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.

Introduction

Dental caries is one of the most common disease globally (1) and its progressing can cause the inflammation of pulp tissue due to bacterial components and byproducts reaching the pulp. The main membrane component of Gram-negative bacteria, lipopolysaccharide (LPS), is one of the most important sources of infection and oxidative stress related to dental caries. LPS elevates blood flow and CO₂, as well as lower pH levels in this environment (2). It has been well known fact that the diseased microenvironment disrupts the functions of mesenchymal stem cells (MSCs) and also influences the fate of dental pulp stem cells (DPSCs), a source of MSC (3). Therefore, in order to provide the appropriate microenvironment for healing and regeneration of the pulp, many compounds or target molecules either alone or in combination with dental materials have been investigated to date and are currently a matter of interest for obtaining the most favorable outcomes. Grape is a fruit rich in phenolic compounds exhibiting antioxidant, anti-microbial, anti-inflammatory, anti-carcinogenic, cardioprotective and anti-aging benefits for human health. Proanthocyanidin (PA) is the most prevalent phenolic compound in grape seeds. It has been reported that PA is an antioxidant, free-radical scavenger, and cardiovascular protector (4, 5). Grape seed compounds have also been evaluated in the field of dentistry in regard to their anti-microbial efficiency as intracanal irrigant in endodontics (6), antioxidant properties against LPS from periodontopathogens in periodontology (7), as a natural collagen crosslinking agent in restorative dentistry (8), and as a cross linker of tissue engineering scaffolds (9). Protective effects of grape seed extracts against LPS induced inflammatory responses and their potent anti-inflammatory impacts in experimental inflammation have been demonstrated in numerous studies (7, 10, 11). The challenges posed by antibiotic-resistant pathogens and infections associated with biofilms have promoted the search of new therapeutic strategies. Natural anti-microbial peptides (AMP) and their peptidomimetics have attracted attention due to their low bacterial resistance and potent anti-microbial activities. Nisin, derived from Lactococcus lactis bacteria, is the only FDA approved (for inhibiting pathogens in food manufacturing) natural anti-microbial peptide. In medical applications, nisin showed efficacy in the treatment of Staphylococcal mastitis and atopic dermatitis, as well as inhibition of bacterial adhesion on implantable materials (12, 13). The inhibitor effects of nisin against intracanal pathogens Streptococcus gordonii, Enterococcus faecalis and dental caries associated microorganism, were also reported for dental applications (14-16). The dental adhesive incorporating nisin demonstrated inhibitory effect on Streptococcus mutans (17). AMPs do not only exhibit antibacterial effects but also regulate immuno-inflammatory responses through their immunomodulatory and wound-healing potential. They stimulate the production of pro-inflammatory cytokines and recruit host defense cells (18). Nisin at certain concentrations demonstrated anti-biofilm effects against saliva derived multi-species biofilms with no cytotoxic effects on the human oral cells (15). Kindrachuk et al. (19) showed that purified nisin induced immunomodulatory responses within both ex vivo and in vivo infection models. Baskaran et al. (20) synthesized Alpha Tricalcium Phosphate (NTCP) incorporated with nisin and assessed the release of nisin from NTCP when it was employed as a pulp capping agent in vitro. Nisin incorporated dental adhesive against Streptococcus mutans has also been developed and exhibited inhibitory effect without no adverse effects (17). Although many beneficial effects of both grape seed oligomeric proanthocyanidins and nisin were reported in the literature, their effects on cell viability and cytokine expressions in both DPSCs and LPS-induced DPSCs have not yet been investigated. The aim of the present study was to evaluate the effects of different concentrations of proanthocyanidin and nisin on DPSCs and also to find the optimal concentration which could enhance tissue regeneration.

