Effects of aqueous and ethanolic extracts of Chinese propolis on dental pulp stem cell viability, migration and cytokine expression

Preparation of propolis extracts

Commercially available propolis (Stakich Bee Propolis Chunks-Pure, Natural, Amazon), collected from bee farms in the Zhejiang Province of mainland China, was crushed with a mortar to obtain a fine powder and extraction was performed as described by Khoshnevisan et al. (2019) with some modifications for the aqueous extract of propolis (AEP) extracts. Fifty milliliters of either Millipore water or ethanol 70% was added to 5 g of propolis powder and incubated for 4 h at 37 °C with agitation. The solution was centrifuged for 10 min at 800× g. The supernatant of AEP was filter-sterilized (polyethersulfone membrane with 0.22 µm pores), aliquoted and stored at −80 °C. The supernatant of ethanolic extract of propolis (EEP) was collected and dehydrated in vacuum desiccator for 24 h to remove ethanol. The dried EEP collected after complete evaporation of the solvent (70% ethanol in water) was re-dissolved with 50 mL of water under agitation for 2 h at 37 °C. Then, the EEP extract was filter-sterilized (Polyethersulfone membrane with 0.22 µm pores), aliquoted and stored at −80 °C. Before use, the propolis stock solutions (100 mg/mL) were warmed up to 37 °C to dissolve any precipitate and diluted in culture media to obtain concentrations from 33 to 0.03 mg of propolis/mL (Elkhenany, El-Badri & Dhar, 2019; Esmaeilzadeh et al., 2022). For experiments testing the effects of propolis solvent, the solvent of each propolis extract was prepared as described above but without propolis powder.

Cell culture

Commercially available human DPSCs (ref: PT-5025; Lonza, Walkersville, MD, USA) were grown in α-Minimum Essential Medium (α-MEM, Sigma-Aldrich, St. Louis, MO, United States) supplemented with 10% fetal bovine serum, L-glutamine (2 mM), ascorbic acid (100 mM), penicillin (100 U/mL), streptomycin (100 mg/mL), and amphotericin B (2.5 mg/mL). Cells were maintained at 37 °C/5% CO₂ and used in experiments at passage 2 to 4. DPSCs were plated at a density of 5,000 cells/cm², and culture medium was changed every 3–4 days (Michot, Casey & Gibbs, 2020).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay

DPSCs were plated in 96-well plates and treated with either control media, AEP or EEP at different concentrations for 3, 4 or 7 days. After treatment, DPSCs were incubated with MTT (0.5 mg/mL) for 2 h at 37 °C. The MTT metabolic product formazan was solubilized with HCl 40 mM in isopropanol for 30 min with agitation. Absorbance was measured at 570 nm using a plate reader (Michot, Casey & Gibbs, 2020).

DAPI staining

DPSC proliferation was evaluated by quantifying the number of cells nuclei, with DAPI staining, after treatment with control media or propolis extracts. DPSCs were plated in 12-well plates and treated with control media, AEP or EEP (0.03–33 mg/mL) for 7 days. After incubation, the cells were washed with phosphate-buffered saline (PBS) and fixed with formalin 4% for 15 min. Non-specific staining was prevented by 1 h incubation in a blocking solution (1% triton + 1% bovine serum albumin in PBS). Cells nuclei were stained with DAPI (1 µM in PBS) for 15 min at room temperature. The cells were washed with PBS three times for 1 min before visualization with a fluorescent microscope (Revolve, Echo). From each cell culture well, five images were collected and the quantification of the number of stained nuclei was performed by a blinded experimenter, using the analyze particle tool of the Image J Software.

Migration assay

Control media, propolis extracts (0.03, 0.1 or 3.3 mg/mL) and TAP (obtained from the Harvard School of Dental Medicine clinic; 10 mg/mL clindamycin, 10 mg/mL metronidazole and 10 mg/mL ciprofloxacin; 30 µL TAP in 0.7 mL media media) were placed in wells of a 12-well plate. DPSCs (10,000 cells/cm²) were seeded on Boyden Chamber Transwell inserts containing a permeable membrane with 8.0 µm pores allowing the migration of cells from the top to the bottom surface of the membrane (MCEP12H48; Millipore). After 2 h resting to allow cell adhesion to the membrane, the transwells with the cells were placed in the 12-well plate containing propolis/TAP/control media and were incubated for 2 days at 37 °C/5% CO₂. The cells on the top face of the membranes, that did not migrate, were removed with a cotton swab and then the cells that migrated on the bottom face of the membrane were fixed with formalin 10% for 15 min. The cells were then stained with crystal violet (1% in water) for 20 min at room temperature. After thorough washing with millipore water, cells were visualized with light microscope (Revolve, Echo), and four images were collected from each transwell. The number of cells was manually counted by a blinded experimenter (Takeshita-Umehara et al., 2023).

