Influence of Ascorbic Acid as a Growth and Differentiation Factor on Dental Stem Cells Used in Regenerative Endodontic Therapies

Abstract

Background: Vitamin C is one of the major extracellular nonenzymatic antioxidants involved in the biosynthesis of collagen. It promotes the growth of fibroblasts, wound healing processes, and enhances the survival and differentiation of osteoblasts. The potential effects of ascorbic acid on human dental pulp cells (DPC) and the cells of the apical papilla (CAP) used in actual regenerative endodontic procedures remain largely unknown. In this study, we investigated the possible employment of ascorbic acid in the differentiation and regenerative therapies of DPC and CAP.

Methods: Nine extracted human wisdom teeth were selected for this study. Subpopulations of stem cells within DPC and CAP were sorted with the mesenchymal stem cell marker STRO-1, followed by treatments with different concentrations (0 mM, 0.1 mM, 0.5 mM, and 1.0 mM) of ascorbic acid (AA), RT-PCR, and Western blot analysis.

Results: FACS analysis revealed the presence of cell subpopulations characterized by a strong expression of mesenchymal stem cell marker STRO-1 and dental stem cell markers CD105, CD44, CD146, CD90, and CD29. Treatment of the cells with defined amounts of AA revealed a markedly increased expression of proliferation marker Ki-67, especially in the concentration range between 0.1 mM and 0.5 mM. Further investigations demonstrated that treatment with AA led to significantly increased expression of common stem cell markers OCT4, Nanog, and Sox2. The most potent proliferative and expressional effects of AA were observed in the concentration of 0.1 mM.

Conclusions: AA might be a novel and potent growth promoter of human dental cells. Increasing the properties of human dental pulp cells and the cells of the apical papilla using AA could be a useful factor for further clinical developments of regenerative endodontic procedures.

Keywords: ascorbic acid; dental pulp stem cells; regenerative endodontic procedures; stem cell markers.

Figure 1

FACS-mediated sorting and expression of mesenchymal stem cell markers in DPC and CAP. (A) DPC and (B) CAP cell populations were sorted according to the expression of stem cell marker STRO-1; STRO-1-positive subpopulations were named as DPSC and SCAP, respectively; representative gates P3 (1.5% of DPSC) and P2 (4.5% of SCAP) demonstrate PE (phycoerythrine) STRO-1-positive cells. (C–G) Expression of mesenchymal stem cell markers in STRO-1-negative (DPC and CAP) and STRO-1-positive (DPSC and SCAP) DPC and CAP. All cell populations were tested for the expression of (A) CD29, (B) CD44, (C) CD90, (D) CD105, and (E) CD146; means ± SD of three independent experiments; * p < 0.05 indicates statistical significance (vs. STRO-1-negative cells).

Figure 2

Proliferation of the cells after treatment with different concentrations of AA, based on the expression of Ki-67. DPC (A), (B) DPSC, (C) CAP, and (D) SCAP were treated with 0.1 mM, 0.5 mM, and 1 mM AA for 24 h, 48 h, and 72 h, and subjected for Ki-67 analyses. (E) Measurement of pH with indicated AA concentrations. Means ± SD of three independent experiments; * p < 0.05 indicates statistical significance vs. 0 mM.

Figure 3

Expression of stem cell markers OCT-4, Sox2, and Nanog in (A) DPC, (B) CAP, (C) DPSC, and (D) SCAP treated with 0.1 mM AA. Means ± SD of three independent experiments; * p < 0.05 indicates statistical significance vs. Ø (0 mM AA).

Figure 4

Protein expression of OCT4, Nanog, and CD105 in (A,B) DPSC and SCAP (C,D) treated with 0.1 mM AA. Means ± SD of three independent experiments; * p < 0.05 indicates statistical significance vs. Ø (0 mM AA).