Materials and methods

Isolation and Characterization of Dental Pulp Stem Cells

The principles of the Declaration of Helsinki were followed in this study. Dental pulp samples were obtained with the approval of Hacettepe University Non-Interventional Clinical Research Ethics Committee (GO22-431). The surgically removed five impacted third molars were gathered from five healthy donors with no history of medicine within the last two weeks and non-smokers aged 18-24. Informed and signed consent was obtained from all individual participants included in the study. After the surgical extractions were conducted in the operating rooms of the Hacettepe University School of Dentistry, teeth were gently wiped with 70% alcohol and were irrigated with distilled water. Teeth were transferred within the storage medium immediately to the Center for Stem Cell Research and Development of Hacettepe University. Dental pulp tissue was extracted by carefully separating it from the pulp chamber using sterile instruments and diamond burs while constantly cooling it with sterile saline solution. The collected tissue was placed in a transport medium containing DMEM-LG (Gibco) with 5% Penicillin-Streptomycin (Pen-Strep, Sigma) and 5% Amphotericin B (Amf B, Biological Industries), and transported to the laboratory. Subsequently, the pulp tissue was transferred to a 35 mm transparent petri dish and rinsed with DMEM-LG (Gibco) containing 2 mL of 5% Pen-Strep and 5% Amf B. The pulp tissue was then finely minced using a scalpel, and enzymatically digested using 0.3 mg/mL collagenase type I (Sigma-Aldrich) for a period of 2 hours at 37°C. It was then centrifuged at 500g for 6 minutes, and subsequently passed through a 40 µm filter (Cell Strainer, BD Biosciences Discovery Labware). The filtered cells were then cultured at 37°C in Alpha-MEM (Biological Industries) supplemented with 20% FBS (BioWest). The culture medium was refreshed every three days. When the cells reached 80.0%-85.0% confluence, adherent DPSCs were detached with trypsin (Grisp) and trypan blue dye exclusion for cell viability. The flow cytometry (Accuri, Becton Dickinson Biosciences) was used for analyzing the surface markers of DPSCs at passage 2 (P2). Cells were incubated in the dark with anti-human antibodies (Becton Dickinson Pharmingen), including CD34-phycoerythrin (PE), CD105-PE, CD45-allophycocyanin (APC), CD90-fluorescein isothiocyanate (FITC), and CD73-FITC. The analysis was performed using BD CSampler Plus Analysis Software (Becton Dickinson Biosciences). To assess their differentiation potential, DPSCs at P2 were seeded in 24-well culture plates (10 000 cells/mL) and cultured until reaching confluence. Osteogenic differentiation was induced by incubating the cells for 21 days in an osteogenic differentiation medium containing (DMEM-LG (Gibco), 10% Fetal Bovine Serum (FBS), 10^-7 M dexamethasone (Sigma), 0.2 mM ascorbic acid (Sigma) and 10 mM glycerol 2-phosphate (Sigma), 1% Penicillin-Streptomycin, 1% L-glutamine). The cells were induced adipogenically by culturing them for 21 days in adipogenic differentiation medium containing (DMEM-LG (Gibco), 10 mM indomethacin (Santa Cruz Biotechnology, Oregon, USA), 0.5 mM 3-isobuthylmethyl-xanthine (Sigma), 1 μM dexamethasone (Sigma), 10 μg/mL insulin (Santa Cruz Biotechnology), 1% Penicillin-Streptomycin, 1% L-glutamine). All cultures were maintained at 37°C in a 5% CO₂ incubator and medium replacement was performed three times per week. Osteogenic and adipogenic differentiation of DPSCs were assessed by microscopic examination after staining with Alizarin red (Sigma) and Oil red-O (Sigma), respectively.

Evaluation the Cytotoxic Effect of Proanthociyanidin and Nisin

DPSCs (P2) were harvested in 96-well plate at concentration of 10.000 cells/ml. After cell adherence, LPS (lipopolysaccharides from Escherichia coli O111:B4; Sigma-Aldrich, St. Louis, MO, USA) was replaced with a medium containing 2 𝜇g/mL and cultured at 37°C in a 5% CO2 incubator for 24h. A culture condition without LPS was prepared as a control group. Different concentration of Nisin (Ni, from Lactobacillus lactis, Sigma Aldrich, USA) and Proanthocyanidin (PA, Sigma Aldrich, USA) were applied to the cell culture plate at 25-50-75-100-200 µg/ml for 24h to evaluate cytotoxicity. A water-soluble tetrazolium-based assay [10% WST-1,4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzenedisulfonate] was used for evaluating the cell metabolic activity for 2 hours. Subsequently, 100 µl of the medium from each condition was transferred to enzyme-linked immunosorbent assay (ELISA) microplates (96-well plates, Corning Life Sciences). The absorbance values of the solutions at 450 nm were determined spectrophotometrically (n=3).

