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  • In contrast to the above the

    2018-11-12

    In cp-690550 to the above, the loose cell network in the +DAG group that was observed via histological staining may be a sign of reduced cell vitality. It is well known that cell death often leads to mineralization that is not combined with the formation of bone (Kirsch, 2006). Inorganic phosphate and glucocorticoids were thought to trigger apoptosis (Meleti et al., 2000; Boyan et al., 2000; Adams et al., 2001; Giachelli, 2005; Weinstein et al., 1998). However, the apoptosis assay performed in the present study showed no signs of apoptosis over the whole period of the experiment, so that a correlation between mineralization and apoptosis in this case was unlikely. According to LeGeros et al., a mineral can be characterized by its lattice structure and chemical composition (LeGeros, 1991). The EDX analysis showed that the mineral material formed mainly consisted of the elements Ca, P and O, providing evidence that the formed mineral was a Ca–P mineral. Furthermore, the spectra showed qualitative similarities to HA spectra (Cheng and Pritzker, 1983; Cheng et al., 1983). But quantitatively, the spectra showed significant differences to the standardized HA and between both groups. There are a lot of different calcium phosphates and each has its specific Ca/P ratio, as proven by other workgroups (LeGeros, 2001; Cheng et al., 1983). Hence, the different Ca/P ratios compared to HA may indicate the presence of different calcium phosphates in the formed mineral. Due to the Ca/P ratios observed (1.1 to 1.4 +DAG/1.3 to 1.6 –DAG), the presence of amorphous Ca–P (ACP), octacalcium phosphate (OCP) and calcium-deficient hydroxyl apatite (CDHA) could be assumed. Different workgroups supposed that Ca–P minerals can follow precursor minerals such as OCP, brushite and monetit (LeGeros, 2001; Lagier and Baud, 2003). The fact that in the present study there were different Ca/P ratios not only between the groups but also in the chronological order of the minerals may be an indicator of the presence of different minerals as well as different stages in the development of these minerals. The fact that there was no continuous increase in the ratio over time in both groups seems to refute this hypothesis. In particular, the +DAG group showed a decrease in the Ca/P ratio after 14days. A possible source for other detected elements (Na, Cl, S, Mg) could be the organic matrix of the cells, the embedding material or the medium. The occurrence of different stages of minerals, intermediate stages of minerals or substitution processes may account for the quantitative differences found (Wiesmann et al., 1993). It is well known that biominerals often differ from the stoichiometric formula because ions in the crystallite lattice may be substituted by others (LeGeros, 2001). In the present study, the EDX analysis in both groups showed a significant peak in Mg; thus, Mg was potentially substituted for Ca, leading to the lower Ca/P ratios over the time period of the experiment. This was confirmed by WDX analysis, indicating that Mg was part of the mineral formed in both groups. The portion of Mg in the mineral increased over time whereas the portion of Ca decreased in the WDX analysis of the +DAG group. In the −DAG group, a minor increase in Mg and a lower Mg/Ca ratio was detected compared to the +DAG group. It seems that DAG amplified the substitution of Mg for Ca during the process of mineralization, although in both groups a substitution of Mg for Ca took place, as already reported by others (Bigi et al., 1996; Wiesmann et al., 1997). A possible source of Mg may be the cultivation medium. The question arises as to why there were differences in the Mg integration between the +DAG and −DAG groups. There was probably a quantitative lack of Ca caused by the accelerated mineralization in the +DAG group, leading to a substitution by Mg. However, the possibility that changes in the elements could have been caused by the preparation of the samples (Wiesmann et al., 1993; Plate et al., 1992) or by mechanical–physical ascendancies, which may lead to measuring errors, cannot be excluded. In particular, the fact that the analyzed minerals were very small suggests that parts of the surrounding tissue were analyzed as well as the mineral, leading to higher P concentrations and therefore lower Ca/P ratios.