Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

Isolation and cultivation of MSCs

Human BM-MSCs were isolated from n=6 healthy Caucasian donors (three men, three women, mean age 31.4 years) after obtaining their informed consent (approval no. EK263122004). BM-MSCs were isolated from the mononuclear cell fraction and expanded as previously described. ³² Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, low glucose; Life Technologies GmbH, Darmstadt, Germany) supplemented with 10% fetal calf serum (Sigma-Aldrich, Hamburg, Germany) and 1% penicillin/streptomycin (Life Technologies GmbH) with or without osteogenic supplements (i.e., 100 nM dexamethasone, 10 mM beta-glycerophosphate, and 50 μM ascorbic acid-2-phosphate; Sigma-Aldrich) and maintained in a humidified atmosphere at 37°C and 5% CO₂. No osteogenic supplements were used for histology, morphology, and all gene expression experiments. Only for biochemical analysis of ALP activity, osteogenic supplements were used to compare the additional effects of supplements and 3D culturing. The study was performed with second-passage BM-MSCs of all donors. After reaching 80–90% confluence, cells were seeded for 2D cultivation at a density of 4.8×10⁴ cells per well in tissue culture plastic six-well plates (Greiner, Frickenhausen, Germany). Peracetic acid-treated human cancellous bone (HCB) cubes with an approximate edge length of 5 mm (Fig. 1B; German Institute for Cell and Tissue Replacement, Berlin, Germany) with a conferred admission for medicinal drugs were used as 3D matrices. ³³ Fifty microliters of cell suspensions with 1×10⁶ BM-MSCs from each donor were seeded per HCB in a drop-wise manner. After a 2 h adherence period in the incubator, the cubes were covered carefully with 6 mL of cell culture medium. Cells were maintained at 37°C in a saturated humidity atmosphere with 5% CO₂. The next day, seeded HCBs were transferred into new six-well plates. Triplicates were used for each 2D and 3D sample. The cells were cultured for 14 days, changing the medium twice a week. Cell morphology and adherence were monitored by light microscopy.

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FIG. 1.

Two-dimensionally (2D) cultured bone marrow-derived mesenchymal stromal cells (BM-MSCs) of one donor after 14 days in culture (A). Macrograph of a human cancellous bone (HCB) cube with an approximate edge length of 5 mm (B). Light microscopic image of 3D cultivated BM-MSCs on HCB, 100× (C). Fluorescence microscopic picture of 2D cultivated BM-MSCs depicting nuclei (Hoechst 33342, blue), cytoskeleton (Alexa 555 phalloidin, red), and the extracellular matrix protein fibronectin (anti-mouse fibronectin-AK and Alexa Fluor^® 488, green), 100× (D). Fluorescence stereoscopic image of HCB (blue) and vital BM-MSCs stained with calcein (green) (E). Histological section of HCB with adherent BM-MSCs after Giemsa stain, 200× (F). Asterisks mark HCB, and arrows highlight BM-MSCs. Color images available online at www.liebertpub.com/tec

RNA isolation and cDNA synthesis

Total RNA was isolated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) after 14 days of cultivation. After rinsing with phosphate-buffered saline (PBS), 2D cultured BM-MSCs were harvested from cell culture plates by using a cell scraper. Cells were centrifuged for 5 min at 2000 g, and the PBS was removed. Three-dimensional samples were rinsed with PBS, and excess PBS was aspirated with a filter paper. All samples were frozen immediately at −80°C until later use. For the isolation of total RNA, BM-MSCs from 3D samples were lysed during a centrifugation step at 16,100 g for 1 min at room temperature (RT) in lysis buffer. After centrifugation, the lysate was transferred into new reaction tubes and further steps were performed according to the manufacturer's instructions. During the isolation, an on-column DNase digestion with RNase-Free DNase Set (Qiagen) was performed. RNA concentration and quality was determined at 260 nm and 260/280 nm using a NanoDrop 2000c spectrophotometer (PEQLAB Biotechnologie GmbH, Erlangen, Germany). The 260/280 nm ratio values were between 1.9 and 2.1. cDNA synthesis from 200 ng RNA for each sample was performed using Superscript II Reverse Transcriptase and with oligo-dT (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. An equal volume of cDNA was pooled from each BM-MSC donor and used as templates in qPCR.