Cytokine Array and ELISA

Detection of cytokines and chemokines released in conditioned medium was performed with the human Cytokine Array C5 (AAH-CYT-5-8) by following the manufacturer’s instructions (RayBiotech, Norcross GA). Densitometry analyses were carried out utilizing the Gel Doc 2000 imaging apparatus and ImageLab software (Bio-Rad, Mississauga, ON). All values were normalized with respect to the mean intensity of the positive and negative controls (n=3). The concentrations of IL-6, IL-8 and TNF alpha in conditioned medium were measured using the human IL-6, IL-8 and TNF alpha ELISA kits following the manufacturer’s instructions (Nephente, Kocaeli, Turkey). The absorbance of the solutions was determined spectrophotometrically at 450 nm (n=3).

Evaluation of Antibacterial Activity of Proanthocyanidin and Nisin

Antibacterial activity of PA and Ni were tested against a Gram (+) strain Staphylococcus aureus (S. aureus, ATTC 6338), and a Gram (-) strain Escherichia coli (E.coli, ATTC 8739) using drop plate method (21). The cultures of bacteria were grown in Nutrient Broth (NB) medium at 37 °C for overnight incubation at 100 rpm. Different concentrations of PA and Ni (25, -50, -75, -100, -200 µg/ml) were added into culture tubes of E. coli and S. aureus (0.5 McFarland) and incubated at 37 °C with continuous shaking at 100 rpm for 24 h. After incubation time, the samples were taken, serially diluted in PBS buffer, subsequently 10 μL were dropped on Nutrient Agar (NA) agar plates. The plates were incubated at inverted position overnight at 37 °C for 24 h. After incubation, the colonies on agar plates were counted. The logarithm to the base 10 (log) of the cell counts was taken for statistical evaluation (n=3).

Statistical Analysis

All experiments were conducted three times (mean ± standard deviation) and analyses were done with SPSS 16.0 (SSP Inc., Chicago, IL, USA). Comparisons between experimental groups were conducted using the Student's t-test. Statistical significance was determined for results with a p-value < 0.05.

Results

Characterization of Dental Pulp Stem Cells

Typical spindle-shaped morphologic characteristics of cultured DPSCs were shown in Figure 1a. Flow cytometry analysis revealed that DPSCs exhibited positive staining for mesenchymal stem cell markers CD73, CD105, CD90 (˃87%) and negative staining for hematopoietic cell markers CD34, CD45 (˂5%), as illustrated in Figure 1b. The multilineage differentiation of DPSCs was confirmed using Alizarin red staining for osteogenesis and Oil red staining for adipogenesis on the 21st days of the culture (Figure 1c).

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

Cell Viability

The cytotoxic effect of PA and Ni on LPS induced or control DPSCs was determined by a water-soluble tetrazolium-based assay. Since 25, 50 µg/ml concentrations of PA and Ni did not show any effects on viability of DPSCs (data not shown), PA and Ni at 75, 100 and 200 µg/ml concentrations were evaluated (Figure 2). DPSCs conditioned with 75 μg/ml PA (1.48-fold, p<0.05) and 75 µg/ml Ni (1.37-fold, p<0.05) showed significantly higher cell viability results compared to DPSCs alone condition (Control). DPSCs showed significantly lower cell viability when conditioned with increasing concentrations of PA (2.80-fold and 2.85-fold for 100 and 200 µg/ml PA respectively, p<0.05). LPS induction of DPSCs for 24h resulted in significantly lower cell viability compared to DPSCs control (1.37- fold, p<0.05). At 100 μg/ml Ni containing condition, LPS-induced DPSCs showed significantly higher cell viability results than control DPSCs (1.83 – fold, p<0.05). Cell viability of LPS induced DPSCs was tended to increase after conditioned with 75 µg/ml PA and 200 µg/ml Ni (1.2- fold, p>0.05).

