Gloria A. Soares

Transformation of monetite to hydroxyapatite in bioactive coatings on titanium

Abstract Calcium phosphates have a wide range of pH stability, depending on their Ca/P ratio. Under physiological conditions (pH ≈7), the most stable calcium phosphate is hydroxyapatite, Ca 10 (PO 4 ) 6 (OH) 2 . Acidic calcium phosphates, like dicalcium phosphate, CaHPO 4 (monetite) and dicalcium phosphate dihydrate, CaHPO 4 ·2H 2 O (brushite), are thermodynamically unstable under pH values greater than 6–7 and undergo transformation into more stable calcium phosphates. It means that, when placed in vivo (pH ≈7), acidic calcium phosphates convert to hydroxyapatite. In the present study, a coating of crystalline monetite oriented along the [112] axis was electrochemically deposited on titanium substrates. This monetite coating was subsequently converted to hydroxyapatite by immersion in alkaline solutions. The result was a crystalline hydroxyapatite coating oriented along the [002] axis. Different alkaline solutions produced the same result. Studying the effect of immersion time on the transformation indicated that 4 h were required to complete the conversion from monetite to hydroxyapatite. The transformation occurred by a dissolution–reprecipitation mechanism, i.e. the monetite coating was continuously dissolved and reprecipitated as hydroxyapatite. This combined electrochemical deposition and chemical conversion process produced hydroxyapatite coatings with satisfactory adhesion to the substrate and a thickness between 10 and 30 μm.

Cytocompatibility of chitosan and collagen-chitosan scaffolds for tissue engineering

In this work, chitosan and collagen-chitosan porous scaffolds were produced by the freeze drying method and characterized as potential skin substitutes. Their beneficial effects on soft tissues justify the choice of both collagen and chitosan. Samples were characterized using scanning electron microscope, Fourier Transform InfraRed Spectroscopy (FTIR) and thermogravimetry (TG). The in vitro cytocompatibility of chitosan and collagen-chitosan scaffolds was evaluated with three different assays. Phenol and titanium powder were used as positive and negative controls, respectively. Scanning electron microscopy revealed the highly interconnected porous structure of the scaffolds. The addition of collagen to chitosan increased both pore diameter and porosity of the scaffolds. Results of FTIR and TG analysis indicate that the two polymers interact yielding a miscible blend with intermediate thermal degradation properties. The reduction of XTT ((2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) and the uptake of Neutral Red (NR) were not affected by the blend or by the chitosan scaffold extracts, but the blend and the titanium powder presented greater incorporation of Crystal Violet (CV) than phenol and chitosan alone. In conclusion, collagen-chitosan scaffolds produced by freeze-drying methods were cytocompatible and presented mixed properties of each component with intermediate thermal degradation properties.

Chemical and topographical influence of hydroxyapatite and β-tricalcium phosphate surfaces on human osteoblastic cell behavior

The objective of this work was to evaluate the relative role of the calcium phosphate surface chemistry and surface topography on human osteoblast behavior. Highly dense phosphate ceramics (single-phase hydroxyapatite HA and beta-tricalcium phosphates TCP) presenting two distinct nano roughnesses were produced. Some samples were gold-sputter coated in order to conveniently mask the surface chemical effects (without modification of the original roughness) and to study the isolated effect of surface topography on cellular behavior. Our results indicated that the nanotopography of the studied ceramics had no effect on the cellular adhesion (cell spreading, focal contacts and stress fibers formation). On the contrary, strong topographical effects were verified on cell proliferation and differentiation. Moreover, the phosphate chemistry was responsible for changes in adhesion, proliferation and cell differentiation. On TCP, it was shown that the main influent parameter was surface chemistry, which negatively affected the initial cell adhesion but positively affected the subsequent stage of proliferation and differentiation. On HA, the main influent parameter was surface topography, which increased cell differentiation but lowered proliferation.

Dissolution of calcium-deficient hydroxyapatite synthesized at different conditions

Abstract The dissolution characteristics of several calcium-deficient hydroxyapatites (HA) have been investigated. Eleven samples were produced by varying synthesis parameters like temperature, pH, digestion time, reagent concentration and velocity of addition. Powder characterization was conducted using X-ray diffraction. Sample crystallinity was variable and samples examined by transmission electron microscopy showed acicular or plate-like morphology. After sample dissolution in Milli-Q water for 168 h, the calcium and phosphate contents in solution were measured by induced coupled plasma optical emission spectrometry (OES). The dissolution behavior of calcium-deficient HA was highly dependent on the sample calcium/phosphorus (Ca/P) molar ratio of the original HA. Dissolution in water was enhanced with decreasing HA sample molar ratio. However, the dissolution process equilibrium was not achieved at the end of 168 h (7 days).

BIOACTIVITY ASSESSMENT OF TITANIUM SHEETS ELECTROCHEMICALLY COATED WITH THICK OXIDE FILM

Bone formation around a metallic implant is a complex process that involves micro- and nano-metric interactions. In order to obtain faster osseointegration it is necessary to develop adequate surface treatments. Recently, the effect of coatings on implants performance has being studied, specially those related to the titanium oxide. In this work, a thick oxide layer on titanium surface was electrochemically produced and characterized. Incubation of this material into simulated body fluids for 7 days showed that a layer containing calcium (Ca) and phosphorus (P) was precipitated on the titanium surface. By XPS analysis the Ca/P ratio was equal to 1.61 and the calcium and phosphorus binding energies were compatible with hydroxyapatite and phosphate, respectively. Fourier-transformed infrared spectroscopy results indicated the presence of carbonate bands besides phosphate ones. Consequently, we can conclude that the oxide film exhibits suitable in vitro behavior due to its ability of inducing the precipitation of a calcium-phosphate layer similar to the bone-like apatite.

