The Phosphate Source Influences Gene Expression and Quality of Mineralization during In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells

For in vitro differentiation of bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells into osteoblasts by 2-dimensional cell culture a variety of protocols have been used and evaluated in the past. Especially the external phosphate source used to induce mineralization varies considerably both in respect to chemical composition and concentration. In light of the recent findings that inorganic phosphate directs gene expression of genes crucial for bone development, the need for a standardized phosphate source in in vitro differentiation becomes apparent. We show that chemical composition (inorganic versus organic phosphate origin) and concentration of phosphate supplementation exert a severe impact on the results of gene expression for the genes commonly used as markers for osteoblast formation as well as on the composition of the mineral formed. Specifically, the intensity of gene expression does not necessarily correlate with a high quality mineralized matrix. Our study demonstrates advantages of using inorganic phosphate instead of beta-glycerophosphate and propose colorimetric quantification methods for calcium and phosphate ions as cost-and time-effective alternatives to X-ray diffraction and Fourier-transform infrared spectroscopy for determination of the calcium phosphate ratio and concentration of mineral matrix formed under in vitro-conditions. We critically discuss the different assays used to assess in vitro bone formation in respect to specificity and provide a detailed in vitro protocol that could help to avoid contradictory results due to variances in experimental design.

Geologie und Hydrogeologie im Raum Bad Pyrmont unter besonderer Berücksichtigung des Quellensystems

The hydrodynamics and hydrochemistry of salt and fresh water from solid rock aquifer systems in the Pyrmont area are described and interpreted on the basis of recent investigations including geoelectrics, isotope hydrology, soil air analysis. Theories on the source of the springs in this area are developed, which explain the different compositions of the springs and make it possible to protect them. Data from new and re-interpretated drill holes, borehole logs and outcrops suggest a revision of the geological structure of the Pyrmont dome. Bad Pyrmont is situated on a wide dome of Triassic rocks in the southern part of the Lower Saxony uplands. Inversion of the relief has caused the development of an erosional basin surrounded by prominent ridges. Deep faults developed at the crest of the dome as this part of the structure was subjected to the strongest tectonic stress. Subrosion of the Zechstein salts in the western part of the dome has caused the main salt bed to wedge out below the western part of the dome along a N-S striking structure; this structure is refered to as the „Salzhang“ (salt slope). West of the „Salzhang“, where subrosion has removed the salt bed that prevents gas rising from below, carbon dioxide of deep volcanic origin can now rise to the surface. Hydraulic cross sections illustrate the presence of extensive and deep-seated groundwater flow within the entire Pyrmont dome. While groundwater flow is directed vertically downwards in the ridges surrounding the dome, centripetal horizontal flow predominates the intermediate area. In the central part of the dome, groundwater rises to join the River Emmer, which is the main receiving water course in the central part of the eroded basin. The depth of the saltwater/freshwater interface is determinated by the weight of the superimposed freshwater body. Hydrochemical cross sections show the shape and position of the interface and document a certain degree of hydrochemical zonation of the gently mineralized fresh water. Genetic relationships between the two main water types and the hydrochemical zones of the freshwater body are discussed. The knowledge of the hydrogeological relationship in the Bad Pyrmont aquifer systems permits a spatially narrow coexistence of wells withdrawing groundwater for different purposes (medicinal, mineral, drinking and industrial water).