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).

Antibacterial Activity of Proanthocyanidin and Nisin

Antibacterial properties of PA and Ni were also evaluated. Figure 3a and c shows the bacteria colony counts results and Figure 3b and 3d shows representative photographs of agar plates with the counts of bacteria after 24 h incubation time for different concentrations of PA. Firstly, 25-50 and 75 µg/ml of PA concentrations did not show antibacterial activity against E. coli. However, in 100 µg/ml PA concentration, live bacteria number began to decrease compared to control bacteria. In the end, 200 μg/mL PA completely killed E. coli. Conversely, PA exhibited significant antibacterial activity against S. aureus at all concentrations. Figure 3e and Figure 3g show the bacteria colony counts results as well as Figure 3f, and Figure 3h shows representative photographs of agar plates with the counts of bacteria after 24 h incubation time for different concentrations of Ni. As shown in Figure 3e and 3f, Ni did not show antimicrobial activity at the 25-50-75-100 µg/ml concentration for E. coli. The number of E. coli bacteria increased until all bacteria were killed at 200 µg/ml concentration. However, as illustrated in Figure 3g and 3h, Ni showed a strong antibacterial property against S. aureus at all concentrations.

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).

TNF-α, IL-6 and IL-8 Expressions of DPSCs

To determine the expression of TNF-α, IL-6 and IL-8 from DPSCs, DPSCs were cultured with or without 2 μg/ml LPS for 24h and were conditioned for 24 hours with 0 (control), 75, 100, and 200 μg/ml PA and Ni concentrations. Figure 4, and 5 shows the expression levels of TNF-α, IL-6 and IL-8 of DPSCs and LPS-induced DPSCs, respectively. According to the results detected by ELİSA methods, for the groups of DPSCs, TNF-α expression of DPSCs significantly decreased at 75 μg/ml PA, 75 and 100 μg/ml Ni concentrations compared to control condition (1.24-fold, 1.49-fold and 1.44-fold, respectively, p<0.05). LPS induction of DPSCs significantly upregulated the TNF-α expression compared to control DPSCs (1.24-fold, p<0.05). For LPS induced DPSCs, TNF-α expression significantly decreased at 100 and 200 μg/ml PA and at 200 μg/ml Ni compared to LPS-induced DPSCs (1.42-fold, 1.36-fold, and 1.44-fold respectively, p<0.05). The TNF-α expression tended to decrease at concentrations of 75 μg/ml PA and at 75-, 100 μg/ml Ni. IL-6 expression of DPSCs increased after PA and Ni conditioning, but it was only statistically significant at 100 μg/ml PA and 75 μg/ml Ni concentrations (1.74- fold and 1.45- fold, respectively, p<0.05). IL-6 and IL-8 expression of DPSCs did not change after LPS stimulation (p>0.05). IL-6 expression of LPS induced DPSCs increased after PA and Ni stimulation but only statistically significant at 100 and 75 μg/ml PA (1.5- fold and 1.46- fold, respectively, p<0.05). IL-8 expression of DPSCs significantly decreased at 75, 100 and 200 μg/ml PA conditioned concentrations compared to controls (1, 41-fold, 1.31-fold and 1.30-fold, respectively, p<0.05). IL-8 expression of LPS induced DPSCs increased significantly at 100 μg/ml PA and decreased at 200 μg/ml Ni conditioned concentration compared to only LPS induced DPSCs (1.06-fold and 1.15- fold, respectively, p<0.05).

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).

