Primary cell culture.
Human dental pulp was collected from immature third molars, and the 1/3 tip of the pulp was cut off and subsequently, the explant outgrowth method was performed (24). For each experiment, three different donors were used (n = 12; 3–4 molars per donor; the male to female ratio of cases was 1:1, age18-25 years,the range of date was from Jan. 2019 to March 2019). Exodontia was performed by the Department of Oral and Maxillofacial Surgery in the Second Affiliated Hospital of Harbin Medical University. The present study was approved by the Institutional Ethics Committee of the Second Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from all donors. The basic culture medium of DPSCs was Human Mesenchymal Stem Cell Growth Medium Kits (RASMX-90011, Cyagen Biosciences Guangzhou, China), including 10% FBS, 1% penicillin and streptomycin and 1% glutamine.
Identification of DPSCs by CD34, CD45, CD73, CD90, CD105 and MSCA-1 using flow cytometry.
Dental pulp cells were cultured in 75cm2 cell culture flasks (Corning, Inc.) and a total of 7 flasks were prepared. Cells were washed three times with PBS and digested using 2 ml pre-warmed 0.25% trypsin. Digestion was terminated and cells were counted. Cells were collected at a density of 1x106 cells/ml into 15 ml centrifuge tubes. Cells were washed three times using PBS and centrifuged for 10 min at 300 x g. at room temperature. The supernatant was discarded and the cell pellets were incubated on ice for 30 min. Cells in one tube served as the control group. Cells in the remaining six tubes were incubated with monoclonal conjugated antibodies (Table I) to identify pulp immunocompetent cells. Antibodies were used at a 1:10 dilution (5 µl antibody with 50 µl buffer). Subsequently, 1–2 ml buffer was added to each tube and centrifuged for 10 min at 300 x g at room temperature to wash the cells. Cells were resuspended in 500 µl buffer. Flow cytometry was performed using the BD FACS Canto flow cytometer (BD Biosciences) and FACS Diva software (version 6.1.3) (BD Biosciences, San Jose, CA, USA).
Experimental groups.
Cells were cultured in a 37 ℃, 5% CO2 incubator and divided into six groups (n = 1x105 cells/group): i) cells treated with basic cell culture medium containing 10 nmol/l dexamethasone, 5 mmol/l β-glycerophosphate and 50 mg/ml vitamin-C-phosphate as control; ii) DPSCs treated with 50 ng/ml recombinant human complement component C5a protein (C5a; R&D Systems, Inc.); iii) DPSCs treated with 100 ng/ml C5a; iv) DPSCs treated with 200 ng/ml C5a; v) DPSCs treated with 300 ng/ml C5a; and vi) DPSCs treated with 400 ng/ml C5a. In addition to basic cell culture medium which included and C5a, 10 nmol/l dexamethasone (Sigma-Aldrich, USA), 5 mmol/l β-glycerophosphate (Sigma-Aldrich, USA) and 50 mg/ml vitamin-C-phosphate (Sigma-Aldrich, USA) were added to each tube to promote odontoblastic differentiation. The culture medium was replaced every other day with fresh culture medium containing the same concentration of C5a.
Multilineage potential of DPSCs into adipocytes, osteoblasts and neurons in vitro.
Cells at passage 3 were plated at a density of 1x105 cells per well. Adipocyte differentiation was induced by culturing DPSCs with 0.5 µM isobutylmethylxanthine (Sigma-Aldrich, USA), 50 µM indomethacin (Sigma-Aldrich, USA) and 0.5 µM dexamethasone (Sigma-Aldrich, USA) in a 37 ℃, 5% CO2 incubator for 3 weeks. Subsequently, cells were fixed in 4% paraformaldehyde for 30 min at room temperature and stained with a fresh Oil Red O solution for 1 h at room temperature. Osteoblast differentiation was induced by culturing DPSCs with 5 mM/l β-glycerophosphate, 10 nM/l dexamethasone and 50 mg/ml vitamin-C-phosphate in a 37 ℃, 5% CO2 incubator for 3 weeks. Subsequently, cells were fixed using 95% ethanol for 30 min at 37˚C and stained with Alizarin Red S for 30 min at 37˚C. The cells for adipocyte and osteoblast differentiation were observed under a light microscope (ZEISS, 37081 Goettingen, Germany)(original magnification, x40). Neuronal differentiation of DPSCs was induced as previously described (27). Briefly, cells were cultured with basic cell culture medium containing 500 mM β-mercaptoethanol (Sigma-Aldrich, USA) and 10 ng/mL βFGF (Sigma-Aldrich, USA) in a 37 ℃, 5% CO2 incubator for 24 h. Subsequently, the culture medium was replaced with serum-free medium containing 2% DMSO and 100 mM butylated hydroxyanisole (Sigma-Aldrich, USA) at 37 ℃, 5% CO2 incubator for 6 h. Cells were fixed in 4% paraformaldehyde at room temperature for 20 min. PBS-T (phosphate buffer saline with 0.1% Triton X-100) were used to permeabilize with at 4℃ for 10min. Then we removed the PBS-T and washed the cells three times with PBS (phosphate buffer saline). Samples were treated with PBS-B (4% bovine serum albumin in PBS) at 37 C for 30 min. Then we incubated the antibodies. The primary antibody solution (1:200, SantaCruz, USA) was added into the samples at 4 ℃ overnight, then washed the samples with PBS for 5 min. The secondary antibody solution (1:200, EarthOx, California, USA) incubated the samples for 60 min at room temperature in the dark. Stained the samples with the nuclear dye 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, Beyotime, CHN) for 5 min, washed with PBS 3 times and then fluorescent images were acquired. Immunofluorescence was detected using a fluorescence microscope (magnification, x20; DMI14000B; Leica Microsystems GmbH, Wetzlar, Germany).
