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A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
A Comparative Analysis of Ascorbic Acid-induced Cytotoxicity and Differentiation between SHED and DPSC
Intan Zarina Zainol Abidin1, Thanaletchumi Manogaran2, Rohaya Megat Abdul Wahab3
1Centre for Research and Graduate Studies, University of Cyberjaya, 63000 Cyberjaya, Selangor, Malaysia.
Aim:
The aim of this study was to compare dental pulp tissue in human exfoliated deciduous teeth (SHEDs) and dental pulp stem cells (DPSCs) in response to ascorbic acid as the sole osteoblast inducer.
Background:
A cocktail of ascorbic acid, β-glycerophosphate, and dexamethasone has been widely used to induce osteoblast differentiation. However, under certain conditions, β-glycerophosphate and dexamethasone can cause a decrease in cell viability in stem cells.
Objectives:
This study aims to determine the cytotoxic effect and potential of ascorbic acid as the sole inducer of osteoblast differentiation.
Methods:
Cytotoxicity analyses in the presence of 10-500 μg/mL ascorbic acid were performed in both cell types using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The concentrations below the IC50 (i.e., 10-150 μg/mL) were used to determine osteoblast differentiation potential of ascorbic acid using the alkaline phosphatase (ALP) assay, von Kossa staining, and reverse transcription-polymerase chain reaction.
Results:
SHEDs and DPSCs proliferated for 21 days, expressed a Mesenchymal Stem Cell (MSC) marker (CD73+), and did not express Hematopoietic Stem Cell (HSC) markers (CD34- and SLAMF1-). SHEDs had a higher range of IC50 values (215-240 μg/mL ascorbic acid), while the IC50 values for DPSCs were 177-211 μg/mL after 24-72 hours. SHEDs treated with 10-100 μg/mL ascorbic acid alone exhibited higher ALP-specific activity and a higher percentage of mineralisation than DPSCs. Both cell types expressed osteoblast markers on day 21, i.e., RUNX2+ and BSP+, in the presence of ascorbic acid.
Conclusions:
SHEDs survive at higher concentrations of ascorbic acid as compared to DPSC. The cytotoxic effect was only exhibited at ≥250 μg/mL ascorbic acid. In addition, SHED exhibited better ALP and mineralization activities, but lower osteoblast marker expression than DPSC in response to ascorbic acid as the sole inducer.