Cytokine Array Analysis

Finally, our objective was to determine the pattern of 80 cytokines/chemokines/growth factors released by the LPS induced, PA or Ni conditioned DPSCs and compared it with the condition of LPS induced DPSCs. This was accomplished using a semiquantitative antibody-based cytokine array (RayBiotech, AAHCYT-5-8). The levels of IL-6, IL-7, IL-8, tissue inhibitor matrix metalloproteinases (TIMP-2) and osteoprotegerin (OPG) of LPS induced DPSCs conditioned with 0, 75 and 100 μg/ml PA, 0, 100 and 200 μg/ml Ni normalized versus those of LPS induced DPSCs control condition (Figure 6). OPG secretion of LPS-induced DPSCs conditioned with 75 and 100 μg/ml PA concentrations was at higher levels (92-fold and 55-fold respectively). IL-8 and TIMP-2 secretion was higher at PA and Ni conditioned DPSCs compared to LPS induced DPSCs condition. IL-7 secretion was higher at 200 and 100 μg/ml Ni, 75 μg/ml PA concentrations (1.63- fold, 1.18- fold and 1.42-fold, respectively).

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).

Discussion

DPSCs are promising candidates for stem cell-based regenerative therapies, from regenerative endodontic applications to central nervous disorders, due to ease of isolation, immunomodulatory, and anti-inflammatory capacities (22). Conditioning stem cells with stress-forming inducers such as LPS, hypoxia, and reactive oxygen radicals for mimicking the possible clinical environment of damaged tissue, and then inducing with a molecule/extract/material to improve cell proliferation, paracrine ability and therapeutic potential is among the stem cell-based studies (23). Using biologically compatible materials and the minimization of the possible adverse impacts on biological tissues is essential for obtaining the most favorable results in biomedical applications, and that may be exerted based on such basic/preclinic research results. Trends for using natural products as a biomaterial in the biomedical field have increased dramatically during the last decade. Natural products are usually either of prebiotic-derived or derived from microbes, plants, and animal sources. In the present study, biological effects of various concentrations of grape seed-derived “proanthocyanidin” and Lactobacillus lactis-derived “nisin” on DPSCs, and also antibacterial effects against gram-positive S. aureus and gram-negative E. coli were evaluated. LPS from E coli was used to as a bacterial inducer for pulpitis because it has been found to be most frequent inducer in the literature (24), although it is not a microorganism involved in caries formation.

Oligomeric proanthocyanidins (OPCs), a group of polyphenols in plants such as grapes and cranberries, are well known for their naturally occurring antioxidant activity and the scavenging of free radicals directly (25). Dos Santos et al. (26) evaluated the metabolism of pulp cells following both direct and indirect contact with the different concentrations of grape extract (0.0065–6.5%) for 1 h. They confirmed the non-cytotoxicity of OPCs in both direct or transdentinal conditions and recommended further experiments for establishing its stimulating potential. Tissue regeneration capacity of OPCs (as a crosslinking agent in Type I collagen membrane) and antibacterial properties have also been evaluated and it has been reported that 10% OPCs-Col membrane was non-toxic, and it stimulated the proliferation of L929 and MG-63 cells in the in vitro study. Cardoso et al. (27) evaluated the cytotoxic and genotoxic effects of various concentrations (100, 50, 10, and 5 μg/mL) of grape seed extract (GSE) on human gingival fibroblasts (HGF). They reported that the highest GSE concentrations (100 and 50 μg/mL) were both genotoxic and cytotoxic on HGF and GSE, while GSE at concentrations of 10 and 5 μg/mL enhanced cell viability after a 24h period. In our study, we found that PA at 75 μg/ml concentration increased the cell viability of both DPSCs (significantly) and LPS-induced DPSCs (not significantly). Ni is considered as a safe, natural food preservative and it has also attention as a potential therapeutic alternative to antibiotics. To date, immunomodulatory role of Ni and effects of this peptide on some oral bacteria species have been evaluated (15, 28). Although its usage together with dental materials was studied or recommended, so far, there has been no available study regarding the biologic effects of Ni to DPSCs. In a study by Eftekhari et al. (29), nisin was found to enhance the differentiation of induced pluripotent stem cells into neuronal lineages. Namjoo et al. (30) showed that preconditioning with Ni improved the viability and the anti-apoptotic capacity of MSCs. Similarly, in our study Ni at 75 and at 100 μg/ml elevated the cell viability of DPSCs and LPS-induced DPSCs significantly.

