Korean J Physiol Pharmacol.  2024 Sep;28(5):423-433. 10.4196/kjpp.2024.28.5.423.

Chios gum mastic enhance the proliferation and odontogenic differentiation of human dental pulp stem cells

• Baek HS
• Park SJ
• Lee EG
• Kim YI
• Kim IR

• KMID: 2559135
• DOI: http://doi.org/10.4196/kjpp.2024.28.5.423

Abstract

Dental pulp stem cells (DPSCs) are a type of adult stem cell present in the dental pulp tissue. They possess a higher proliferative capacity than bone marrow mesenchymal stem cells. Their ease of collection from patients makes them well-suited for tissue engineering applications, such as tooth and nerve regeneration. Chios gum mastic (CGM), a resin extracted from the stems and leaves of Pistacia lentiscus var. Chia, has garnered attention for its potential in tissue regeneration. This study aims to confirm alterations in cell proliferation rates and induce differentiation in human DPSCs (hDPSCs) through CGM treatment, a substance known for effectively promoting odontogenic differentiation. Administration of CGM to hDPSC cells was followed by an assessment of cell survival, proliferation, and odontogenic differentiation through protein and gene analysis. The study revealed that hDPSCs exhibited low sensitivity to CGM toxicity. CGM treatment induced cell proliferation by activating cell-cycle proteins through the Wnt/β-catenin pathway. Additionally, the study demonstrated that CGM enhances alkaline phosphatase activation by upregulating the expression of collagen type I, a representative matrix protein of dentin. This activation of markers associated with odontogenic and bone differentiation ultimately facilitated the mineralization of hDPSCs. This study concludes that CGM, as a natural substance, fosters the cell cycle and cell proliferation in hDPSCs. Furthermore, it triggers the transcription of odontogenic and osteogenic markers, thereby facilitating odontogenic differentiation.

Keyword

Cell proliferation; Chios gum mastic; Dental pulp stem cells; Odontogenic differentiation

Figure

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  Fig. 1 Cytotoxic effect of Chios gum mastic (CGM) on human dental pulp stem cells (hDPSCs). hDPSCs were treated with CGM at concentrations of 0–50 μM for 24–96 h. Cell viability was expressed as % based on the 24-h control group (0 μM). The results were expressed as mean ± SD and were derived from three independent experiments (*p < 0.05, **p < 0.01).

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  Fig. 2 Effects of Chios gum mastic (CGM) on the cell cycle progression of human dental pulp stem cells (hDPSCs). Cells were treated with 0, 1, 2.5, 5, 10, 25, and 50 μM CGM for 48 h. (A) After staining the cells with propidium iodide, the cell cycle was analyzed using FACS. (B) The percentage ratios of G1 (green symbols), S (blue symbols), and G2 (purple symbols) of the histogram shown in A are shown as line category graphs. (C) The protein expression of cell cycle-related factors (Cyclin A, Cyclin E, and CDK2) was confirmed through Western blot analysis. β-actin was used as a loading control.

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  Fig. 3 Effect of Chios gum mastic (CGM) on cell proliferation in human dental pulp stem cells (hDPSCs). (A) Using a transwell chamber, hDPSCs were treated with 5 and 10 μM of CGM, and cell migration was confirmed for 48 h (×40). (B) To determine the cell proliferation rate by CGM, hDPSCs were treated with 5 and 10 µM of CGM for 7 days. Cell proliferation was confirmed by staining the cells with 1% crystal violet (×40). (A, B) The cell migration and proliferation rates were expressed based on the control group and displayed as a histogram. The results were expressed as mean ± SD and were derived from three independent experiments (*p < 0.05, **p < 0.01). (C) mRNA expression patterns of Wnt3a, β-catenin, and AXIN in hDPSC cells following CGM treatment. Cells were treated with 0, 1, 2.5, 5, 10, 25, and 50 μM CGM for 48 h. The results were expressed as mean ± SD and were derived from three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001). (D) After treating the cells with 0, 5, and 10 μM CGM for 48 h, the expression of Ki-67 was observed using a confocal microscope through immunofluorescence. Actin was stained with rhodamine phalloidin (red), Ki-67 was stained with Alexa 488 (green), and nuclei were stained with DAPI (blue) (×400). (E) The protein expression of Wnt signal-related factors (Wnt3a, β-catenin, GSK3β, p-GSK3β, AXIN1, and Ki-67) was confirmed by Western blot analysis. β-actin was used as a loading control.

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  Fig. 4 Osteogenic differentiation effect of Chios gum mastic (CGM) in human dental pulp stem cells (hDPSCs). (A) After treatment with 0, 5, and 10 μM of CGM in hDPSCs for 48 h, the expression of collagen type I was observed using confocal microscopy through immunofluorescence. Actin was stained with rhodamine phalloidin (red), collagen type I was stained with Alexa 488 (green), and nuclei were stained with DAPI (blue) (×400). (B) To determine the cell alkaline phosphatase (ALP) activity by CGM, hDPSCs were treated with 5 and 10 µM of CGM for 7 and 14 days. The ALP activity ratio was calculated in relation to the control group (for each 7 and 14 days), and the results were presented as a histogram. Results were expressed as mean ± SD and were derived from three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001). (C, D) To measure ALP activity, we treated CGM under the same conditions as alizarin red staining and cultured the cells for 7 and 14 days. The color development observed after alizarin red staining was quantified based on the control group (for each 7 and 14 days) and expressed accordingly. Results were expressed as mean ± SD and were derived from three independent experiments (*p < 0.05, ***p < 0.001) (×40). (E) In order to confirm the effect of CGM on the odontogenic differentiation process at the mRNA level, hDPSCs were treated with 5 and 10 µM of CGM for 7 days, and the expression of bone and dentine-related factors (ALP, BSP, DMP-1, DSPP, OP, and RUNX2) in the cells was quantified by qPCR. The results were expressed as the mean ± SD. Data were derived from three independent experiments (**p < 0.01, ***p < 0.001).

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