Alizarin red staining

DPSCs were treated with either control media, odontogenic differentiation media (ODM; β-glycerophosphate 5 mM + dexamethasone 100 nM in supplemented αMEM), AEP (0.1 mg/mL) or EEP (0.1 mg/mL). After 21 days treatment DPSCs were fixed with 10% formalin in PBS for 15 min. DPSCs were washed with PBS, incubated with alizarin red (2% in water, pH 4.1, 15 min) and washed with deionized water before microscopic visualization of calcium nodule formation. Then, the stain was extracted by 16 h incubation with 5% isopropanol + 10% acetic acid in water and absorbance was measured at 405 nm (Michot, Casey & Gibbs, 2020; Baldión, Velandia-Romero & Castellanos, 2018).

RNA extraction and quantitative Polymerase Chain Reaction (qPCR)

DPSCs were treated for 14 days with control media, ODM, AEP (0.1 mg/mL) or EEP (0.1 mg/mL) for differentiation assays, or 2 days with control media, lipopolysaccharide (LPS; 0.1 µg/mL), LPS + AEP (0.1 mg/mL) or LPS + EEP (0.1 mg/mL) for the quantification of cytokine expression. Total RNA was extracted using RNAzol per manufacturer’s instructions, and the quality and quantity were measured using a Nanodrop. cDNA synthesis from 1 μg of total RNA and elimination of genomic DNA were performed using RT2 First Strand Kit (Qiagen). The PCR amplification was performed using 10 ng of cDNA, TaqMan Fast Advanced Master Mix (Applied Biosystem) and Assays-on-Demand Gene Expression probes (final concentration of 900 nM for primers and 250 nM for the probe; Applied Biosystems) for the following target genes: Dentin Sialophosphoprotein (DSPP, Hs00171962_m1), Dentin matrix protein (DMP1, Hs01009391_g1), Matrix Extracellular Phosphoglycoprotein (MEPE, Hs00220237_m1), alkaline phosphatase (ALP, Hs03046558_s1), Interleukin 1 beta (IL1b, Hs01555410_m1), Interleukin 6 (IL6, Hs00174131_m1), Interleukin 8 (IL8, Hs00174103_m1) and chemokine (C-C motif) ligand 2 (CCL2, Hs00234140_m1). The PCR reaction, in triplicate for each sample, was performed with a StepOnePlus Real-Time PCR System (Applied Biosciences) for 40 amplification cycles as follows: 15 s denaturation at 95 °C and 60 s annealing/elongation at 60 °C. Specific RNA levels were calculated using the ΔΔCT method (Livak & Schmittgen, 2001) and normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Hs02758991_g1). The efficiency of the primers was evaluated as previously described by Dankai, Pongpom & Vanittanakom (2015) (Fig. S1). The validity of the results was checked with appropriate controls including reverse transcription control and negative PCR control (Michot, Casey & Gibbs, 2020).

Data analysis

Statistical analyses were performed and figures were generated using GraphPad Prism 5 Software. Data are expressed as the mean and standard error of the mean (SEM) (Lee, In & Lee, 2015). The homogeneity of variances was tested with the Brown–Forsythe test. The effects of AEP and EEP in the MTT assay were analyzed with two-way ANOVA followed by the Bonferroni’s post-hoc test. All other data were analyzed using one-way ANOVA followed by the Dunnett’s post-hoc test for comparison to the control group or Tukey’s test for multiple comparisons. Kruskal-Wallis test followed by Dunn’s test was used when normality test failed. Significance was set at p < 0.05.

Effects of propolis extracts on DPSC viability/proliferation

DPSC viability/proliferation was evaluated using the MTT assay and it was first determined that the majority of the solvent dilutions of both AEP and EEP did not affects cell viability. Only the highest concentration of solvents (1/3 dilution, corresponding to the solvent dilution contained into the propolis extract solution at 33 mg/mL) slightly decreased DPSC viability compared to control group (Fig. S2). Aqueous extract of propolis (AEP) induced a dose-dependent decrease in DPSC viability/proliferation compared to the control group. A significant decrease in DPSC viability/proliferation was observed after 4 days treatment with AEP doses higher than 10 mg/mL (p = 0.008 and p < 0.001 for AEP 10 and 33 mg/mL respectively; Fig. 1A). Longer treatment times further affected DPSC viability, as the AEP doses higher than 3.3 mg/mL decreased DPSC viability/proliferation. AEP at doses lower than 1 mg/mL did not change DPSC viability/proliferation for up to 7 days treatment (Fig. 1A).