TNF-α, IL-6 and IL-8 expressions of DPSCs and LPS-induced DPSCs after exposed to distinct concentrations of PA and Ni were analyzed by ELİSA. TNF-α is a prominent proinflammatory cytokine known to elevate protein expressions associated with cellular response to stress and apoptosis (31). It has been reported that oligomerized grape seed polyphenols at 20 μg/ml showed decreased inflammatory changes by lower TNF-α expression in adipocyte and macrophage co-culture condition (32). Another study, it was highlighted that decreased IL-6, IL-8 and TNF-α expression was obtained in human colorectal adenocarcinoma cell line Caco-2 after treatment of grape seed extract (GSE), with or without LPS (25 μg/mL). As for the effect of Ni on TNF-α expression, it was exhibited in a previous study (19) that Ni Z, a class of lantibiotics, at 50 μg/ml induced the secretion of IL-8 and reduced TNF-α expression in response to bacterial LPS in the human peripheral blood mononuclear cells. Ni Z may selectively modulate host immune responses and contribute to protective host immunity with its immunomodulatory activities (19). Similar to what has been found in the literature, PA at 75μg/ml and Ni at 75 and 100μg/ml decreased TNF-α expression of DPSCs significantly in our study. As for LPS-induced DPSCs conditions, PA at 100 and 200 μg/ml and Ni at 200 μg/ml decreased TNF-α significantly. Ni at 75 μg/ml, PA at 75 and 100 μg/ml have significantly increased IL-6 synthesis in DPSCs, and an upregulation trend in LPS-induced DPSCs was also observed, although not significantly. This may be explained by the immunoregulatory role of IL-6, a dual function that possess both pro-inflammatory and anti-inflammatory (or regenerative) properties (33). It is a biologically active factor secreted by MSCs and has many biological functions such as regulating migration as well as stimulating mitosis and angiogenesis (34). It was observed that osteogenically differentiated DPSCs showed remarkably higher IL-6 expression than undifferentiated DPSCs (35). Similarly, IL-6-stimulated DPSCs exhibited higher osteogenic differentiation and stronger osteogenic markers than non-stimulated DPSCs (36). In our study, although LPS induction did not alter the IL-6 expression in DPSCs, higher expression of IL-6 due to PA may be explained by the induction of the regenerative process, which increased TIMP-2, and OPG. Besides, after material induction, increased cytokine releasing such as IL-1𝛼, IL-1𝛽, IL-6, and IL-8 from mineralizing cells, mild and acute inflammatory responses may also contribute to pulp, and hence the clinical repair (37). IL-8 is one of the pro-inflammatory and immunomodulatory mediators that were defined as a chemoattractant of neutrophils, recruited in acute inflammation, as well as chemotactic of endothelial cells with a major role in angiogenesis. It has also a major role in odontoblast defense against dentin-invading bacteria. Increasing IL-8 expression was reported in osteogenically differentiated MSCs and also in LPS-stimulated odontoblasts (37). The results obtained in our study show that IL-8 expression in DPSCs decreased at all PA concentrations, but after LPS stimulation they increased at PA 100 significantly, and at Ni 200 vice versa. However, they did not change at other concentrations. Previous studies (38, 39) reported anti-inflammatory effects of bioflavonoids due to lower IL-8 expression but is difficult to compare the studies since there were differences in doses, cells and types of bioflavonoid source. Kindrachuk et al. (19) reported that Ni Z at 50 μg/ml induced the IL-8 expression in PBMCs, hence it may modulate the host immune response. Only Ni at 100 μg/ml (not significantly) increased IL-8 expression of DPSCs in our study. IL-8 expressions regulate in stimulus-specific and cell type-specific manner (40). PBMCs, hematopoietic stem cell-derived, and DPSCs may show distinct expression patterns to Ni. It has been stated in a review regarding the immunomodulatory properties of Ni that it has anti-inflammatory effects on the infected organism. However, the discrepancies in the results of studies with regard to the effect of Ni on cytokine production were reported as resulting in the differences in the experimental models (different types of cells, concentrations of nisin, or incubation times) (41). To our knowledge, our study represents the first report on the effects of Ni on DPSCs.

