The in vivo process of biological mineralization can be successfully simulated in vitro by a slow crystallization of a nonstoichiometric carbonated apatite from revised simulated body fluid (rSBF) carried out in a constant-composition double-diffusion (CCDD) device under physiological conditions (temperature 37 °C, solution pH within 7.2-7.4). The substitution of an organic buffer (Hepes) by permanent carbon dioxide bubbling to keep the pH constant has no strong influence on the crystallization. By increasing the overall concentration, calcification can be easily induced on porous bioinert surfaces such as cellulose or cholesterol that normally cannot be coated with calcium phosphates from supersaturated solutions without additional promoters.

Three types of poorly crystallized calcium-deficient hydroxyapatites (CDHA) with Ca/P molar ratios 1.50, 1.58, and 1.67 were prepared from CaHPO4·2H2O and KOH. These were sintered at 1050 °C for 4 h. Well-crystallized β-tricalcium phosphate (β-TCP), biphasic calcium phosphate (BCP), and stoichiometric hydroxyapatite (HA) were produced, respectively. The sintered and unsintered calcium phosphates were studied by X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. In addition, Ca/P molar ratios were determined by a chemical analysis. Thermogravimetric analysis (TGA) revealed a loss of water for the period of sintering. On the basis of the experimental data, a structure of BCP was suggested. After applying the numerical values of ionic (OH-, Ca2+, and PO4 3-) diffusion coefficients at 1000 °C, the solid-state transformation mechanism of CDHA into BCP was proposed.

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Precipitation experiments with aqueous solutions of the Kokubo's revised simulated body fluid (rSBF) equal to 2, 4, 8, and 12 times the ionic concentration of human blood plasma were performed. Instead of Hepes, solution pH was adjusted to the desired value of 7.40 +/- 0.02 by either bubbling of CO2 or addition of HCl. The experiments were performed in tightly closed plastic vessels kept at 37.0 +/- 0.2 degrees C for 72 h under permanent shaking. Afterward, the suspensions were filtrated, and the precipitates were collected and analyzed. The results revealed that increasing the concentration of rSBF resulted in great changes in both the structure and the chemical composition of the precipitates. Phosphate substitution for carbonate (although the amounts of calcium and magnesium remained unchanged) and crystallinity decreasing were the most important modifications found in the precipitates formed from the highly condensed solutions of rSBF.

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