DPSC proliferation was also evaluated by quantifying cell numbers using DAPI to stain cell nuclei. Seven days treatment with the four lowest doses of AEP (0.03–1 mg/mL) did not affect the number of cells compared to the control group (p > 0.95; Figs. 1B, 1C) indicating a similar cell proliferation in these experimental groups. However, the number of DPSC treated with the highest doses of AEP is decreased compared to the control group (cell number: 125+/−10; 78+/−11; 61+/−10; 25+/−2 for control, AEP 3.3, 10, and 33 mg/mL respectively; p < 0.01; Fig. 1B).

Similar results were obtained in DPSCs treated with EEP. Both MTT and DAPI staining data show that the EEP doses from 3.3 to 33 mg/mL dose-dependently decreased DPSC viability/proliferation (p < 0.05) and the lowest EEP doses (0.03 to 1 mg/mL) did not affect viability/proliferation compared to control group (Fig. 2).

Interestingly, when comparing the effects of AEP and EEP to each other, our data shows that although both propolis extracts decreased DPSC viability/proliferation, the AEP was less cytotoxic than EEP (Fig. 3). This difference was more marked with quantification of cell numbers with DAPI staining, as EEP 0.33–10 mg/mL treated groups have a lower number of DPSC than AEP 0.33–10 mg/mL treated groups (p < 0.002; Fig. 3B).

Effects of propolis on DPSCs migration and differentiation

DPSC migration and differentiation are two key processes for regenerative endodontics. First, the effects of propolis extracts were tested on DPSC migration using Boyden Chamber Transwell inserts. A 48 h treatment with AEP significantly increased the number of DPSC that migrated to the lower surface of the transwells, with a maximum effect observed for the 0.1 mg/mL dose (number of cells: 2.3 ± 0.5, 14.7 ± 3.0, 25.4 ± 4.2, in control, AEP 0.03 and 0.1 mg/mL treated groups respectively, p < 0.002, Fig. 4). Similar effects were found after treatment with EEP, although EEP 0.1 mg/mL is slightly less effective than AEP 0.1 mg/mL to induce DPSC migration (Fig. 4). In addition, the effects of TAP were evaluated on stem cell migration. TAP, one of the most used intra-canal medications, and propolis have been shown to have antibacterial effects but whether TAP would be of potential interest for stem cell migration compared to propolis is not known. We show that TAP did not induce DPSC migration compared to control group (p > 0.99) suggesting propolis might have a better biological profile as an intracanal medication for regenerative endodontic procedures.

The effects of propolis on DPSC differentiation into mineralizing cells were evaluated using Alizarin red staining. DPSCs treated with odontogenic differentiation media for 21 days, which serves as positive control, shows formation of numerous calcium nodules, whereas both AEP and EEP did not induce the formation of calcium deposits in DPSCs (Figs. 5A, 5B). Similarly, gene expression analysis showed that alkaline phosphatase (ALP) was not expressed (Ct value >37.98 for each experimental groups at day 7 and 14; a CT value >35 indicates the absence of expression) and the expression of the odontoblast markers dentin matrix protein (DMP1), matrix extracellular phosphoglycoprotein (MEPE) and dentin sialophosphoprotein (DSPP) were not increased by treatment with AEP or EEP (Figs. 5C–5E), suggesting that propolis does not affect DPSC differentiation into mineralizing cells.

Effects of propolis on pro-inflammatory cytokine expression

To determine whether propolis extracts influence DPSC inflammatory response, DPSC were stimulated for 2 days with the bacterial toxin lipopolysaccharide (LPS; 0.1 µg/mL; L3129; Sigma-Aldrich, St. Louis, MO, United States) in presence or absence of AEP (0.1 mg/mL) or EEP (0.1 mg/mL) and evaluated cytokine mRNA expression. LPS alone increased the expression of IL1b, IL6, IL8 and CCL2 (fold increase: ×10, ×19, ×62, and ×37 respectively compared to control media treated group; p < 0.001; Fig. 6). This increase in cytokine expression was only marginally prevented by AEP as only IL1b expression was significantly reduced (9.9+/−4.0 vs. 1.8+/−0.4 in LPS and LPS + AEP treated group, p = 0.03; Fig. 6). However, EEP treatment induced a clear reduction in the expression of IL1b and IL6 (about two-fold decrease, p < 0.05) but no significant decrease in IL8 and CCL2 expression (Fig. 6), indicating that EEP might have a more potent anti-inflammatory effect than AEP on DPSCs.