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.
Highlights
Bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells (MSCs) have long been recognized as a powerful source for the study of osteoblast behaviour and the regeneration of bone tissue
In order to induce osteoblast differentiation in mesenchymal stem cells, the growth medium needs to contain a certain level of free inorganic phosphate that allows for the mineralization to occur
The X-ray diffraction (XRD) patterns of samples obtained with mM bGP and 10 mM NaxH3-xPO4 show a weak broad signal between 30 and 32u 2H which hints at the presence of small amounts of amorphous calcium phosphate that may have formed by spontaneous precipitation due to the high concentration of phosphate ions
Summary
Bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells (MSCs) have long been recognized as a powerful source for the study of osteoblast behaviour and the regeneration of bone tissue. During the in vitro differentiation into mature osteoblasts the downregulation of proliferation, secretion of an organic extracellular matrix and mineralization of this matrix by deposition of hydroxyapatite pose important developmental steps. These are accompanied by a stage-specific expression of bone-related genes [1]. It is worth to note that many different sources of FBS are used for the cultivation of MSCs, and that the source, but even the batch of FBS can influence the cells, resulting in an incalculable variability in experimental design [2,3]. A considerable diversity has developed in respect to protocols used for two-dimensional in vitro osteogenesis and in respect to the methods and methodology of analysis making it difficult to transfer and compare results between different studies
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