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

Abstract

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 β-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.

Prolonged Exposure to Hypoxic Milieu Improves the Osteogenic Potential of Adipose Derived Stem Cells

“…This finding was confirmed by molecular analysis, since the low oxygen tension promoted the expression of noncollagenous and collagenous proteins which are related to the MSC differentiation and mineralization [Granchi et al, ]. In addition, the up‐regulation of ALPL transcripts suggests that the mineralization process is facilitated by hypoxic conditions [Schäck et al, ].…”

Calcium phosphate nanoparticles are associated with inorganic phosphate-induced osteogenic differentiation of rat bone marrow stromal cells

“…Therefore, investigating the physicochemical properties of CaP nanoparticles and their possible interactions with BMSCs will likely broaden our understanding of high Pi-related bone impairment in vivo. In this study, the concentration of Pi was based on that used in the literature [5,12,13]. Our results indicated that CaP nanoparticles formed in culture media were associated with the process of high Pi mediated osteogenic differentiation BMSCs and the physicochemical properties of CaP nanoparticles played an important role in modulating the biological response of BMSCs.…”

Chondrocytes Promote Vascularization in Fracture Healing Through a FOXO1-Dependent Mechanism

“…Cells were cultured in 50% DMEM (Gibco, Gaithersburg, MD, USA) and 50% F12 (Gibco) with 5% FBS. Differentiation to chondrocytes was performed over 6 days with ascorbic acid (50 μg/mL) and increasing concentrations of NaH 2 PO 4 0.5mM, 1mM, and 2mM as described . Human microvascular endothelial cells (HMVECs) were purchased from Cell Systems (Kirkland, WA, USA) and cultured in EGM2‐MV medium (Lonza, Walkersville, MD, USA) with 5% FBS and 1% antibiotics in an incubator with 5% CO 2 at 37°C.…”