin, were studied after immersion in HBSS. In this case only XPS was able to detect traces of calcium and phosphate ions in the surface layer which were invisible to the DCA analysis.

The enthalpy of sublimation of diphenylacetylene from Knudsen effusion studies

The enthalpy of sublimation of diphenylacetylene at 298.15 K, ΔgcrH⊖m(C2(C6H5)2] = 95.1 ± 1.1 kJmol−1, was derived from vapour pressure-temperature data, obtained with two different Knudsen effusion apparatus, and from heat capacity measurements obtained by differential scanning calorimetry. The molybdenum-diphenylacetylene bond dissociation enthalpy in Mo(η5-C5H5)2[C2(C6H5)2] was reevaluated as 115 ± 26 kJ mol−1, on the basis of the new value for ΔcrgH⊖m[C2(C6H5)2].

Interaction of local and general anaesthetics with liposomal membrane models: A QCM-D and DSC study

The behaviour of four local anaesthetics (lidocaine, levobupivacaine, ropivacaine and tetracaine) and one general anaesthetic (propofol) is compared when interacting with two types of model membranes: supported layers of liposomes and liposomes in solution. Several liposomal compositions were tested: dimyristoylphosphatidylcholine (DMPC), binary mixtures of DMPC with cholesterol (CHOL), and ternary mixtures of dipalmitoylphosphatidylcholine (DPPC), DMPC, and CHOL. A quartz crystal microbalance with dissipation, QCM-D, was used to assess changes in the properties of supported layers of liposomes. The effect of the anaesthetics on the phase behaviour of the liposomes in suspension was determined by differential scanning calorimetry. Both techniques show that all anaesthetics have a fluidizing effect on the model membranes but, apparently, the solid supported liposomes are less affected by the anaesthetics than the liposomes in solution. Although the different anaesthetics were compared at different concentrations, tetracaine and propofol seem to induce the strongest perturbation on the liposome membrane. The resistance of the liposomes to the anaesthetic action was found to increase with the presence of cholesterol, while adding DPPC to the binary mixture DMPC+CHOL does not change its behaviour. The novelty of the present work resides upon three points: (1) the use of supported layers of liposomes as model membranes to study interactions with anaesthetics; (2) application of QCM-D to assess changes of the adsorbed liposomes; (3) a comparison of the effect of local and general anaesthetics interacting with various model membranes in similar experimental conditions.

Phase behaviour of oleanolic acid, pure and mixed with stearic acid: Interactions and crystallinity.

The phase behaviour of pure oleanolic acid (OLA) and in mixtures with stearic acid (SA) was characterized by differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and nuclear magnetic resonance (NMR). The crystalline OLA as received (OLA(ar)) becomes amorphous after being dissolved in chloroform and vacuum-dried at 50 degrees C (OLA(50)). Upon heating, both forms transform to the needle shape crystalline form (OLA(220)). Dimerization through H-bonding between COOH groups was detected both in OLA(ar) and OLA(220). Dimers are stronger in OLA(220), where H-bonding also involves the alcohol groups and plays a role in the crystalline organization. A eutectic type phase diagram was established for mixtures, with the eutectic composition close to pure SA. Mixtures rich in SA are miscible in the liquid and in the amorphous solid states, where the presence of SA-OLA co-dimers, formed through H-bonding between carboxyl groups, was detected. Miscibility and SA crystallinity decrease drastically with the OLA content.

Mineralisation of two calcium phosphate ceramics in biological model fluids

Two ceramic phosphates, commercial hydroxyapatite and synthesised biphasic hydroxyapatite–14 wt.% α-tricalcium phosphate, were incubated in Hanks balanced salt solution (HBSS) and Kokubos simulated body fluid (SBF) at 37 °C and the respective surface modifications investigated. The static contact angles (SCA) of water and diiodomethane on the surfaces of both incubated and non-incubated materials were measured as a function of time, showing that surface modifications were different for each type of phosphate and depended on the incubating solution. Dynamic contact angle (DCA) hysteresis provided information on the characteristics of possible deposits. These studies were complemented with SEM/EDS, FTIR and XRD analysis of the surfaces. Compositional changes in the incubating solutions with time were monitored by ICP spectroscopy. The main conclusions are (1) the presence of α-tricalcium phosphate in the synthesised biphasic hydroxyapatite enhances the deposition of calcium phosphate, (2) the precipitation of calcium phosphate is favoured by the pH increase of the incubating solution; consequently, buffer Tris inhibits the deposition because it avoids the pH increase and forms soluble complexes with Ca2+ ions. Another conclusion is that wettability measurements, especially DCA, provide a most sensitive method to detect surface transformations leading to mineralisation.

Accuracy and precision of heat capacity measurements using a heat flux differential scanning calorimeter

Abstract The accuracy and precision of a Setaram DSC 121 for the measurement of heat capacities of solids in the continuous mode was tested using sapphire and benzoic acid. The influence of the heating rate, mass of sample, and position of the crucible in the measuring tube on the obtained results is discussed. In the temperature range studied (320–440 K for sapphire and 320–370 K for benzoic acid) the accuracy and precision of the determination, based on the average results of three independent experiments, are better than 1% provided that adequate experimental conditions (large sample masses and low heating rates) and data handling procedures are used.