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

J Appl Oral Sci. 2019 Feb 21:27:e20180317. doi: 10.1590/1678-7757-2018-0317.

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.

Publication types

  • Evaluation Study

MeSH terms

  • Alkaline Phosphatase / analysis
  • Alkaline Phosphatase / drug effects
  • Analysis of Variance
  • Blotting, Western
  • Bone Morphogenetic Protein 2 / drug effects*
  • Bone Morphogenetic Protein 2 / metabolism
  • Cell Differentiation / drug effects*
  • Cell Differentiation / physiology
  • Cells, Cultured
  • Humans
  • Mesenchymal Stem Cells / drug effects*
  • Mesenchymal Stem Cells / metabolism
  • Osteoblasts / drug effects
  • Osteocalcin / analysis
  • Osteocalcin / drug effects
  • Osteogenesis / drug effects*
  • Osteogenesis / physiology
  • Osteonectin / analysis
  • Osteonectin / drug effects
  • Reference Values
  • Reproducibility of Results
  • Time Factors
  • Tretinoin / pharmacology*

Substances

  • Bone Morphogenetic Protein 2
  • Osteonectin
  • Osteocalcin
  • Tretinoin
  • Alkaline Phosphatase