Quantitative real-time polymerase chain reaction

Human Endogenous Control TaqMan^® assays with 32 FAM labeled probes (Table 1) spotted in triplicate in a 96-well format were used for quantitative gene expression analysis of RGs (Life Technologies GmbH; Applied Biosystems, Darmstadt, Germany). No osteogenic supplements were used for all gene expression experiments. qPCRs were performed with TaqMan Fast Universal PCR Master Mix (2×) according to the manufacturer's instructions using a 7500 Real-Time PCR Fast System (Applied Biosystems). A volume of 0.96 μL template cDNA corresponding to 19.2 ng of RNA was used for each well of the qPCR array. All reactions were carried out in a total volume of 10 μL with a thermal profile of an initial cycle for 20 s at 95°C for enzyme activation followed by 40 cycles with 3 s at 95°C denaturation, 30 s at 60°C for annealing and elongation, and terminated by a holding stage at 4°C. Technical triplicates were run for all samples. For the no template control (NTC), the three wells with the probe of 18 s RNA was chosen as this assay does not provide extra wells for the NTC.

Expression of the osteogenic marker genes alkaline phosphatase (ALP), liver/bone/kidney (ALPL, Hs01029144_m1), bone gamma-carboxyglutamate (gla) protein (BGLAP, Hs00609452_g1), integrin-binding sialoprotein (IBSP, Hs00173720_m1), runt-related transcription factor 2 (RUNX-2, Hs00231692_m1), and secreted phosphoprotein 1 (SPP1, Hs00959010_m1) were analyzed as target genes using the same cDNA pool, protocols, and instrument as mentioned earlier with TaqMan Gene Expression Assays (Life Technologies GmbH; Applied Biosystems). Data analysis was performed using the 7500 software v2.0.5 (Applied Biosystems). Gene expression of a total of n=31 RGs was evaluated on the basis of cycle threshold (Ct) values from BM-MSCs cultivated for 14 days under 2D versus 3D culture conditions. Results were exported to Microsoft Excel for analysis and presented as mean±SD. SD of ΔCt values were calculated using an equation with SD1 and SD2 being the SD, and n₁ and n₂ being the sample sizes of 3D (1) and 2D (2), respectively. \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}SD_ { pooled } = \sqrt { \frac { SD_1^2 } { n_1 } + \frac { SD_2^2 } { n_2 } } \end{align*} \end{document}

Gene expression of osteogenic target genes was calculated as a relative fold difference using the delta-delta Ct method, where the 3D cultivated BM-MSCs represent the sample and the 2D cultivated BM-MSCs represent the control. ³⁴ \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}\rm\hbox{Fold difference} = 2^{ - [ \Delta { \rm Ct \ sample}\ ( 3{ \rm D} ) - \Delta {Ct\ control}\ ( 2{ \rm D} ) ] }\end{align*} \end{document}

The 3D and 2D gene expression levels were paired by original wells. For each experimental unit, three wells (triplicate) were generated. The three ratios were paired for the calculation from those three wells (delta Ct 3D_1 and delta Ct 2D_1; delta Ct3D_2 and delta Ct 2D_2; and delta Ct 3D_3 and delta Ct 2D_3) and then, the average was obtained from the three ratios. A fold difference of one equates to no regulation. Downregulation of gene expression corresponds to values below one. Values above one mean an upregulation of gene expression.

