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. 2021 Sep 14;11(9):915.
doi: 10.3390/jpm11090915.

Adipogenic Stimulation and Pyrrolidine Dithiocarbamate Induced Osteogenic Inhibition of Dental Pulp Stem Cells Is Countered by Cordycepin

Affiliations

Adipogenic Stimulation and Pyrrolidine Dithiocarbamate Induced Osteogenic Inhibition of Dental Pulp Stem Cells Is Countered by Cordycepin

Shankargouda Patil et al. J Pers Med. .

Abstract

Background: dental pulp-derived stem cells are easy to access and collect and are an excellent source of stem cells for regenerative therapy. These cells can interact with many biomolecules and scaffolds and can pass on the instructive signals to the sites of regeneration where they are used. In this regard cordycepin, a potential biomolecule derived from medicinal mushrooms with a spectrum of bioactive properties such as antioxidant, anti-inflammatory, and anticancer has not yet been tested for its effect on human dental pulp stem cells.

Objective: the objective of the present study was to assess the in vitro adipogenic and osteogenic differentiation potential of human dental pulp stem cells with or without induction after administration of cordycepin.

Materials and methods: human dental pulp stem cells DPSCs were isolated from a healthy permanent tooth extracted for orthodontic purposes after obtaining informed consent. Flow cytometry technique was used to assess the surface markers of these cells such as CD73, CD90, and CD105, CD34, CD45, and HLA-DR. Further, an MTT assay was performed on the cells after subjecting them to various concentrations of cordycepin. Following this, the adipogenic and osteogenic potential of the dental pulp stem cells was assessed with or without induction under the influence/absence of 5 µM of cordycepin. The results obtained were statistically analyzed and documented.

Results: it was found that the dental pulp stem cells showed strong positive expression for CD73, CD90, and CD105 and faint expression of CD34, CD45, and HLA-DR. MTT assay revealed that 5 µM was the optimum concentration of cordycepin for all the assays. Concerning adipogenesis experiments, there was a statistically significant lowering of all the 4 adipogenesis-related genes PPARγ, FABP4, LPL, and C/EBPα following cordycepin treatment in the presence of induction compared to the only induction group and untreated control cells (p < 0.05). In connection with osteogenesis, was found that there was a statistically significant increase in the expression of RUNX2, COL1A1, OSX and OCN genes along with the increase in alkaline phosphatase and alizarin red staining in the DPSC treated with cordycepin along with the presence of induction and simultaneous addition of PDTC compared to the control untreated cells and cells treated with induction and simultaneous addition of PDTC (p < 0.05).

Conclusion: cordycepin can be exploited for its osteopromotive properties and can be used as a bioactive molecule alongside the administration of dental pulp stem cells in the area of regenerative biology and medicine.

Keywords: cordycepin; dental pulp stem cells; osteogenesis; regeneration.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Mesenchymal stem cell (MSC) markers and the MTT assay were used to characterize DPSCs. (A) Passage 2 photomicrograph of DPSCs. Scale bar = 100 μm; (BG) MSC-specific positive markers CD73, CD90, and CD105, as well as MSC-specific negative markers CD34, CD45, and HLA-DR, were examined in DPSCs; (H) For 48 h, DPSCs were treated with various concentrations of cordycepin (0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, and 50 μM) and a comparative study was performed to determine the cytotoxic effect of cordycepin to DPSCs. Cpn: cordycepin. * p < 0.05, ** p < 0.01.
Figure 2
Figure 2
Adipogenic differentiation of DPSCs and quantitative RT-qPCR study of gene expression. (AC) Adipogenic differentiation was induced in DPSCs with and without cordycepin administration, and oil red O was used for functional staining. Scale bar = 100 μm; (DG) Comparative gene expression study of genes involved in adipogenesis PPARγ, FABP4, LPL, C/EBPα in with and without cordycepin treated adipogenesis induced DPSCs. ns not significant, ** p < 0.01. ADIP-induced: adipogenic induction, Cpn: cordycepin, PPARγ: peroxisome proliferator-activated receptor-gamma, FABP4: fatty acid-binding protein 4, LPL: lipoprotein lipase, C/EBPα: CCAAT/enhancer-binding protein alpha.
Figure 3
Figure 3
Osteogenic differentiation of DPSCs, quantification of mineralization, and alkaline phosphatase activity. (AH) Osteogenic differentiation was induced in DPSCs with and without treatment with cordycepin and inhibition of osteogenesis was induced with PDTC. Functional staining was done with alizarin red S. Scale bar = 100 μm; (I) Comparative quantification of mineralization with and without cordycepin treated osteogenesis induced and osteogenesis inhibited DPSCs; (J) Comparative alkaline phosphatase activity with and without cordycepin treated osteogenesis induced and osteogenesis inhibited DPSCs. ns not significant, * p < 0.05, ** p < 0.01. OST-induced: osteogenic induction, Cpn: cordycepin.
Figure 4
Figure 4
Osteogenic differentiation of DPSCs and quantitative RT-qPCR study of gene expression. (AD) Comparative gene expression analysis of osteogenesis-related genes RUNX2, COL1A1, OSX, and OCN in with and without cordycepin treated osteogenesis induced and osteogenesis inhibited DPSCs. ns not significant, ** p < 0.01. OST-induced: osteogenic induction, Cpn: cordycepin, RUNX2: Runt-related transcription factor 2, COL1A1: collagen, type I, alpha 1, OSX: transcription factor Sp7/Osterix, OCN: bone gamma-carboxyglutamic acid-containing protein/osteocalcin.

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