The Effects of Grape Seed Oligomeric Proanthocyanidin and Nisin on Dental Pulp Stem Cells

Abstract

Objective: This study aimed to evaluate the biological effects of "proanthocyanidin" (PA), and "nisin" (Ni), on dental pulp stem cells (DPSCs) and LPS-induced DPSCs as well as their antimicrobial effects against S. aureus and E. coli.

Materials and methods: After characterization of DPSCs, cytotoxicity of PA and Ni on DPSCs were evaluated using a water-soluble tetrazolium salt (WST-1). The cytokines and chemokines released by DPSCs and the expression levels of IL-6, IL-8, and TNF alpha were detected with human Cytokine Array C5 and enzyme-linked immunosorbent assay (ELİSA), respectively. The antibacterial activities of PA and Ni were tested using the drop plate method.

Results: PA at 75 μg/ml increased cell viability, decreased TNF-α expression of DPSCs, did not show any cytotoxic effects on LPS-induced DPSCs, and also showed a tendency to decrease TNF-α expression. PA at 75 μg/ml exhibited higher expressions of TIMP-2, OPG, IL-7, and IL-8 in LPS-induced DPSCs compared to DPSCs. Ni at 100 μg/ml decreased TNF-α expression in DPSCs with no cytotoxic effects. It provided increased cell viability and a downregulation trend of TNF-α expression in LPS-induced DPSCs. Both Ni and PA provided strong antibacterial effects against S. aureus. Ni at 200μg/ml had strong antibacterial effects against E. coli without affecting negatively the viability of both DPSCs and LPS-induced DPSCs and showed anti-inflammatory activity by decreasing TNF-α expression. PA provided strong antibacterial effects against E. coli at 200 μg/ml but affected DPSCs viability negatively.

Conclusion: PA and Ni at specific concentrations exhibited immunomodulatory activity on DPSCs and LPS-induced DPSCs without any cytotoxic effects and strong antibacterial effects on S. aureus.

Keywords: Antimicrobial; Antimicrobial Agents; Dental pulp stem cells; Grape seed; Immunomodulating Agents; MeSH Terms: Grape Seed Extract; Nisin; Proanthocyanidin; Proanthocyanidins.

Figure 1

Morphology and characterization of dental pulp stem cells. Phase-contrast microphotographs showing dental pulp stem cells (a), scale bar = 200 µm. Representative FACS analysis of dental pulp stem cells (b). Cells highly expressed CD90, CD73 and CD 105 (>87.0%) and lacked CD34 and CD45 (<5.0%) markers. Determination of adipogenic and osteogenic differentiation capacities of human dental pulp stem cells by Oil Red-O and Alizarin Red staining methods (c). 21st day of the culture demonstrating the formation of lipid droplets, bone like mineralization was observed with red staining at day -21. Scale bar: 200 µm

Figure 2

The cell viability analysis of DPSCs (a) and LPS-induced dental pulp stem cells (DPSCs) (b) which were conditioned with different concentrations of Proanthocyanidin (PA) and Nisin (Ni) for 24h. The control group is DPSCs, and the LPS group is LPS-induced DPSCs (2 µg/ml for 24h). Significant differences were found by paired t test * p<0.05 (mean ± std, n=3).

Figure 3

Antibacterial activity of Proanthocyanidin (a, b, c, d) and Nisin (e, f, g, h) against a Gram (-) strain Escherichia coli and a Gram (+) strain Staphylococcus aureus, using drop plate method. Proanthocyanidin and nisin showed a strong antibacterial property against E. coli at 200 µg/mL concentration (a) and against S. aureus at all concentrations (c).

Figure 4

Effects of Proanthocyanidin (PA) and Nisin (Ni) on the expression of TNF-α in DPSCs and LPS induced-DPSCs (a,b). DPSCs treated with 2 µg/ml LPS for 24 h are the LPS induced DPSCs (b). Subsequently, DPSCs were conditioned with different concentrations of PA and Ni for 24 h. TNF-α expression levels in the culture medium of PA and Nisin conditioned DPSCs/ LPS induced DPSCs were measured using ELISA. Significant differences were determined by paired t test. * p<0.05 (mean ± std, n=3).

Figure 5

Effects of Proanthocyanidin (PA) and nisin on the expression of IL-6 (a,b) and IL-8 (c,d) in DPSCs and LPS induced-DPSCs. DPSCs treated with 2 µg/ml LPS for 24 h are the LPS induced DPSCs (b,d). Subsequently, DPSCs were conditioned with different concentrations of PA and Ni for 24h. IL-6 and IL-8 expression levels in the culture medium of PA and nisin conditioned DPSCs/ LPS induced DPSCs were measured using ELISA. Significant differences were found by paired t test. * p<0.05 (mean ± std, n=3).

Figure 6

Cytokine/chemokine secretions of LPS-induced DPSCs which were conditioned with Proanthocyanidin (PA) and nisin (Ni) after normalization based on PA and Ni-supplemented conditions. Different secretion profile of IL-6, IL-7, IL-8 and TIMP-2 in LPS induced DPSCs at 75, 100 µg/ml PA and 100,200 µg/ml Ni concentrations (a). OPG secretion profile of LPS induced DPSCs at 75, 100 µg/ml PA (b), (mean ± std, n=3).