Determination of cell-specific ALP activity

Osteogenic differentiation was validated by measurement of cell-specific ALP activity. The activity of the enzyme was measured in cell extracts of 2D and 3D samples using p-nitrophenylphosphate (pNpp) and quantified by colorimetric measurement (SpectraFluorPlus, Tecan, Switzerland). Cells were washed with PBS and lysed in 500 μL lysis buffer (1.5 M Tris–HCl, pH 10, containing 1 mM ZnCl₂, 1 mM MgCl₂, and 1% Triton X-100) at 4°C for 20 min. The 3D samples were additionally sonicated for 1 min for better lysis results. After incubation, the lysates of 2D samples were transferred from six-well plates into new reaction tubes and centrifuged at 20,000 g and 4°C for 30 min. The lysates of 3D samples were transferred after centrifugation into new reaction tubes to separate them from HCB cubes. A fifty-microliter lysate was incubated with 100 μL 3.6 mM pNpp in 100 mM diethanolamine, pH 9.8, containing 0.1% Triton X-100 at 37°C for 30 min. The reaction was stopped with 100 μL 100 mM NaOH, and the amount of p-nitrophenol (pNp) was determined spectrophotometrically at 405 nm (SpectraFluorPlus). The final cell-specific ALP activity was calculated as μmol pNp/30 min per 1×10⁶ cells. Triplicates were used for each experimental unit. Total DNA quantification was assessed by Pico Green Assay (Invitrogen). The same lysate as for the determination of cell-specific ALP level was used for DNA quantification. One hundred fifty microliters of Pico Green solution were incubated with 50 μL of cell lysate in a black microtiter plate. After 5 min of incubation in the dark, the fluorescence was measured at an excitation of 490 nm and an emission of 520 nm with a plate reader (SpectraFluorPlus). Relative fluorescence units were correlated with the cell number using a calibration line. Triplicates were used for each experimental unit.

Immunofluorescence staining

To represent cell morphology, 2D cultured BM-MSCs were characterized by immunofluorescence staining of cytoskeleton by Alexa Fluor^® 555 Phalloidin selectively labels F-actin (Life Technologies GmbH, Molecular Probes; Darmstadt, Germany) and extracellular matrix protein fibronectin with anti-mouse fibronectin-AK (Sigma-Aldrich, Munich, Germany) and Alexa Fluor 488. After 7 days of cultivation, BM-MSCs were washed with PBS and fixed with 4% paraformaldehyde for 10 min at RT. Thereafter, cells were permeabilized for 15 min with 0.4% Triton at RT and then incubated in 3% goat serum in PBS for 1 h to block unspecific binding sites. Then, the incubation was carried out with the antibody anti-mouse fibronectin (dilution 1:1000) overnight at 4°C. The next day, the cells were washed with PBS and incubated with Alexa Fluor 488 for 60 min at RT on an orbital shaker in the dark. Staining of nuclei was performed with Hoechst 33342 (7.5 mg/mL final concentration) for 5 min in the dark (Life Technologies GmbH, Molecular Probes). Images were taken with a computer-driven digital camera on an inverted microscope Leica DMI4000B (Leica Microsystems, Wetzlar, Germany).

Determination of cell viability

For determination of cell viability, the LIVE/DEAD^® Viability/Cytotoxicity assay was used (Invitrogen). Three-dimensional samples were washed with PBS, and BM-MSCs on HCB were stained with 4 μM calcein-AM and 8 μM ethidium homodimer-1 for 45 min at RT in the dark. The cell-permeable calcein leads to green fluorescence due to the presence of intracellular esterase activity in living cells. Ethidium homodimer-1 enters cells with damaged membranes and binds to nucleic acids, resulting in a 40-fold enhancement of red fluorescence on binding to nucleic acids of dead cells. After staining, the dye was removed and the sample was carefully rinsed with PBS. Viability was visualized via fluorescence microscopy analysis using a stereoscope Zeiss Discovery V.20 (Zeiss, Jena, Germany) with Ex/Em of 494/517 nm and 528/617 nm.

Histology

To demonstrate cell adherence of BM-MSCs on the HCB cubes, samples were analyzed histologically. Fourteen days after seeding, specimens were washed with PBS and fixed in 70% ethanol at RT. After dehydration in increasing ethanol concentrations (2 h in 70%, 80%, 96%, and 100%) and xylol (1 h) at RT in the Leica tissue processor (Leica Microsystems), the samples were embedded in Technovit 9100N (Heraeus-Kulzer, Friedrichsdorf, Germany). A low-temperature (–5°C) polymerization was performed in plastic vessels (Heraeus-Kulzer, Hanau, Germany). Slices of 10 μm thickness were obtained from the cured specimen blocks using an RM2155 microtome (Leica, Nussloch, Germany). The mounted sections were deacrylated twice for 20 min with 2-methoxyethylacetate (Merck Biomaterials, Darmstadt, Germany) followed by incubation for 20 min in xylol twice, 5 min in acetone, 10 min in 80% ethanol, and rehydration with distilled water.

