Biomimetic Mg- and Mg,CO3-substituted hydroxyapatites: synthesis characterization and in vitro behaviour

Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 +/- 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime.

The environment of CO3(2-) ions in the bone mineral of chickens of different ages and in bone fractions of different density have been investigated by resolution-enhanced Fourier Transform Infrared (FTIR) Spectroscopy. Three carbonate bands appear in the upsilon 2 CO3 domain at 878, 871, and 866 cm-1, which may be assigned to three different locations of the ion in the mineral: in monovalent anionic sites of the apatitic structure (878 cm-1), in trivalent anionic sites (871 cm-1), and in unstable location (866 cm-1) probably in perturbed regions of the crystals. The distribution of the carbonate ions among these locations was estimated by comparing the intensities of the corresponding FTIR spectral bands. The intensity ratio of the 878 and 871 cm-1 bands remains remarkably constant in whole bone as well as in the fractions obtained by density centrifugation. On the contrary, the intensity ratio of the 866 cm-1 to the 871 cm-1 band was found to vary directly and decreased with the age of the animal. In bone of the same age, the relative content of the unstable carbonate ion was found to be highest in the most abundant density centrifugation fraction. A resolution factor of the CO3(2-) band (CO3 RF) was calculated from the FTIR spectra which was shown to be very sensitive to the degree of crystallinity of the mineral. The crystallinity was found to improve rapidly with the age of the animal. The CO3 RF in the bone samples obtained by density centrifugation from bone of the same animal was found to be essentially constant.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel synthesis route has been developed to produce a high-purity mixed AB-type carbonate-substituted hydroxyapatite (CHA) with a carbonate content that is comparable to the type and level observed in bone mineral. This method involves the aqueous precipitation in the presence of carbonate ions in solution of a calcium phosphate apatite with a Ca/P molar ratio greater than the stoichiometric value of 1.67 for hydroxyapatite (HA). The resulting calcium-rich carbonate-apatite is sintered/heat-treated in a carbon dioxide atmosphere to produce a single-phase, crystalline carbonate-substituted hydroxyapatite. In contrast to previous methods for producing B- or AB-type carbonate-substituted hydroxyapatites, no sodium or ammonium ions, which would be present in the reaction mixture from the sodium or ammonium carbonates commonly used as a source of carbonate ions, were present in the final product. The chemical and phase compositions of the carbonate-substituted hydroxyapatite was characterized by X-ray fluorescence and X-ray diffraction, respectively, and the level and nature of the carbonate substitution were studied using C-H-N analysis and Fourier transform infrared spectroscopy, respectively. The carbonate substitution improves the densification of hydroxyapatite and reduces the sintering temperature required to achieve near-full density by approximately 200 degrees C compared to stoichiometric HA. Initial studies have shown that these carbonate-substituted hydroxyapatites have improved mechanical and biologic properties compared to stoichiometric hydroxyapatite. Copyright 2001 John Wiley & Sons, Inc. J Biomed Mater Res 59: 697-708, 2002