Culture of DPCs

This study was approved by the Institutional Review Board of Taipei Veterans General Hospital (approval no. 2017–07-023 AC). All adult participants provided written informed consent. All child participants provided written informed assent (Both child and parent). The primary human DPCs used in this study were established from a third molar from a 22-year-old female after obtaining informed consent. Pulp tissues were collected from the sectioned tooth, rinsed with PBS twice, and then minced into small fragments. The tissues were treated with trypsin/EDTA (Sigma–Aldrich, St. Louis, MO, USA) at 37 °C for 15 min and neutralized with DMEM (Corning, New York City, NY, USA) containing 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit-Haemek, Israel), 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B (Pen-Strep-Amp antibiotics, Biological Industries). The tissues were placed onto the surface of a 100 μm cell strainer (BD Bioscience, San Jose, CA, USA) and cultured in the presence of MEM-α (Lonza, Basel, Switzerland) containing 16.6% FBS and Pen-Strep-Amp antibiotics at 37 °C in a humidified atmosphere in a 5% CO2 incubator [11]. In this system, single cells were able to pass through the mesh and attached to the plate below. When confluent, the cells were treated with trypsin/EDTA and passaged. All pulp cells used in this study were between passage 4 and 10.

Culture of THP-1 and HL-60 cells

The THP-1 and HL-60 cell lines were purchased from Bioresource Collection and Research Centre (BCRC, Hsinchu, Taiwan). THP-1 was maintained in RPMI 1640 (Sigma–Aldrich) supplemented with 10% FBS, Pen-Strep-Amp antibiotics, and 0.05 mM 2-mercaptoethanol (Sigma–Aldrich) at 37 °C in a humidified atmosphere in a 5% CO2 incubator [12]. HL-60 was maintained in Iscove’s modified Dulbecco’s medium (Sigma–Aldrich) supplemented with 20% FBS and Pen-Strep-Amp antibiotics at 37 °C in a humidified atmosphere in a 5% CO2 incubator [13]. These cell lines were passaged every 3 to 4 days.

The settings of each group

Seeded DPCs in 96 or 12-well plates and incubated at 37 °C in a humidified atmosphere in a 5% CO2 incubator overnight.

THP-1: DPCs = 1:0 – only seeded THP-1 cells in empty wells without DPCs.

THP-1: DPCs = 0:1 – only added fresh medium.

THP-1: DPCs = 1:1000 – added THP-1 equivalent to 1/1000 of the number of DPCs.

THP-1: DPCs = 1:100 – added THP-1 equivalent to 1/100 of the number of DPCs.

THP-1: DPCs = 1:10 – added THP-1 equivalent to 1/10 of the number of DPCs.

THP-1: DPCs = 1:2 - added THP-1 equivalent to 1/2 of the number of DPCs.

THP-1: DPCs = 1:1 – added THP-1 equivalent to 1/1 of the number of DPCs.

Cell proliferation assay and cell viability assay

The alamarBlue assay was used to determine the proliferation of DPCs [14]. Briefly, DPCs were seeded in 96-well plates and incubated at 37 °C in a humidified atmosphere in a 5% CO2 incubator overnight. The next day, the medium was removed, and THP-1 cells with different cell ratios and 100 nM phorbol-12-myristate-13-acetate (PMA, Sigma–Aldrich) were added to THP-1 culture medium for 24 h [15]. At preset times, ten μl AlamarBlue® solution (Thermo Fisher Scientific) was added to the cultivated cells for two h and measure the fluorescence with Excitation wavelength at 530-560 nm and Emission wavelength at 590 nm by GloMax® Explorer Multimode Microplate Reader (Promega Corporation, Madison, WI, USA).

The cell viability of coculture DPCs and THP-1 cells were detected by MTT assay [11]. Briefly, DPCs were seeded in 6-well plates and then cocultured with THP-1 cells. At preset times, 1 mg/mL MTT substrate was added to the cultivated cells for 1 h and then the cell morphology was photographed.

Expression levels of IL-6 and IL-8 by RT–qPCR

Used TRI reagent (Sigma–Aldrich) according to the manufacturer’s instructions to extract total RNA, and synthesized cDNA by a ReverTra Ace Set (TOYOBO Ideas & Chemistry, Osaka, Japan). The mRNA expression of IL-6, IL-8 and GAPDH was examined by real-time PCR using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) with gene-specific primers. The primer sequences were as follows: IL-6, 5′-AAC CTG AAC CTT CCA AAG ATG G-3′ (sense) and 5′-TCT GGC TTG TTC CTC ACT ACT-3′ (antisense); IL-8, 5′-CAT ACT CCA AAC CTT TCC ACC CC-3′ (sense) and 5′-TCA GCC CTC TTC AAA AAC TTC TCC A-3′ (antisense); GAPDH, 5′-AGA AGG CTG GGG CTC ATT TG-3′ (sense) and 5′-AGG GGC CAT CCA CAG TCT TC-3′ (antisense) [16]. The thermal cycling conditions were as follows: predenaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 15 s, and annealing and extension at 60 °C for 60 s. These reactions were carried out using a QuantStudio I system (Thermo Fisher Scientific). The comparative threshold cycle (Ct) method was used to measure relative changes in expression. The 2-ΔΔCt values were used to represent the fold changes in mRNA expression between the sample groups and between the various experimental setups [17].

Expression levels of IL-6, TNF-α and MMP-9 by ELISA

The protein levels of IL-6, TNF-α and MMP-9 in cocultured medium were detected by Lumit™ IL-6 (Human) immunoassay (Promega), Lumit™ TNF-α (Human) immunoassay (Promega) and Human MMP-9 ELISA Kit (Thermo Fisher Scientific). Briefly, DPCs were seeded in 96-well plates and incubated at 37 °C in a humidified atmosphere in a 5% CO2 incubator overnight. The next day, the medium was removed, and DPCs were treated with 0.1 μg/mL LPS or cocultured with THP-1 cells (THP-1: DPCs = 1:10). After 24 and 48 h, IL-6, TNF-α and MMP-9 expression were detected according to the manufacturer’s instructions.

Statistical analysis

The data are presented as the mean ± standard deviation from at least two independent observations. The results were analyzed using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA). The Mann–Whitney test and unpaired t-test were used for comparisons between two groups, and two-way ANOVA was applied for comparisons among multiple groups. Differences were considered statistically significant when p < 0.05.