Giemsa staining was used to show cell morphology and adherence. Nuclei appear dark purple and cytoplasm appear light pink. Slices were stained at 60°C for 30 min. The stain was differentiated with acetic acid for 30 s, followed by a short incubation in 96% and 6 min in 100% ethanol. Dehydration was finished with xylol, and slices were mounted in DePex mounting medium.

Measurement of RG stability

The RG stability was calculated using two well-established algorithms, geNorm and NormFinder software packages. geNorm enables a ranking of suitable RGs according to their stability measured by the stability value M. ²⁴ The algorithm used is the “average pairwise variation” of a control gene with all other control genes in the study. The lowest M-value corresponds to the gene with the least expression variation and represents the most stable internal control in the experiment.

NormFinder applies a robust mathematical model that attempts to find the optimal RG out of a group of candidate genes. ²¹ The model can analyze expression data obtained through any quantitative method; for example, real-time RT-PCR and microarray-based expression analysis. The analysis resulted in variability values of the most stable RG and in an optimum pair of RGs. The resulting pair might have compensating expression, so that one gene, for example, is slightly overexpressed in one group, but the other gene is correspondingly lower expressed in the same group. So, the optimum pair may not include the optimum single gene. In contrast to geNorm, NormFinder takes information of groupings of samples into account, such as untreated and treated or sick and healthy.

Ct-values of all samples were entered in the respective program, and the stability values were calculated for each RG. The analysis was performed twice, first for Ct-values of 2D and 3D samples and second for 3D samples alone.

As a third method, the coefficient of variation (CV) was calculated for each gene. The CV equals the standard deviation divided by the mean Ct-value and is expressed as a percentage. The CV is a statistical tool for comparing the degree of variation between genes, even if the mean values drastically differ from each other. ³⁵

Statistics

Statistical data analysis was calculated using GraphPad Prism 5.0 software (La Jolla, CA). Gene expression data were analyzed as follows. Shapiro–Wilk normality test was used to test whether the values come from a Gaussian distribution. If the values were normally distributed, unpaired t-test with Welch's correction was used. Nonparametric Mann–Whitney test was applied when values were not normally distributed. Data are presented as mean±standard deviation (SD).

Biochemical data were analyzed with two-way ANOVA with Bonferroni post-tests. Data are presented as mean±standard error of the mean (SEM). Statistical significance of all data was considered if p<0.05.

Cell morphology, adherence, and viability of 2D and 3D cultured BM-MSCs

Cell morphology, adherence, and viability of BM-MSCs were analyzed after 14 days under 2D or 3D cultivation conditions without osteogenic supplements. Two-dimensional cultured BM-MSCs of all six donors showed a typical spindle-shaped cell morphology (Fig. 1A, D, example of one donor). BM-MSCs were able to adhere and proliferate on the HCB (Fig. 1C, E, F) over a period of 14 days. After 14 days of cultivation, a dense vital cellular network was formed (Fig. 1E), which partially closed the pores of the HCBs (Fig. 1F). BM-MSCs were able to assume the organization of a 3D structure on the HCBs.