Matrix metalloproteinases (MMPs), a group of host-derived proteolytic enzyme are responsible for breaking down extracellular matrix (ECM) components in both physiologic and pathologic conditions (42). The activation of MMPs from proenzymes and their tissue inhibitors (TIMP) control the catalytic activity of MMPs. Inhibiting the secretion of MMPs could be an effective strategy to prevent and manage pathological tissue damage (42). La et al. (11) observed no obvious cytotoxic effects (cell viability up to 90%) after the 24h induction of macrophages with up to 100 μg/ml GSE that contains 52% PA. They treated macrophages with different concentrations of GSE (0, 25, 50, and 100 μg/ml) for 2h and then stimulated the cells with LPS (1μg/ml) for 24h to assess the impact of this extract on MMP secretion. Their findings indicated that non-toxic concentrations of GSE inhibited the secretion of MMP-1, -3, -7, -8, -9, and -13 by LPS-stimulated macrophages, thus indicating that the polyphenols in GSE may assist in preventing the excessive accumulation of MMPs. According to our cytokine array results, both Ni at 100, 200 and PA at 75, 100 μg/ml concentrations expressed higher TIMP-2. TIMPs are specialized inhibitors that form a stable complex with MMPs. TIMPs can directly promote ECM deposition by inhibiting the MMPs, hence ECM proteolysis, and indirectly regulate ECM turnover (43). In the present study, it was observed that when DPSCs are exposed to stressful conditions, they secrete greater amounts of neurotrophic factors, such as BDNF, GDNF, as well as TIMP-2 which helps protect the tissue integrity by inhibiting MMP activity (44). Higher expressions of TIMP-2 were also observed in inflamed dental pulp compared to the healthy dental pulp (45). Similarly, DPSCs exposed to LPS are under stressful conditions and have less cell viability and more TNF-α expression in our study. Higher TIMP-2 expression of LPS-induced DPSCs after exposure to Ni at 100, 200, and PA at 75, 100 μg/ml concentrations may show increasing ECM accumulation by which these molecules provide a favorable environment. Interleukin-7 (IL-7) is a multipotent cytokine that plays a vital role in maintaining the homeostasis of the immune system by regulating T-cell development, proliferation, differentiation and B-cell maturation (46). Its inhibitor effect on osteoclastogenesis was also shown in previous studies (47, 48). The human IL-7 overexpression in the osteoblast lineage of mice resulted in higher trabecular bone volume by mCT in vivo and lower osteoclast formation (48). Trubiani et al. (49) showed that IL-7 expression increased during osteogenic differentiation of the periodontal ligament-derived MSCs, and they highlighted its function in promoting autocrine growth and delivering survival and differentiation signals to the adjacent odontogenic structures. However, controversial results are present regarding the effects of IL-7 in bone metabolism. There are studies reporting its roles in osteoclastogenesis activity or in suppressing osteogenic differentiation (50, 51). As for the expression of IL-7 in dental pulp, Elmeguid et al. (52) observed lower IL-7 expression in both pulp tissues with reversible and irreversible pulpitis compared to that in healthy controls. In the present study, higher IL-7 expression was observed in Ni at 200, PA at 75 μg/ml compared to only LPS-induced DPSCs. The effect of OPCs and Ni on IL-7 expression needs to be investigated in a greater number of studies. Higher OPG expression of LPS-induced DPSCs when conditioned with PA 75 and 100 μg/ml in our study may be attributed to the potential of PA promoting the odontoblastic differentiation and mineralization process. It has been reported that OPG inhibits osteoclast differentiation through binding to the receptor activator of nuclear factor-kB ligand (RANKL) and preventing RANKL from interacting with RANK, and thereby the OPG/RANKL ratio is an indicator of bone health and reflects the balance between bone formation and resorption (53). High expression of OPG in the odontoblastic layer of healthy and inflamed peripheral pulp samples has been shown (54). OPG and ALP expressions are commonly considered markers of odontoblastic differentiation. Huang et al. (55) reported that platelet-rich fibrin enhanced the cell proliferation and differentiation of DPSCs by up-regulating OPG and ALP expression. As a result, it might have potential in reparative dentin formation. Similarly, OPG expression slightly increased in DPSCs under tension (56). Belisibakis et al. (57) suggested that the increased expression of OPG could provide protection against dentine or bone resorption, and homeostatic balance might shift to tissue formation. Kwak et al. (10) found that grape seed proanthocyanidin significantly descends (RANKL)-induced osteoclast differentiation, hence the activity of mature osteoclasts in bone resorption. They also showed that GSPE protects against LPS-induced bone loss in mice. According to the results of our study, PA at 75 μg/ml concentrations may offer a favorable environment for dentin formation by inducing OPG and TIMP-2 expression and increasing the proliferation of LPS-induced DPSCs.

