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. 2021 Apr-Jun;11(2):143-148.
doi: 10.1016/j.jobcr.2021.01.002. Epub 2021 Jan 13.

Retinoic and ascorbic acids induce osteoblast differentiation from human dental pulp mesenchymal stem cells

Lina M Escobar  1   2 , José Daniel Escobar  1 , Zita Bendahan  3 , Jaime E Castellanos  2
Affiliations

Retinoic and ascorbic acids induce osteoblast differentiation from human dental pulp mesenchymal stem cells

Lina M Escobar et al. J Oral Biol Craniofac Res. 2021 Apr-Jun.

Abstract

Previous studies have suggested an important role of retinoic acid (RA) and ascorbic acid (AA) in the stimulation of osteoblastic differentiation; however, the function of RA and AA in the osteogenic differentiation from human dental pulp (hDPSCs) remains unclear.

Objective: This in vitro study investigated the effects of RA and AA on the differentiation of osteoblast from hDPSCs.

Methods: hDPSCs were treated with different doses of RA and AA, separately or in combination (RA ​+ ​AA). Morphology and cell proliferation were assessed. Osteoblast differentiation was evaluated by alizarin red, alkaline phosphatase staining, and RUNX2 gene expression.

Results: A significant reduction was observed in the number of cells treated with RA (26%) and RA ​+ ​AA (30%) after 12 days of treatment. AA treatment alone induced a 12% reduction in the number of cells. Morphologically, the cells treated with RA and RA ​+ ​AA were larger and more elongated than the control cells. A mesh pattern was observed in cells treated with AA. Numerous calcified nodules were present in cells treated with RA, AA, and RA ​+ ​AA. This coincided with increased expression of RUNX2 and high alkaline phosphatase staining levels.

Conclusions: hDPSCs treated with RA and RA ​+ ​AA showed significant reduction in proliferation, detectable morphological changes, and expression of the key differentiation gene RUNX2, consistent with an osteoblast phenotype. AA induced morphological changes and early formation of calcified nodules. RA had a predominant effect when AA and RA were used together.

Keywords: Ascorbic acid; Cell differentiation; Mesenchymal stem cells; Osteoblasts; Retinoic acid.

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Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

Fig. 1
Fig. 1
Isolation and characterization of hDPSC. (A) Photomicrograph of mesenchymal cells with fibroblastoid appearance after 7 days of culture. Flow cytometry histograms with positive surface markers for CD105 (B), CD90 (C), CD73 (D) and negative for CD34 (E), CD45 (F) and CD 146 (G). Bar: 200 ​μm.
Fig. 2
Fig. 2
Quantification of cell number in cultures treated with AA and RA. (A) RA produced a decrease in cell number after 5 days of treatment with 10−5 ​M and at 7 days with 10−5 ​M and 10−6 ​M, compared with the control group. (B) AA reduced the number of hDPSC treated with 1 ​mM and 0.5 ​mM doses after 5 and 7 days of treatment. The 0.25 ​mM dose did not show significant differences in comparison with the control group. Asterisks indicate significance (p ​< ​0.05). Data are is expressed as mean ​± ​SD.
Fig. 3
Fig. 3
Cell proliferation in response to RA ​+ ​AA treatment. (A) Changes in cell number induced by treatment with AA (0.5 ​mM), RA (10−5 ​M) and (RA ​+ ​AA) for 12 days. All three treatments produced a significant reduction in the number of cells from day 6 of treatment relative to the control group. ∗p ​< ​0.05. The data are expressed as mean ​± ​SD. (B) Photomicrograph of hDPSC cultures. The cells were treated with RA (10−5 ​M), AA (0.5 ​mM) and RA ​+ ​AA for 7, 14 and 21 days. Bar: 200 ​μm.
Fig. 4
Fig. 4
Quantification of mineralization. (A) Mineralization determined by Alizarin red S staining. Strong matrix staining was observed in the photomicrographs, which indicated the apparent formation of calcification nodules. Bar: 200 ​μm. (B) Measurement of absorbance of Alizarin red S stain extracted from cells under different treatments at 7, 14, and 21 days. Differentiation medium (DM). ∗p ​< ​0.05. Data are expressed as mean ​± ​SD.
Fig. 5
Fig. 5
Characterization of the osteoblast differentiation. (A) Staining of hDPSC cells for alkaline phosphatase. Cells without treatment (Control), and treated with RA, AA and RA ​+ ​AA, were immunohistochemically stained for alkaline phosphatase at 7 and 14 days of treatment. Bar: 200 ​μm. (B) Quantification of RUNX2 relative gene expression. Quantification of RUNX2 expression was performed in cells treated with RA, AA and RA ​+ ​AA during 7, 14, and 21 days. The data are normalized to GAPDH; cells treated with differentiation medium (DM) were used as a positive control. ∗p ​< ​0.05.
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