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 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

Calcium phosphate ion products.

<p>Calcium phosphate products in the different osteogenic induction media assuming 100% hydrolysis of βGP and 2 mM Ca²⁺ in the serum supplement (resulting in 2 mM Ca²⁺ in the basal medium).</p

FT-IR spectra.

<p>FT-IR spectra (450–1750 cm⁻¹) of samples cultured in different osteogenic induction or control media (n = 1) show the characteristic hydroxyapatite bands and the bands characteristic of the organic matrix of bone.</p

XRD patterns.

<p>XRD patterns of cells after osteogenic differentiation for 28 days supplemented with different sources of phosphate. Additionally, a fragment of human hip bone has been used as a reference sample of human bone. With second-derivation curve fitting analysis discrete reflections at 25.9 (002), 31.77 (211), 32.18 (112), 32.9 (300), 34.04 (202), and 39.79° (310) were detected for all samples supplemented with phosphate as well as for the bone reference sample. These reflections correspond to prominent peaks of hydroxyapatite <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065943#pone.0065943-Markovic1" target="_blank">[50]</a>.</p

Rate of collagen synthesis.

<p>CICP assay of the supernatant during osteogenic induction. Data are mean of days 14 and 21± SD for n = 4 donors.</p

mRNA expression levels of osteogenic marker genes.

<p>mRNA expression levels of osteocalcin, osteopontin, TNAP and IBSP after 28 days of osteogenic differentiation depending on the phosphate source. The relative mRNA expression (Δ Ct) is plotted against the phosphate supplementation for five different genes involved in the formation of bone (n = 4). Plotted as mean ± SD.</p

Free phosphate concentration.

<p>Concentration of free phosphate in the medium over the course of differentiation depending on the different phosphate sources shown for donors A and B respectively. Plotted as mean ± SD (small SD are not visible). A. Donor A (53-year old female). B. Donor B (47-year old male). Donors were chosen for relatively high difference in TNAP mRNA expression.</p

Calcium to phosphate ratios and ion concentration in the cell layer.

<p>A. Calcium to phosphate ratio. Calcium and phosphate ions were extracted from the cell layer of four different donors and quantified. Data are mean ± SD for n = 4 donors. B. Calcium and phosphate content of the cell layer after 28 days of osteogenic induction with different phosphate additives. Data are mean ± SD for n = 4 donors.</p

βGP hydrolysis and TNAP mRNA expression.

<p>A. Differences in phosphate hydrolysis over 3 days depending on the donor cells with (*) or without 14 days of pre-incubation in osteo-inductive medium containing 10 mM βGP with normal changes of medium. The free P_i concentration generated under cell-free conditions has been subtracted. Plotted is mean ± SD. B. Real-time PCR mRNA expression data of TNAP for donor B relative to donor A on days 7, 14, 21 and 28 of the osteogenic differentiation. C. Stability of βGP in the medium with cells and under cell-free conditions (no changes of medium) without pre-incubation of cells. Data plotted as mean ± SD (small SD not visible) D. Stability of βGP in the medium with cells and under cell-free conditions (no changes of medium) with 14 days of pre-incubation in osteo-inductive medium containing 10 mM βGP with normal changes of medium.</p

Matrix and mineral characteristics derived from FT-IR data.

<p>Representative data of one donor for collagen crosslinks, mineral maturity and mineral to matrix ratios dependent on different phosphate additives in the osteo-induction medium.</p