Linking changes in mineral chemistry to reactivity: a study on carbonate incorporation in synthetic hydroxyapatite and its effect on solubility.

Abstract

The human skeleton is an important contributor to homeostasis in our bodies, as chemical elements can either be stored in the bone material or extracted when needed anywhere else. More than two-thirds of human bone mass consists of a mineral called hydroxyapatite. Ions such as carbonate can be incorporated into the apatite crystal structure by replacing other ions. Such substitution processes occur in every healthy body, but they can have significant effects on the bone mineral reactivity when excessive amounts of carbonate are incorporated, as is the case in patients with X-linked hypophosphatemia. The aim of this study was to find out how the incorporation of carbonate affects the solubility of hydroxyapatite minerals. Five samples were synthesised at 37 C by a double decomposition reaction between a calcium chloride solution and a carbonate phosphate solution, which had a carbonate concentration ranging between 0 and 0.48 M. The precipitated minerals were imaged using atomic force microscopy and a Leica microscope and analyzed by Fourier transform infrared spectroscopy and Raman spectroscopy. Next, the samples were (partly) dissolved in a buffered sodium chloride solution with pH 6.5, while samples were collected at regular time intervals. Colourimetric determination of the phosphate concentration of these extracted sample solutions was done using the molybdenum blue method. Results from this study indicate the precipitation of octacalcium phosphate in the sample without added carbonate, instead of the expected apatite. This can be explained by the solution pH of 5.2 during sample synthesis and the fact that octacalcium phosphate is stable between pH 4 and 6.5, while hydroxyapatite only forms at pH values above 7.4. Furthermore, the spectroscopic data indicated that there was a positive correlation between the carbonate concentration during synthesis and the amount of incorporated carbonate for samples CO1 to CO3. Sample CO4 had the highest initial carbonate concentration, but this did not result in the highest carbonate content. Instead, sample CO2 and CO4 had similar amounts of incorporated CO32-, despite their different initial carbonate concentrations. This observation was attributed to a pH increase of the synthesis solution in response to the increasing amount of carbonate in solution. It is suggested that the carbonate content is related to synthesis pH and that a maximum amount of CO32- incorporation occurs around pH 10-10.5. This can be attributed to the speciation of carbonate, as CO32- becomes more abundant than HCO3- around a pH of 10.33, which could have inhibited the carbonate incorporation. Furthermore, the sample crystallinity decreases with increasing carbonate content. This is related to the increasing amount of structural defects that occurs in the apatite lattice as more carbonate is taken up in the structure. Carbonate often replaces hydroxide (A-type substitution) or phosphate (B-type substitution), but can also be incorporated in the external, less crystalline apatite layers (labile substitution). The spectroscopic data shows that B-type substitution is dominant in all samples, but relatively more A-type and labile carbonate is found in samples that were synthesised at higher pH. This subsequently resulted in a lower crystallinity for these samples. When exposed to the buffered solution with pH 6.5, a significant amount of phosphate was released during the first 10-20 minutes of the experiment. The sample without carbonate showed the highest dissolution rate during this phase, which is attributed to the high surface area of the platy minerals and the instability of octacalcium phosphate at pH values above 6.4. For the carbonated samples, CO3 experienced significantly higher dissolution rates than CO2 and CO4, while they had a similar crystallinity index. This can be attributed to the positive correlation between dissolution rate and carbonate content. Although crystallinity is likely to affect the dissolution process as well, it seems that the carbonate content is dominant in determining the mineral solubility. Therefore, this study concludes that bone minerals with a relatively high carbonate content will experience a significant increase in solubility. This is likely to speed-up the bone remodelling process, as the dissolution of the hydroxyapatite minerals occurs faster. For patients with X-linked hypophosphatemia, this will only contribute to the problem, as more carbonate will be incorporated to make up for the loss of phosphate during the faster bone remodelling process, which will subsequently increase the bone mineral solubility.