Label-free Quantitative Proteomic Analysis of Ascorbic Acid-induced Differentially Expressed Osteoblast-related Proteins in Dental Pulp Stem Cells from Deciduous and Permanent Teeth.

BACKGROUND: Proteomic is capable of elucidating complex biological systems through protein expression, function, and interaction under a particular condition. OBJECTIVE: This study aimed to determine the potential of ascorbic acid alone in inducing differentially expressed osteoblast-related proteins in dental stem cells via the liquid chromatography-mass spectrometry/ mass spectrometry (LC-MS/MS) approach. METHODS: The cells were isolated from deciduous (SHED) and permanent teeth (DPSC) and induced with 10 µg/mL of ascorbic acid. Bone mineralisation and osteoblast gene expression were determined using von Kossa staining and reverse transcriptase-polymerase chain reaction. The label-free protein samples were harvested on days 7 and 21, followed by protein identification and quantification using LC-MS/MS. Based on the similar protein expressed throughout treatment and controls for SHED and DPSC, overall biological processes followed by osteoblast-related protein abundance were determined using the PANTHER database. STRING database was performed to determine differentially expressed proteins as candidates for SHED and DPSC during osteoblast development. RESULTS: Both cells indicated brownish mineral stain and expression of osteoblast-related genes on day 21. Overall, a total of 700 proteins were similar among all treatments on days 7 and 21, with 482 proteins appearing in the PANTHER database. Osteoblast-related protein abundance indicated 31 and 14 proteins related to SHED and DPSC, respectively. Further analysis by the STRING database identified only 22 and 11 proteins from the respective group. Differential expressed analysis of similar proteins from these two groups revealed ACTN4 and ACTN1 as proteins involved in both SHED and DPSC. In addition, three (PSMD11/RPN11, PLS3, and CLIC1) and one (SYNCRIP) protein were differentially expressed specifically for SHED and DPSC, respectively. CONCLUSION: Proteome differential expression showed that ascorbic acid alone could induce osteoblastrelated proteins in SHED and DPSC and generate specific differentially expressed protein markers.

A Comparative Analysis of Ascorbic Acid-induced Cytotoxicity and Differentiation between SHED and DPSC.

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, ß-glycerophosphate, and dexamethasone has been widely used to induce osteoblast differentiation. However, under certain conditions, ß-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.

A Perspective on Stem Cells as Biological Systems that Produce Differentiated Osteoblasts and Odontoblasts.

Stem cells (SCs) are capable of self-renewal and multilineage differentiation. Human mesenchymal stem cells (MSCs) and haematopoietic stem cells (HSCs) which can be obtained from multiple sources, are suitable for application in regenerative medicine and transplant therapy. The aim of this review is to evaluate the potential of genomic and proteomic profiling analysis to identify the differentiation of MSCs and HSCs towards osteoblast and odontoblast lineages. In vitro differentiation towards both of these lineages can be induced using similar differentiation factors. Gene profiling cannot be utilised to confirm the lineages of these two types of differentiated cells. Differentiated cells of both lineages express most of the same markers. Most researchers have detected the expression of genes such as ALP, OCN, OPN, BMP2 and RUNX2 in osteoblasts and the expression of the DSPP gene in odontoblasts. Based on their cell-type specific protein profiles, various proteins are differentially expressed by osteoblasts and odontoblasts, except for vimentin and heterogeneous nuclear ribonucleoprotein C, which are expressed in both cell types, and LOXL2 protein, which is expressed only in odontoblasts.