Human osteoblasts adhesion and proliferation on magnesium-substituted tricalcium phosphate dense tablets

Tricalcium phosphate (TCP) is recognized as a promising bone replacement material due to its high bioactivity and resorbable properties. To mimic biological apatites, incorporation of magnesium (Mg) in TCP was proposed. Mg-substituted TCP (β-TCMP) and β-TCP dense tablets were obtained by pressing and sintering at 1,000°C Mg-substituted calcium deficient apatite (Mg-CDA) and commercial TCP, respectively. The materials were characterized using X-ray diffraction, infrared spectroscopy and electron microscopy. Human osteoblast cells (SaOs2) were seeded onto the sintered tablets for 4xa0h, 24xa0h and 7xa0days. Results showed that Mg-CDA was completely transformed into β-TCMP. Moreover, β-TCMP stimulated adhesion and proliferation of human osteoblast cells. Consequently, the magnesium incorporation on calcium deficient apatites followed by sintering at 1,000°C seems to be a useful path to obtain biocompatible and non cytotoxic dense tablets with TCP structure with potential application on bone engineering.

Surface topography modulates the osteogenesis in human bone marrow cell cultures grown on titanium samples prepared by a combination of mechanical and acid treatments

Titanium samples of different roughness Ra and morphology were prepared using a combination of mechanical (grinding with a SiC paper or blasting with aluminum oxide particles with 65 or 250 μm) and chemical (attack with a sulphuric acid based solution or a hydrofluoric acid based solution) treatments. The biological performance of the prepared surfaces was evaluated using human bone marrow osteoblastic cell cultures. Mechanically treated samples presented different Ra values and surface morphology. The hydrofluoric acid solution was more effective than the sulphuric acid solution in smoothing titanium surface and also in eliminating aluminum contamination resulting from the blasting process. Bone marrow cells seeded on the different titanium samples showed a similar pattern of behavior during cell attachment and spreading. Cells proliferated very well on all the titanium surfaces and cell growth was observed during approximately two to three weeks. The samples treated with the hydrofluoric acid solution presented higher alkaline phosphatase activity. Only the blasted samples treated with the acid solutions allowed seeded bone marrow cells to form a mineralized extracellular matrix. The best biological performance was found in the blasted samples treated with the hydrofluoric acid solution, which could be related to the characteristic microtopography of these samples that presented a homogeneous and smooth roughness.

Synthesis and characterization of biocomposites with different hydroxyapatite–collagen ratios

Hydroxyapatite (HA)-type I collagen (Col) composite is a tissue-engineered bone graft which can act as a carrier or a template structure for cells or any other agents. In this paper, the effect of Col ratio on the scaffold structure and composition was analyzed. Scaffolds composed by HA/Col with different weight ratios (80:20; 50:50; 20:80, and 10:90) were produced by the precipitation method at pH 8–9, 37°C and 6xa0h of ripening. Using X-ray diffraction data, the Rietveld structure refinement showed that the size of HA crystals along the c-axis direction (002) decreases significantly in the presence of Col. Thus, the HA crystal shape turned from needle-like in pure HA, into spherical, in the 10:90 composite due to Col fibrillogenesis. The homogeneity of the composite was significantly dependent on the amount of Col in it. HA/Col 20/80 composite presented HA particles in a more homogenous way. Such a biocomposite was successfully produced in a rapid way and it is potentially useful for both small tissue repairs and engineering.

Importance of dynamic culture for evaluating osteoblast activity on dense silicon-substituted hydroxyapatite.

This paper reports an investigation on human osteoblast-like cells (SaOs-2) seeded onto pure hydroxyapatite (HA) and silicon-substituted HA (SiHA) tablets under static and dynamic culture conditions. The biological characterizations were conducted in classical static conditions in multi-wells plates, and in a perfusion bioreactor that permits continuous circulation of culture medium at 2 mL/h. The morphology, proliferation and differentiation of osteoblastic cells were examined for the two types of samples in the both culture conditions after 1, 3 and 8 days. Under dynamic conditions, cells cultured on SiHA surfaces showed a faster adhesion process and the formation of longer and thinner focal adhesions than in static conditions. The number of cells grown onto both ceramic surfaces was higher in dynamic conditions when compared with static conditions. Moreover, a higher activity of alkaline phosphatase was found for cells seeded under dynamic conditions. Our findings suggest that the application of perfusion culture system on cells cultured on dense substrates is valuable for predicting in vivo behaviour of cells on biomaterials.

Surface transformation of silicon-doped hydroxyapatite immersed in culture medium under dynamic and static conditions

A comparative study of in vitro bioactivity of hydroxyapatite (HA) and silicon-doped hydroxyapatite (SiHA) has been carried out by immersion in a cell culture medium with or without fetal bovine serum during 14 days in static and dynamic conditions. A specific bioreactor was developed for the experiments in dynamic conditions. Ceramic surface transformations were characterized by electron microscopy, atomic force microscopy and X-ray photoelectron spectroscopy before and after immersion. The monitoring of calcium, phosphate and proteins in immersion medium was also done during the experiment. The two hydroxyapatite surfaces immersed in cell culture medium under dynamic conditions were found to be more probably covered by a new Mg-enriched Ca-deficient apatite layer than surfaces immersed under static conditions. These results suggest that dynamic procedure and medium with serum macromolecules seem to be more adequate to predict the in vivo activity of bioceramics. Moreover, SiHA presented a higher capacity of protein adsorption.