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    • StemRegenin 1 Apparently it is generally accepted that

    2018-10-29

    Apparently, it is generally accepted that the addition of DAG to the culture medium stimulates the osteogenic differentiation of stem StemRegenin 1 (Jaiswal et al., 1997; Bourne et al., 2004). However, most of the studies neglected to analyze the minerals formed and the process of mineralization. The aim of this study was to analyze the process of mineralization and to characterize the mineral formed using histological staining, scanning electron microscopy (SEM), energy-dispersive diffraction analysis (EDX), wavelength dispersive diffraction analysis (WDX), transmission electron microscopy (TEM) with selected area diffraction analysis (SAED), Raman spectroscopy and an apoptosis assay. Because of the abovementioned advantages of the USSCs compared to other stem cells and the results of our previous studies, we decided to use USSCs. In order to simulate the in vivo situation more closely, we used the 3D microsphere culture technique.
    Results
    Discussion The FESEM analysis of the spheres in this study verified a morphological development of the cells to cuboidal, osteoblast-like cells after 28days, especially in the +DAG group. According to different workgroups, the morphological changes towards an osteoblast-like appearance and the mineralization are regarded as indicators of osteoblastic differentiation (Bielby et al., 2004; Handschel et al., 2008; Jaiswal et al., 1997; Pittenger et al., 1999; Jager et al., 2004; Vats et al., 2005). Toluidine blue and alizarin red staining demonstrated that mineralization occurred in the spheres of both groups, with and without the supplementation of DAG (dexamethasone, ascorbate, β-glycerophosphate). Despite these similarities, several differences between the two groups were observed by analyzing the histological staining, for example regarding the time point of the beginning of mineralization, the quantity of minerals formed and the distribution the mineralization within the spheres. The distribution of the mineral substance was more homogenous in the −DAG group, and in this group the mineral material formed had a more microcrystalline structure compared to the +DAG group. Furthermore, in the +DAG group, smaller mineral globules merged to form to larger aggregations of the mineral substance and an association was observed between the mineral material and the collagen fibers of the ECM. Interestingly, no differences were detected between the groups regarding the quantity of mineralization after 21days of cultivation. All of these facts may be signs of an acceleration of mineralization due to the DAG in the spheres. A possible reason for the differences in the distribution of mineral formation may be a simple problem of diffusion. Only the outer cell layers of the spheres were in direct contact with the ambient medium and were therefore exposed to a higher concentration of DAG compared to the inner cells. Consequently, only the outer cells could become stimulated by DAG and mineralization started earlier and was more pronounced in these cells. In addition, previously published results of our work showed that cell migration out of USSC microspheres is reduced by prolonged osteogenic induction (DAG) over time (Langenbach et al., 2010). Following the fact that mineral material was present even in spheres of the −DAG group in this study there must be further factors that trigger the process of mineralization. It is well known that osteogenic differentiation occurs subsequently to proliferation and therefore high seeding densities of cells are suggested to support osteogenic differentiation. Wilson and colleagues demonstrated that higher seeding densities resulted in increased bone formation in vivo and that during cell proliferation osteogenic differentiation was restricted or inhibited (Wilson et al., 2002). Furthermore, high cell seeding densities led to a higher proportion of osteocalcin-positive tissue compared to low seeding densities (Holy et al., 2000). Additionally, as a consequence of high cell densities, both expression of the important osteogenic regulatory factor RUNX2 (runt related nuclear transcription factor 2) and transcription of osteonectin (secreted protein, acidic, cystein rich — SPARC) were up-regulated in vitro (Bitar et al., 2008). The presence of mineral material even in the −DAG group spheres of this study may be caused by high cell densities in the centers of these spheres which may lead to osteogenic differentiation of these cells. Another reason for the spontaneous differentiation may lie in the communication between the cells and the interaction between the cells and the extracellular matrix (Langenbach et al., 2010). Cells interact with the ECM via integrins, which span the plasma membrane. These integrins provide binding sites for ECM proteins like collagen type I and they signal bi-directionally across the plasma membrane, thereby influencing many important cellular processes, such as proliferation, migration and gene expression (Hervy et al., 2006; Schwartz and Assoian, 2001). The de novo synthesis and deposition of ECM proteins by mesenchymal stem cells were shown to change the chemical identity of substrate materials, as well as integrin expression and signal transduction, and they were also shown to influence osteogenesis (Kundu et al., 2009). The mitogen-activated protein kinase (MAPK) cascade plays a critical role in osteogenic differentiation by activating the RUNX2 transcription activator (Xiao et al., 2000; Franceschi and Xiao, 2003). Salasnyk and colleagues demonstrated that the adhesion to collagen type I and vitronectin is sufficient for inducing osteogenic differentiation by mesenchymal stem cells. In this process, upon ECM molecules, the focal adhesion kinase (FAK) pathway is activated, which in turn phosphorylates the extracellular signal-related kinase (EKR), which then regulates RUNX2 transcription. Our group was able to show the importance of different ECM proteins such as osteonectin and collagen type I in the osteogenic differentiation of USSCs (Naujoks et al., 2011). In addition, in another experiment by our workgroup, the expression of osteonectin, a matrix-associated protein influencing the synthesis of ECM proteins (Bradshaw et al., 2003), and the expression of collagen type I by DAG-stimulated USSCs on different biomaterials were investigated (Naujoks et al., 2011). We found an increased expression of osteonectin on collagen type I sponges compared to other biomaterials. The expression of these matrix proteins may prove the osteogenic differentiation of USSCs.