Effects of Cirsium setidens (Dunn) Nakai on the osteogenic differentiation of stem cells

Abstract

Cirsium setidens (Dunn) Nakai, commonly known as gondre, is a perennial herb that grows predominantly in South Korea. It contains several bioactive phytochemicals with antioxidant, anti‑cancer, anti‑tumor and anti‑inflammatory properties. The present study aimed to investigate the effects of methanolic extracts of gondre on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). As characterized by nuclear magnetic resonance spectroscopy and matrix‑assisted laser deposition/ionization (time‑of‑flight) mass spectrometry, the methanol extract of gondre was found to be enriched with pectolinarin. After 48 h, enhanced viability of hPDLSCs was observed in the presence of gondre compared with under control conditions, suggesting the biocompatibility of gondre. Notably, biocompatibility was markedly affected by gondre concentration in cultured media. Relatively high cell viability was observed in medium containing 0.05% gondre. Furthermore, mineralization was significantly higher in hPDLSCs in the presence of gondre compared with that in control cells, indicating their mineralization potential. Increased expression of various transcription markers, such as collagen 1, runt‑related transcription factor 2, bone sialoprotein and alkaline phosphatase, was also detected when hPDLSCs were stimulated with gondre compared with in the control groups, further confirming the superior osteogenic potential of gondre extract for tissue engineering applications, particularly in bone tissues.

Keywords: Cirsium setidens; pectolinarin; cell viability; osteogenesis; tissue engineering.

Figure 1.

The ¹H-nuclear magnetic resonance spectrum of the methanol-extracted bioactive phytochemicals from Cirsium setidens (gondre), indicating the presence of pectolinarin chemical moiety.

Figure 2.

MALDI-TOF mass spectrum of the methanol-extracted sample of gondre, using 2,5-DHB as a matrix. The arrows indicate the gap between the two fragmented peaks in Da. The fragmented ions appeared at ~98, 106, 139, 253, and 288 Da. MALDI-TOF, Matrix-assisted laser desorption/ionization (time-of-flight).

Figure 3.

Isolation and characterization of hPDLSCs. (A) Schematic presentation for the presence of hPDLSCs in the human molar tooth, (B) The morphologies of the cultured stem cells after different time intervals, (C) Fluorescence-activated cell sorting analysis of hPDLSCs. (D) The multi-lineage differentiation potential of hPDLSCs after 21 days of treatment in different induction media. hPDLSCs, human periodontal ligament stem cells.

Figure 4.

Evaluation of the cytotoxicity and migration potential of hPDLSCs. (A) The cell viability data of hPDLSCs in the presence of different concentrations of gondre at indicated time intervals. (B and C) The scratch healing assay in the presence of gondre at indicated time intervals (magnification, ×10). The data are mean ± SD of triplicate experiments (n=3). *P<0.05 vs Control group. hPDLSCs, human periodontal ligament stem cells.

Figure 5.

In vitro differentiation potential of hPDLSCs in the presence of gondre extract. (A) The mineralized nodule formation potential of hPDLSCs in the presence of different gondre concentrations at indicated time intervals (magnification, ×10). (B) Alkaline phosphatase activity of hPDLSCs in the presence of the indicated gondre concentration after 14 days of incubation (magnification, ×10). hPDLSCs, human periodontal ligament stem cells.

Figure 6.

Evaluation of the expression of osteogenesis-specific gene markers and proteins in the presence of gondre powder. (A and B) The relative expression level of mRNA in the presence of different concentrations of gondre after 7, and after 14 days of treatment, respectively. Data were normalized to the housekeeping gene HPRT. (C) Western blot analysis of Runx2 and OSX following gondre (0, 0.01, 0.05, and 0.1%) treatment. (D) Western blotting was quantified using the ImageJ software, and data were normalized to α-tubulin. The data are mean ± SD of triplicate experiments (n=3). *P<0.05, **P<0.01 vs. Control. Col1, collagen 1; Runx2, runt-related transcription factor 2; BSP, bone sialoprotein 2; ALP, alkaline phosphatase; OSX, osterix.

Figure 7.

A hypothetical drawing for gondre-induced odontoblastic differentiation of hPDLSCs. hPDLSCs, human periodontal ligament stem cells; RUNX2, runt-related transcription factor 2; BSP, bone sialoprotein; ALP, alkaline phosphatase; OSX, osterix.