Expression levels of RGs from 2D and 3D cultivated BM-MSCs

The expression levels of 31 known and potential RGs from 2D and 3D cultivated BM-MSCs without osteogenic supplements were analyzed and represented in Figure 2. Table 2 summarizes the mean Ct-values with their standard deviations of all RGs from both cultivation methods, including the ΔCt-values. The RG with the highest expression level of 2D and 3D cultivated BM-MSCs was the gene for mitochondrially encoded ATP synthase 6 (MT-ATP6) with a mean Ct-value of 18.58±0.22 and 18.85±0.18, respectively. The lowest gene expression was measured for the RG tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide and (YWHAZ) with a mean Ct-value of 29.67±0.15 and 29.61±0.15 for BM-MSCs in 2D and 3D culture, respectively. Notably, the RGs MT-ATP6, GAPDH, phosphoglycerate kinase 1 (PGK1), growth arrest and DNA-damage-inducible, alpha (GADD45A), TFRC, proteasome (prosome, macropain) 26S subunit, ATPase, 4 (PSMC4), and E74-like factor 1 (ets domain transcription factor) (ELF1) showed significant differences in gene expression of 2D and 3D cultured BM-MSCs (Fig. 2). The largest gene expression difference between both cultivation methods was detected for GADD45A with a delta Ct-value of 0.71 (2D: 24.37±0.21; 3D: 25.08±0.23) followed by GAPDH with 0.5 (2D: 19.66±0.4; 3D: 19.16±0.16). The RG candidate IPO8 showed the lowest gene expression difference between 2D and 3D cultivated BM-MSCs with a delta Ct-value of only 0.0097. Eukaryotic translation initiation factor 2B, subunit 1 alpha, 26 kDa (EIF2B1), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) had the second and third lowest delta Ct-values of 0.013 and 0.022, respectively. The gene ribosomal protein L37a (RPL37A) was the RG with the highest standard deviation, and thus with the greatest variation of the mean Ct-values of 2D and 3D cultivated BM-MSCs with 18.66±0.95 and 19.12±0.41, respectively.

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FIG. 2.

Cycle threshold (Ct) values of n=31 reference genes in two-dimensionally and three-dimensionally cultivated bone marrow-derived mesenchymal stromal cells (mean±standard deviation). Significance with *p<0.05, **p<0.01, and ***p<0.001.

Effect of 3D cultivation of BM-MSCs on the stability of RG expression

The expression stability of the n=31 RG in BM-MSCs was measured with geNorm and NormFinder after 14 days of 2D and 3D cultivation (Fig. 3). The stability values were calculated separately for the 3D cultivation data and for combined gene expression data of 2D and 3D cultivated BM-MSCs to provide all data depending on which experimental design will be chosen from the research community. The stability values of geNorm, NormFinder and the calculation of the CV after 3D cultivation are ranked and summarized in Table 2.

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FIG. 3.

Gene expression stability values of putative reference genes. Calculation of reference gene stability in three-dimensionally (top) and two- and three-dimensionally (button) cultivated bone marrow-derived mesenchymal stromal cells using the geNorm (A, B) and NormFinder (C, D). Low stability values mean high gene expression stability.

Using the algorithms of geNorm and NormFinder, we identified TATA box binding protein (TBP), transferrin receptor (p90, CD71) (TFRC), and HPRT1 as the three most stable RGs in 3D-cultivated BM-MSCs. HPRT1 was the most stable RG using CV calculation method followed by CASC3 and CDNK1 with CV of 0.28%, 0.35%, and 0.37%, respectively (Table 3). Comparing the three methods of stability calculation, the three RGs with the highest stability, TBP, TFRC, and HPRT1, are within the first eight ranks. The RGs RPL37A, ACTB, and ABL1 were the least stably expressed genes in all three calculation methods. The order of stability ranks within the 31 RGs is almost identical, on comparing geNorm and NormFinder analysis (Table 3). There is little difference with a maximum shift of two positions. The calculation of the CV showed more variations in the RG order compared with geNorm and NormFinder analysis except for the last five genes.

When the stability of RG expression of 2D and 3D cultured BM-MSCs was analyzed in combination, geNorm detected HPRT1, HMBS, and peptidylprolyl isomerase A (cyclophilin A) (PPIA) as the three most stably expressed RGs (Fig. 3). The NormFinder analysis revealed HPRT1, PPIA, and mitochondrial ribosomal protein L19 (MRPL19) as the three most stably expressed RGs when analyzing both Ct-values of 2D and 3D cultures. Surprisingly, the two most widely used RGs GAPDH and ACTB were among the three least stably expressed genes in the geNorm analysis. Again, the RG RPL37A had the lowest gene expression stability when analyzed with geNorm and NormFinder. GADD45A and GAPDH were the second and third least stable RGs in the NormFinder analysis.

