Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells

Abstract

Background: Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields.

Objective: Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved.

Material and methods: ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software.

Results: RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone.

Conclusions: In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.

Figure 1. Extracellular matrix evaluation. A) Alkaline phosphatase (ALP) activity expressed as mg of rNitrophenol (rNP) per mg of protein per ml at days 7, 14, 21, and 28. Different letters refer to statistically significant differences (r<0.001) (ANOVA/Tukey). Statistical analyses were performed comparing all treatments at each time point separately. B) von Kossa staining of ASCs cultured for 21 days. Bar = 500 µm. Black areas show the presence of phosphate and indicate extracellular matrix mineralization, suggesting differentiation into osteoblasts. Cells were treated with OM (B1), OM supplemented with BMP-2 (B2), OM with RA (B3), or OM supplemented with BMP-2 and RA (B4). MC3T3-E1 was used as a positive control (B5). Images are representative of three independent experiments; C) Calcium quantification expressed as mg per dl at days 12, 23, and 32. Bars represent the mean ± SD of three independent experiments. Different letters represent statistically significant differences (r<0.05) (ANOVA/Tukey) among treatments at each time point.

Figure 2. mRNA relative expressions. ASCs were treated according to the groups: OM (osteogenic medium); BMP-2 (OM and BMP-2); RA (OM and retinoic acid, RA); BMP-2+RA (OM, BMP-2, and RA). Results were normalized against human osteoblasts (positive control). Different letters represent statistically significant differences among treatments at each time point (ANOVA/Tukey, ρ<0.05). Bars represent the mean ± SD of three independent experiments

Figure 3. Expression of osteoblast-related proteins. Proteins expressions evaluated by western blotting at days 7, 14, 21, and 28 of ASCs treated according to the groups: OM (osteogenic medium); BMP-2 (OM and BMP-2); RA (OM and retinoic acid, RA); BMP-2+RA (OM, BMP-2, and RA). MC3T3-E1 was used as positive control. Bands are representative of three independent experiments. Band intensities were quantified. Different letters represent statistically significant differences (r<0.05) (ANOVA/Tukey) among treatments at each time point