When an antimicrobial agent confronts its target, it must initially break down through the microbial cell wall. Considering the differences in the cell wall structures, the effect of the antimicrobial agent to be applied can be different due to these two different cell wall characteristics (58). From this point of view, in this study one Gram (+) bacterium strain, Staphylococcus aureus and one Gram (-) bacterium strain, Escherichia coli were evaluated in antibacterial activity. PA at 75 and 100 μg/ml concentrations showed antibacterial activity against E. coli and both PA and Ni at 200μg/ml concentrations completely killed E. coli. However, the number of bacteria increased when exposed to Ni at concentrations lower than 200 μg/ml. Since Ni is well known for its potent antibacterial activity against Gram-positive bacteria, but not against Gram-negative bacteria, the number of E. coli bacteria increased until all bacteria were killed at 200 µg/ml concentration. The reason for the insensitivity of Gram-negative bacteria to Ni could be attributed to the relatively large size (1.8–4.6 kDa) of Ni, hindering its passage through the outer membrane of Gram-negative bacteria. The polyphenols, likewise proanthociyanidin within the GSE, can eradicate Gram-positive and Gram-negative bacteria by modifying the microbial cell permeability and thereby affecting pathways such as nucleic acid synthesis, cell cytoplasmic membrane function, and bacterial metabolism (59). Grape seed extract was used as an endodontic irrigant solution in a previous study (60), and antibacterial activity of 6.5% GSE was exhibited against Gram positive bacteria E. faecalis biofilm through confocal laser scanning microscopy. In a recent review on the antimicrobial activity of GSE in endodontic disinfection, it has been reported that GSE exhibited a noteworthy antimicrobial activity, and the effectiveness of GSE was found to be associated with factors such as concentration, physical state, and exposure duration (59).

The present study has several limitations: it might have been more beneficial to evaluate the expression levels of IL-6, IL-8, and TNF-α by normalizing to the cell counts and to assess the cytokines with anti-inflammatory properties. In addition to S. aureus and E. coli, investigating more predominant bacterial species associated with caries and endodontic infections (61, 62) would allow for a more comprehensive assessment of the antibacterial activity of PA and Ni.

Conclusion

The impacts of PA and Ni on both healthy DPSCs and on LPS-induced DPSCs, with the aim of mimicking the harsh conditions of inflamed pulp, were evaluated in this study. According to the 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. Besides, it exhibited higher expressions of TIMP-2, OPG, and IL-7 in LPS-induced DPSCs, thus suggesting that PA at 75μg/ml may induce DPSCs toward osteoblastic/odontoblastic differentiation and further hard tissue formation. Ni at 100 μg/ml showed a tendency to increase DPSCs viability and it decreased TNF-α expression. As for the LPS-induced DPSCs, it increased cell viability and showed a tendency to decrease TNF-α expression. Also, Ni at 200 μg/ml decreased TNF-α expression significantly. As natural compounds, both Ni and PA have strong antibacterial effects on Gram-positive bacteria, which could provide a decrease the microbial load. Ni at 200μg/ml provided strong antibacterial effects against Gram-negative bacteria without negatively affecting the viability of both DPSCs and LPS-induced DPSCs. Also, it showed anti-inflammatory activity by decreasing TNF-α expression. Although PA at 200 μg/ml provided strong antibacterial effects against Gram-negative bacteria, its negative effect on DPSCs viability should be considered. Further study is needed to evaluate the osteogenic/odontogenic properties of PA stimulated DPSCs by checking the odontogenic and osteogenic gene expression profiles.