Effect of RG expression stability on the expression of osteogenic target genes

Gene expression levels of the osteogenic target genes RUNX-2, ALPL, SPP1, IBSB, and BGLAP were measured after 14 days in 2D and 3D cultures without osteogenic supplements. The gene expression levels were then calculated with each of the 31 RGs as an internal control, with a combination of the three most stable RGs after analysis with geNorm and NormFinder and with a combination of all 31 RGs (Fig. 4). The fold difference was calculated with the 2D cultured BM-MSCs as a control and the 3D cultivated BM-MSCs representing the sample.

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FIG. 4.

Gene expression levels of osteogenic marker genes in three-dimensionally cultivated bone marrow-derived mesenchymal stromal cells after 14 days of cultivation. Relative fold differences of runt-related transcription factor 2 (RUNX-2), alkaline phosphatase, liver/bone/kidney (ALPL), secreted phosphoprotein 1 (SPP1), integrin-binding sialoprotein (IBSP), and bone gamma-carboxyglutamate (gla) protein (BGLAP) were calculated using the ΔΔCt method with two-dimensionally cultured MSCs as a control. Normalization was performed with 31 single reference genes (RGs), the combination of the three most stable RGs from the geNorm and NormFinder analysis, and the mean of all 31 RGs (bars are represented in dark gray). Error bars represent the standard deviation of the mean from three individual experiments in triplicate. Significance was calculated with *p<0.05, **p<0.01, and ***p<0.001.

The osteogenic target genes SPP1 and IBSP were generally upregulated under 3D conditions with whatever normalization combination was chosen. It should be noted that the upregulation of SPP1 and IBSP was due only to the 3D cultivation; no osteogenic supplements were used in the culture medium. The fold difference values for SPP1 ranged from 4.7±1.37 to 10.71±2.4. If normalization with all 31 RGs was performed, the fold difference was 6.48±1.04 and for the three most stable RGs of the geNorm and NormFinder normalization, the fold difference was 6.4±1.03 and 6.44±1.03. The results show a clear and significant underestimation of the SPP1 expression in the normalization with GAPDH (fold change of 4.7±1.37) and a strong and highly significant overestimation in the normalization with GADD45A (fold change of 10.71±2.4). The fold changes in the normalization with RPL37A, TFRC, and MT-ATP6 were also significantly higher than in the normalization with all RGs or with the three most stable RGs of the geNorm and NormFinder analysis.

Depending on which RG was normalized, the fold changes of IBSP ranged from 3.17±0.78 to 7.37±1.91 after 3D cultivation. The normalization with the three most stable RGs of the geNorm and NormFinder analysis and all RGs resulted in a fold change of 4.47±1.04, 4.42±1.04, and 4.44±1.05, respectively. Again in the normalization with GAPDH, a significant underestimation of the fold change was found with 3.17±0.78 compared with the normalization with all RGs or with the three most stable ones of geNorm and NormFinder. A significant overvaluation in gene expression was found when normalization was performed with GADD45A (7.37±1.91) and TFRC (5.75±0.99) compared with the normalization with all RGs or with the three most stable ones of geNorm and NormFinder.

The osteogenic target genes RUNX-2 and BGLAP (Fig. 4) were not regulated by 3D cultivation compared with 2D cultivated BM-MSCs. Fold differences of 1.01±0.05, 1.00±0.05, and 1.01±0.06 were measured for RUNX-2 when normalized to all RG, geNorm, or NormFinder, respectively. A significant (p<0.05) underestimation of RUNX-2 expression was detected when normalization with GAPDH (0.74±0.21), ELF1 (0.85±0.12), and PSMC4 (0.91±0.1) was chosen compared with the normalization with all RGs or with the three most stable ones of geNorm and NormFinder. A highly significant (p<0.001) overestimation of the fold difference was measured when normalized with GADD45A (1.68±0.32), TFRC (1.32±0.17), and MT-ATP6 (1.24±0.19) compared with fold differences of the normalization with all RGs or with the three most stable ones of geNorm and NormFinder. When RPL37A (1.44±0.38) was taken for normalization, a significant difference of the fold change was measured compared with the normalization with all RGs (with p<0.01) or with the three most stable ones of geNorm and NormFinder (with p<0.05). A significant difference (p<0.05) of RUNX-2 expression was measured when normalization was performed with TBP and B2M but only compared with the normalization with the three most stable RG of the geNorm analysis.

