Discerning the role of topography and ion exchange in cell response of bioactive tissue engineering scaffolds

Surface topography is known to have an influence on osteoblast activity. However, in the case of bioactive materials, topographical changes can affect also ion exchange properties. This makes the problem more complex, since it is often difficult to separate the strictly topographical effects from the effects of ionic fluctuations in the medium. The scope of this paper is to analyze the simultaneous effect of topography and topography-mediated ion exchange on the initial cellular behavior of osteoblastic-like cells cultured on bioactive tissue engineering substrates. Two apatitic substrates with identical chemical composition but different micro/nanostructural features were obtained by low-temperature setting of a calcium phosphate cement. MG63 osteoblastic-like cells were cultured either in direct contact with the substrates or with their extracts. A strong and permanent decrease of calcium concentration in the culture medium, dependent on substrate topography, was detected. A major effect of the substrate microstructure on cell proliferation was observed, explained in part by the topography-mediated ion exchange, but not specifically by the ionic Ca(2+) fluctuations. Cell differentiation was strongly enhanced when cells were cultured on the finer substrate. This effect was not explained by the chemical modification of the medium, but rather suggested a strictly topographical effect.     

Silicon-stabilized α-tricalcium phosphate and its use in a calcium phosphate cement: characterization and cell response

α-Tricalcium phosphate (α-TCP) is widely used as a reactant in calcium phosphate cements. This work aims at doping α-TCP with silicon with a twofold objective. On the one hand, to study the effect of Si addition on the stability and reactivity of this polymorph. On the other, to develop Si-doped cements and to evaluate the effect of Si on their in vitro cell response. For this purpose a calcium-deficient hydroxyapatite was sintered at 1250°C with different amounts of silicon oxide. The high temperature polymorph α-TCP was stabilized by the presence of silicon, which inhibited reversion of the β→α transformation, whereas in the Si-free sample α-TCP completely reverted to the β-polymorph. However, the β-α transformation temperature was not affected by the presence of Si. Si-α-TCP and its Si-free counterpart were used as reactants for a calcium phosphate cement. While Si-α-TCP showed faster hydrolysis to calcium-deficient hydroxyapatite, upon complete reaction the crystalline phases, morphology and mechanical properties of both cements were similar. An in vitro cell culture study, in which osteoblast-like cells were exposed to the ions released by both materials, showed a delay in cell proliferation in both cases and stimulation of cell differentiation, more marked for the Si-containing cement. These results can be attributed to strong modification of the ionic concentrations in the culture medium by both materials. Ca-depletion from the medium was observed for both cements, whereas continuous Si release was detected for the Si-containing cement.     

Material characterization and in vivo behavior of silicon substituted alpha-tricalcium phosphate cement

The possibility and biological effects of substituting silicon in alpha-tricalcium phosphate (alpha-TCP) by way of solid-state reaction have been evaluated. alpha-TCP powders with varying substitution amounts (1 and 5 mol % Ca2SiO4) were synthesized by reacting mixtures of CaCO3, Ca2P2O7, and SiO2, at a rate of 4 degrees C(min)(-1) to 1100 degrees C, left to dwell for 2 h and then heated to 1325 degrees C at 4 degrees C(min)(-1) and left to dwell for a period of 4 h. The powders were then rapidly quenched in air. Si incorporation could be verified by X-ray diffraction analysis, indicating an increase of the lattice volume with increasing Si content from 4284.1(8) to 4334(1) A3 for pure alpha-TCP and alpha-Si5%TCP, respectively. The hydrolysis of milled alpha-SiTCP powders was monitored by isothermal calorimetry, and the compressive strength of set cements was tested. The results showed changes in speed and amount of heat released during reactivity tests and a decrease in mechanical strength (60, 50, and 5 MPa) with increasing Si content. In vitro bioactivity of the set cements after soaking in simulated body fluid for 4 weeks was also tested. The formation of a bonelike apatite layer on the surface of the set cements could be observed and was thickest for 1%Si (20 microm). These results were in good agreement with the in vivo studies performed, which showed strong evidence that the cement containing 1% silicon doped alpha-TCP enhanced mesenchymal cell differentiation and increased osteoblast activity compared with alpha-TCP.     

Porous hydroxyapatite and gelatin/hydroxyapatite microspheres obtained by calcium phosphate cement emulsion

Hydroxyapatite and hybrid gelatine/hydroxyapatite microspheres were obtained through a water in oil emulsion of a calcium phosphate cement (CPC). The setting reaction of the CPC, in this case the hydrolysis of α-tricalcium phosphate, was responsible for the consolidation of the microspheres. After the setting reaction, the microspheres consisted of an entangled network of hydroxyapatite crystals, with a high porosity and pore sizes ranging between 0.5 and 5 μm. The size of the microspheres was tailored by controlling the viscosity of the hydrophobic phase, the rotation speed, and the initial powder size of the CPC. The incorporation of gelatin increased the sphericity of the microspheres, as well as their size and size dispersion. To assess the feasibility of using the microspheres as cell microcarriers, Saos-2 cells were cultured on the microspheres. Fluorescent staining, SEM studies, and LDH quantification showed that the microspheres were able to sustain cell growth. Cell adhesion and proliferation was significantly improved in the hybrid gelatin/hydroxyapatite microspheres as compared to the hydroxyapatite ones.