Formation of hydroxyapatite precursors at 37 °C in urea- and enzyme urease-containing body fluids

Abstract

Calcium hydroxyapatite (HA: Ca10(PO4)6(OH)2) bioceramics, owing to their similarity with the human bone and dentin minerals, attract significant interest for orthopaedic and dental applications. Biological apatites, on the other hand, were observed [1] to be carbonatesubstituted and calcium-deficient. HA powders for bioceramic applications have usually been chemically synthesized [2–9] via aqueous solutions. It is known [6] that HA is the least soluble and the most stable compound of calcium phosphate phases in aqueous solutions at pH values higher than 4.2. HA powders synthesized in highly alkaline (pH > 0) media [2–9] were typically recognized by their relatively high thermal stability and phase purity even after high temperature (1100– 1300 ◦C) sintering. However, formation of HA powders in neutral and/or slightly acidic aqueous media is known to be a more complicated and difficult task [7, 10]. Simulated body fluids (SBF), with ion concentrations resembling those of human blood plasma, were first used by Kokubo et al. [11, 12], to prove the similarity between in vitro and in vivo behavior of certain glass-ceramic compositions. In these studies, the glassceramic samples were soaked in SBF solutions, and their surfaces were observed to be coated with a poorly crystallized calcium deficient apatite, which was similar to bone apatite [12]. Biomimetic HA powders were previously synthesized by Tas [13] from calcium nitrate tetrahydrate and di-ammonium hydrogen phosphate salts dissolved in synthetic body fluid solutions (instead of distilled water), under the physiological conditions of 37 ◦C and pH 7.4. The maintenance of pH value at or slightly above 7.4 during precipitation required [13] a controlled but continuous addition of a base, such as diluted solutions of NH4OH. The addition of urea (H2NCONH2) into the body fluids just before the HA precipitation experiments was found [14] to cause a significant reduction in the amount of the base which needed to be continuously added to keep the pH at 7.4. The procedure of addition of enzyme urease (which is known [15] to accelerate the decomposition rate [16] of urea, especially at low temperatures) into the ureacontaining body fluids, and the use of these in the synthesis of biomimetic HA precursors at 37 ◦C and pH value of 7.4 are hereby presented. Body fluids were prepared by dissolving reagent-grade chemicals of NaCl, NaHCO3, KCl,