Gene expression of BGLAP was not regulated by 3D cultivation as seen by a fold difference of 1.05±0.06, 1.04±0.06, and 1.05±0.06 when normalized to all RGs, geNorm, or NormFinder, respectively (Fig. 4). A significant underestimation of the fold difference was measured for the normalization with GAPDH (0.77±0.2) and CASC3 (0.91±0.15) compared with the normalization with all RGs or with the three most stable RGs of geNorm and NormFinder. When normalized to ELF1 (0.89±0.2) and PSMC4 (0.95±0.12), a significant underestimation of the fold difference was detected compared with the normalization with the three most stable RGs of NormFinder and all RGs. A highly significant (p<0.001) overestimation of the fold difference was measured when normalized with GADD45A (1.75±0.32), TFRC (1.38±0.17), and MT-ATP6 (1.29±0.39) compared with fold differences of the normalization with all RGs or with the three most stable ones of geNorm and NormFinder. The normalization to MT-ATP6 (1.29±0.39, p<0.01) and RPL37A (1.51±0.45, p<0.05) resulted in a significant higher expression of BGLAP compared with the three other normalization methods. Compared with the normalization with geNorm, a significant overvaluation of the fold change with the normalization of TBP and B2M was detected.

The osteogenic target gene ALPL was downregulated by the 3D cultivation method compared with conventional 2D cultured BM-MSCs when normalized to all RG (0.74±0.06), geNorm (0.73±0.06), and NormFinder (0.74±0.06) (Fig. 4). An overestimation of downregulation was detected for the normalization with GAPDH with a fold change of 0.54±0.15, p<0.01 when compared with the normalization methods mentioned earlier. When ELF1 was used as an internal control, a significant difference in the fold change of ALPL (0.62±0.14) was measured compared with the normalization with all RGs and the three most stable RGs of the NormFinder analysis. When normalized to CASC3 (0.64±0.12) and PSMC4 (0.67±.09), a significant overestimation of downregulation was detected compared with the normalization with all RGs. A highly significant and significant overestimation of the fold changes for ALPL was measured when normalized to the potential RGs GADD45 (1.23±0.25, p<0.001), TFRC (0.97±0.13, p<0.001), RPL37A (1.06±0.31, p<0.05), and MT-ATP6 (0.91±0.15, p<0.01 for GeNorm and NormFinder, p<0.05 for all RG) compared with the fold differences of the normalization with all RGs or with the three most stable ones of geNorm and NormFinder. The normalization with GADD45 even resulted in a seemingly upregulation of expression of the osteogenic target gene ALPL.

Effect of 3D cultivation on the level of cell-specific ALP activity

The level of cell-specific ALP activity was measured after 1, 7, 14, and 21 days of 3D and 2D cultivation with or without osteogenic supplements (Fig. 5). Three-dimensional cultivation itself did not increase the ALP activity level significantly (day 14 and 21: 0.4±0.12 and 0.51±0.09 μmol pNp/30 min/1×10⁶ cells) compared with the ALP activity level in 2D cultivated BM-MSCs (day 14 and 21: 0.31±0.02 and 0.44±0.01 μmol pNp/30 min/1×10⁶ cells). A highly significant increase of ALP activity was detected after 7 (1.03±0.4 μmol pNp/30 min/1×10⁶ cells), 14 (1.8±0.18 μmol pNp/30 min/1×10⁶ cells), and 21 days (2.1±0.52 μmol pNp/30 min/1×10⁶ cells) in 3D culture with the osteogenic supplements in the medium compared with all other culture conditions. On day 21, we could measure a significant increase of the ALP activity level in 2D osteogenically stimulated BM-MSCs (1.26±0.07 μmol pNp/30 min/1×10⁶ cells) compared with 2D BM-MSCs without osteogenic supplements (0.44±0.01 μmol pNp/30 min/1×10⁶ cells).

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FIG. 5.

Activity of alkaline phosphatase in two- and three-dimensionally cultured bone marrow-derived mesenchymal stromal cells over a cultivation period of 21 days with and without osteogenic supplements in culture medium (mean±standard deviation). Significance was calculated with 2-way ANOVA and Bonferroni post-tests with ***p<0.001.