Cytotoxicity (MTT) assay cultured by different concentrations of C5a.
The effect of different concentrations of C5a on cell viability was assessed using an MTT assay. Cells were seeded (1x104 cells/well) into 96-well plates. After 24 h, cells were treated with 50, 100, 200, 300 or 400 ng/ml C5a in a 37 ℃, 5% CO2 incubator for 24 h. The control group was incubated with normal medium. Subsequently, cell cytotoxicity was assessed using the Cell Proliferation Kit I (MTT; Roche Applied Sciences), according to the manufacturer’s protocol. Dimethyl sulfoxide (200 µl) was added to dissolve the formazan crystals. The absorbance of each well was measured at a wavelength of 490 nm using an ELISA reader (Thermo Fisher Scientific, Inc.). Cell viability was calculated according to the following formula: Cell proliferation rate (%) = [mean optical density (OD) of the group - mean of OD of zero-set group] / (mean OD of untreated group - mean of OD of zero-set group) x100.
Mineralization induction of all 6 groups.
Cells at passage 3 were seeded (5x104 cells/well) into 6-well plates, and cultured with different concentrations of C5a (50, 100, 200, 300 or 400 ng/ml) and mineralized medium (5 mM/l mll β-glycerophosphate, 10 nM/ml l dexamethasone and 50 mg/ml vitamin-C-phosphate). The control group was cultured in mineralized medium without C5a. Following culture in a 37 ℃, 5% CO2 incubator for 28 days, cells were fixed using 95% ethanol for 30 min at 37˚C. Subsequently, cells were stained with 0.1% Alizarin Red S (Sigma-Aldrich; Merck KGaA) at 37˚C for 30 min to detect calcium accumulation. The mineralized nodules were observed under a light microscope (ZEISS, 37081 Goettingen, Germany)(original magnification, x40).
Reverse transcription-quantitative PCR (RT-qPCR) of DSP and Actin expressions.
Following culture for 28 days, total RNA was extracted from the cells using the RNeasy Mini kit used according to the manufacturer’s protocol (Qiagen GmbH). Total RNA was reverse transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis kit used according to the manufacturer’s protocol (Roche Diagnostics GmbH). The following thermocycling conditions were used for reverse transcription: 50˚C for 60 min, 85˚C for 5 min and 4˚C for 10 min. Subsequently, qPCR was performed using FastStart Universal SYBR Green Master Rox Mix (Roche Diagnostics GmbH). The following thermocycling conditions were used for qPCR: 95˚C for 2 min; 40 cycles of 95˚C for 15 sec and 60˚C for 30 sec. The following primer pairs were used for qPCR: DSP forward, 5’-TTTCCGCTTGTCATCATCTCC-3’ and reverse, 5’- GGTGTCCTGGCACTACTGCAT-3’; and actin, forward 5’-GGGAAATCGTGCGTGACATT-3’ and reverse, 5’-GGAACCGCTCATTGCCAAT-3’. mRNA expression levels were quantified using the 2−∆∆Cq method (28) and normalized to the internal reference gene actin.
Western blotting of DSPP and Actin expression.
Following culture for 28 days, total protein was extracted from the cells using cell lysis buffer, which consisted of 150 mM NaCl, 20 mM Tris (pH 7.5), 2.5 mM sodium pyrophosphate, 1% Triton X-100, 1% Na3CO4, 0.5 µg/ml leupeptin, 1 mM EDTA and 1 mM phenylmethanesulfonyl fluoride. Total protein was quantified using the Pierce™ BCA Protein Assay kit used according to the manufacturer’s protocol (Thermo Fisher Scientific, Inc.). Protein samples (20 µg/lane) were separated via 12% SDS-PAGE and transferred onto PVDF membranes by Trans-Blot SD Cell Semi-Dry Transfer (Bio-Rad Laboratories, Inc.). Subsequently, the membranes were blocked with 5% milk at room temperature for 1 h with gentle agitation. The membranes were incubated at 4˚C overnight with the following primary antibodies: Mouse anti-human DSPP (LFMB-21; cat. no sc-73632; 1:200; Santa Cruz Biotechnology, Inc.) and β-actin (cat. no. 20536-1-AP; 1:5,000; ProteinTech Group, Inc.). Following primary incubation, the membranes were incubated with the following secondary antibodies at room temperature for 1 hour.: goat anti-mouse IgG (cat. no. SA00001-1; 1:10,000; ProteinTech Group, Inc.) and goat anti-rabbit IgG (cat. no. SA00001-2; 1:10,000; ProteinTech Group, Inc.). Protein bands were visualized using the Clarity MaxTM Western ECL substrate (Bio-Rad Laboratories, Inc.) and photographed using the Tanon 1000 digital image gel analytical system (Tanon Science & Technology Co., Ltd.). β-actin was used as the loading control. The bands were quantified using Image J (NIH, USA).
Statistical analysis.
One-way ANOVA followed by Bonferroni’s multiple comparisons was performed using SPSS software (version 13.0; SPSS, Inc.). P < 0.05 was considered to indicate a statistically significant difference. Data are expressed as the mean ± standard error of the mean.