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.

Cytotoxicity effect of degraded and undegraded kappa and iota carrageenan in human intestine and liver cell lines.

BACKGROUND: Carrageenan is a linear sulphated polysaccharide extracted from red seaweed of the Rhodophyceae family. It has broad spectrum of applications in biomedical and biopharmaceutical field. In this study, we determined the cytotoxicity of degraded and undegraded carrageenan in human intestine (Caco-2; cancer and FHs 74 Int; normal) and liver (HepG2; cancer and Fa2N-4; normal) cell lines. METHODS: Food grade k-carrageenan (FGKC), dried sheet k-carrageenan (DKC), commercial grade k-carrageenan (CGKC), food grade i-carrageenan (FGIC) and commercial grade i-carrageenan (CGIC) were dissolved in hydrochloric acid and water to prepare degraded and undegraded carrageenan, respectively. Carrageenan at the concentration range of 62.5 - 2000.0 µg mL(-1) was used in the study. MTT assay was used to determine the cell viability while the mode of cell death was determined by May-Grunwald Giemsa (MGG) staining, acridine orange-ethidium bromide (AO/EtBr) staining, agarose gel electrophoresis and gene expression analysis. RESULTS: Degraded FGKC, DKC and CGKC showed IC50 in 24, 48 and 72 hours treated Caco-2, FHs 74 Int, HepG2 and Fa2N-4 cell lines as tested by MTT assay. Degraded FGIC and CGIC only showed its toxicity in Fa2N-4 cells. The characteristics of apoptosis were demonstrated in degraded k-carrageenan treated Caco-2, FHs 74 Int, HepG2 and Fa2N-4 cells after MGG staining. When Caco-2 and HepG2 cells were undergone AO/EtBr staining, chromatin condensation and nuclear fragmentation were clearly seen under the microscope. However, DNA ladder was only found in HepG2 cells after gel electrophoresis analysis. Degraded k-carrageenan also inactivated PCNA, Ki-67 and survivin gene in HepG2. On the other hand, undegraded FGKC, DKC, CGKC, FGIC and CGIC treated cells showed no cytotoxic effect after analyzed by the same analyses as in degraded carrageenan. CONCLUSION: Degraded k-carrageenan inhibited cell proliferation in Caco-2, FHs 74 Int, HepG2 and Fa2N-4 cell lines and the anti-proliferative effect was related to apoptosis together with inactivation of cell proliferating genes as determined by morphological observation and molecular analysis. However, no cytotoxic effect was found in undegraded carrageenan towards normal and cancer intestine and liver cell lines.

Differentiation analyses of adult suspension mononucleated peripheral blood cells of Mus musculus.

BACKGROUND: The purpose of this study is to determine whether isolated suspension mouse peripheral mononucleated blood cells have the potential to differentiate into two distinct types of cells, i.e., osteoblasts and osteoclasts. RESULTS: Differentiation into osteoblast cells was concomitant with the activation of the Opn gene, increment of alkaline phosphatase (ALP) activity and the existence of bone nodules, whereas osteoclast cells activated the Catk gene, increment of tartrate resistant acid phosphatase (TRAP) activity and showed resorption activities via resorption pits. Morphology analyses showed the morphology of osteoblast and osteoclast cells after von Kossa and May-Grunwald-Giemsa staining respectively. CONCLUSIONS: In conclusion, suspension mononucleated cells have the potentiality to differentiate into mature osteoblasts and osteoclasts, and hence can be categorized as multipotent stem cells.