Taurine Augments Telomerase Activity and Promotes Chondrogenesis in Dental Pulp Stem Cells

Abstract

Background: Stem cell therapy has become an advanced and state-of-the-art procedure to regenerate lost tissues of the human body. Cartilage repair is a challenging task in which stem cells find potential application. One of the important biologic modifiers that can cause chondrogenic differentiation of stem cells is taurine. However, taurine has not been investigated for its effects on dental pulp derived stem cell (DPSC) chondrogenic differentiation.

Objective: The objective of the study was to investigate if taurine administration to DPSCs heralds chondrogenic differentiation as ascertained by expression of SOX9, COL2A1, ACAN, ELN, and COMP. The study also investigated if the differentiated cells synthesized glycosaminoglycans, a marker of cartilage formation. The study also aimed to assess proliferative activity of the cells after taurine administration by measuring the hTERT gene and protein expression.

Materials and methods: DPSCs were obtained from a molecular biology laboratory and characterization of stem cell markers was done by flow cytometry. The cells were subjected to a MTT assay using various concentrations of taurine. Following this, hTERT gene and protein estimation was done in the control, telomerase inhibitor treated DPSC (TI-III), 10 μM taurine treated DPSC, and TI-III + 10 μM taurine treated DPSCs. A polymerase chain reaction was done to assess gene expression of SOX9, COL2A1, ACAN, ELN, and COMP genes and glycosaminoglycans were estimated in control cells, Induced DPSCs, induced and TI-III treated DPSCs, and 10 μM taurine treated DPSCs.

Results: DPSCs expressed CD73, CD90, and CD105 and did not express CD34, CD45, and HLA-DR, which demonstrated that they were mesenchymal stem cells. The MTT assay revealed that various concentrations of taurine did not affect the cell viability of DPSCs. A concentration of 10 μM of taurine was used for further assays. With regard to the hTERT gene and protein expression, the taurine treated cells expressed the highest levels that were statistically significant compared to the other groups. Taurine was also found to restore hTERT expression in telomerase inhibitor treated cells. With regard to chondrogenesis related genes, taurine administration significantly increased the expression of SOX9, COL2A1, ACAN, and ELN genes in DPSCs and caused a significant increase in glycosaminoglycan production by the cells.

Conclusions: Taurine can be regarded a biologic modifier that can significantly augment chondrogenic differentiation of DPSCs and can find potential applications in regenerative medicine in the area of cartilage regeneration.

Keywords: chondrogenesis; dental pulp; mesenchymal stem cells; regenerative medicine; taurine; telomerase.

Figure 1

Characterization of DPSCs for mesenchymal stem cell (MSC) markers and trilineage differentiation. (A) Photomicrograph of DPSCs at passage 4. (B–I) DPSCs were checked for MSC-specific positive markers CD73, CD90, and CD105; and MSC-specific negative markers CD34, CD45, and HLA-DR. (J–L) DPSCs were differentiated into adipocytes, osteoblasts, and chondrocytes. Scale bar = 100 μm. DPSCs: dental pulp stem cells, ADIPO: adipogenic differentiation, OSTEO: osteogenic differentiation, CHONDRO: chondrogenic differentiation.

Figure 2

MTT assay to assess the cytotoxicity of taurine to DPSCs. (A,B) DPSCs were treated with various concentrations of taurine (1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, and 50 μM) for 48 h and seven days and comparative analysis was done to check the cytotoxicity of taurine to DPSCs. ns: not significant, * p < 0.05, ** p < 0.01. Tau: Taurine.

Figure 3

Taurine upregulates gene expression of TERT and protein levels of hTERT in DPSCs. (A) Comparative gene expression analysis of TERT with and without taurine treated DPSCs and telomerase inhibited DPSCs. (B,C) Whole cell extracts were evaluated by using the western blot method to decide the protein expression levels of hTERT in DPSCs. The graphs show the ratios of band densities of hTERT. ns: not significant, * p < 0.05, ** p < 0.01. Tau: Taurine, TI-III: telomerase inhibitor III, TERT/hTERT: human telomerase reverse transcriptase.

Figure 4

Chondrogenic differentiation of DPSCs and quantification of toluidine blue intensity. Chondrogenic differentiation was induced in DPSCs with and without treatment with telomerase inhibitor and DPSCs treated with taurine alone were assessed for chondrogenesis. Functional staining was done with (A–D) toluidine blue, (E–H) Alcian blue, and (I–L) safranin O. Scale bar = 100 μm, (M) Comparative quantification of toluidine blue intensity day 21. * p < 0.05, ** p < 0.01. Induction: Chondrogenic differentiation, Tau: Taurine, TI-III: Telomerase Inhibitor III, TB: Toluidine blue.

Figure 5

Chondrogenic differentiation of DPSCs and the analysis of chondrogenesis related gene expression by quantitative RT-qPCR. (A–E) Comparative gene expression analysis of chondrogenesis-related genes SOX9, COL2A1, ACAN, ELN, and COMP in chondrogenesis induced DPSCs with and without treatment with telomerase inhibitor and DPSCs treated with taurine alone. ns: not significant, * p < 0.05, ** p < 0.01. Induction: chondrogenic differentiation, Tau: taurine, TI-III: telomerase Inhibitor III, SOX9: SRY-box transcription factor 9, COL2A1: collagen, type II, alpha 1, ACAN: aggrecan, ELN: elastin, COMP: cartilage oligomeric matrix protein.

Figure 6

Taurine treatment to DPSCs and immunostaining. (A–H) DPSCs treated with taurine alone were assessed for chondrogenesis related proteins SOX-9 and aggrecan at day 21. Scale bar = 200 μm. Tau: taurine.