NAC powder (Aladdin Chemical, Shanghai, China) was dissolved in HEPES buffer (Solarbio, Beijing, China) for an NAC stock solution (500 mM stock, pH = 7.0) stored at 4℃ and protected from light.
DFSC isolation and culture
The sample contained 5 fresh human dental follicles from 5 individuals who were selected to volunteer in this study according to the inclusion criteria: age between 13 and 22 years, healthy subjects, with good oral health conditions, referred for third mandibular molar extraction due to orthodontic or embed-impacted reasons in Tianjin Medical University Stomatology Hospital, unerupted teeth with low density of the dental follicle tissues around crowns observed on panoramic radiographs (Additional file 1: Fig. S1B and C). For this purpose, approval from the Ethics Committee of the Affiliated Stomatological Hospital of Tianjin Medical University (permission no. TMUSH-hMEC2016082) and written consent from each patient were obtained. Isolation and characterization of human DFSCs were performed according to our previous study (28). Cells were cultured in minimum essential medium-α (α-MEM; HAKATA, Shanghai, China) containing 10% heat-inactivated fetal bovine serum (FBS; HAKATA, Shanghai, China) and 1% penicillin/streptomycin (Solarbio, Beijing, China) in a humidified atmosphere of 5% CO2 at 37℃. When adherent cells reached approximately 80% confluent, they were washed with phosphate buffer solution (PBS; Solarbio, Beijing, China), detached using 0.25% trypsin with 1 mM EDTA-4Na (Gibco, MA, USA) and seeded for passage culture. For NAC treatment, each culture medium supplemented with or without NAC stock solutions (final concentrations of 0, 2.5, 5, or 10 mM) was added immediately after seeding and renewed every 2 days. For PI3K inhibition, cells were treated with LY294002 (10 µM; Beyotime, Shanghai, China) for 2 days. Cells from passage 6 to 12 were used for all experiments. This study was conducted according to World Medical Association Declaration of Helsinki, ethical principles for medical research involving human subjects.
Oxidant and antioxidant assays
DFSCs were cultured as above for 2–4 days before assays. According to a ROS assay kit (Beyotime, Shanghai, China), after washed three times with PBS, DFSCs were incubated in 10 µM DCFH-DA solution at 37ºC for 20 minutes and observed by laser confocal microscope (Zeiss, Oberkochen, Germany) at the wavelength of 488/525 (excitation/emission). For quantitation, DFSCs were dissociated by 0.25% trypsin-EDTA and incubated as described above and the relative mean fluorescence intensity (MFI) was analyzed and normalized to the control using flow cytometry (BD, NY, USA) and FlowJo (BD, NY, USA). A superoxide assay kit (Beyotime, Shanghai, China), a hydrogen peroxide (H2O2) assay kit (Solarbio, Beijing, China), a total glutathione assay kit (Beyotime, Shanghai, China), a total superoxide dismutase (SOD) assay kit (Beyotime, Shanghai, China) and a catalase (CAT) assay kit (Beyotime, Shanghai, China) were used respectively. Absorbance was measured by a microplate reader (Tecan, Männedorf, Switzerland). Protein concentration of each sample was determined at A280 using Nanodrop2000 (Thermo Scientific, MA, USA).
Cell viability assays
DFSCs were seeded immediately at a density of 4*103/well in a 96-well plate and incubated with NAC at 37℃ and 5% CO2. At defined end points (24, 48, 72 hours). cells were rinsed with PBS three times to remove residual NAC and cell viability was quantified using a cell counting kit-8 (CCK-8; Solarbio, Beijing, China) following the manufacture’s protocol for 90 minutes. A microplate reader (Tecan, Männedorf, Switzerland) was used to measure absorbance at 450 nm. Cells were observed at 72 hours using an inverted microscope (Zeiss, Oberkochen, Germany).
The osteogenic-inducing medium was α-MEM supplemented with 10% FBS, 1% penicillin/streptomycin, varying concentrations of NAC, 10 mM Na-β-glycerophosphate (Solarbio, Beijing, China), 100 nM dexamethason (Sigma-Aldrich, MO, USA) and 50 µg/mL vitamin C (Solarbio, Beijing, China). Alkaline phosphatase (ALP) assays were conducted at day 7 of osteogenic induction. ALP staining was carried out using a BCIP/NBT alkaline phosphatase color development kit (Beyotime, Shanghai, China). ALP activity was performed using alkaline phosphatase assay kit (Beyotime, Shanghai, China). Protein concentration of each sample was as described above. Alizarin red S (ARS) staining was performed at day 17 of osteogenic induction using 0.2% ARS (Solarbio, Beijing, China) at 37℃ for 30 minutes. For semi-quantitive analysis, PBS containing 10% cetylpyridine chloride (Sigma-Aldrich, MO, USA) was added into each well to dissolve precipitation after staining and the absorbance was measured using a microplate reader (Tecan, Männedorf, Switzerland) at 542 nm. Cells were observed using an inverted microscope (Zeiss, Oberkochen, Germany).
Detection of apoptosis
The percentage of apoptotic cells was examined after culture for 4 days using an Annexin V-FITC apoptosis detection kit (KeyGEN, Jiangsu, China). Briefly, cells were harvested, washed three times in cold PBS, resuspended in binding buffer at a concentration of 1*105 cells/mL and incubated with 5 µL Annexin V-FITC and 5 µL PI for 15 minutes away from light at room temperature. Viable cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), late apoptotic cells (Annexin V-/PI+) and necrotic cells (Annexin V+/PI+) were analyzed using flow cytometry (BD, NY, USA) and FlowJo (BD, NY, USA).
Cell cycle and aneuploidy assay
With a cell cycle detection kit (KeyGEN, Jiangsu, China), cells reached 70% confluence were used for the detection of cell cycle and cells reached 90% confluence were used for the evaluation of aneuploidy. After trypsin digestion and centrifugation, precipitates were washed twice and resuspended with PBS into a single-cell suspension. Cold absolute alcohol was quickly added in the cell suspension and fixed cells at 4℃ for 24 hours (final alcohol concentration of 70%). Finally, fixed cells were rinsed twice with PBS and stained with PI following instructions. Cell cycle and DNA aneuploidy were analyzed using flow cytometry (BD, NY, USA) and Modfit LT (VSH, USA).
Cell senescence assay
The activity of senescence-associated β-galactosidase (SA-β-Gal) was evaluated using a cell senescence detection kit (Beyotime, Shanghai, China). DFSCs (from passage 9 to passage 12) with or without NAC were cultured in 6-well plates and stained following the manufacturer’s protocol. Positive cells were observed in blue and calculated from 5 randomly selected fields using an inverted microscope (Zeiss, Oberkochen, Germany). Thereafter, the percentage of positive cells to whole cell number was calculated.
Surface marker expression analysis
After culture for 4 days, DFSCs were washed 3 times with PBS and collected using 0.25% trypsin with 1 mM EDTA at passage 6. Cell suspension (100,000 cells) was mixed with 5 µL of FITC-anti-CD44 (cat#338803, BioLegend, CA, USA) and PE-anti-CD90 (cat#328109, BioLegend, CA, USA) according to the manufacturer’s instructions. After incubation for 30 minutes in the dark, the cells were washed 3 times with PBS and then acquired by flow cytometry (BD, NY, USA) and the MFI was analyzed by FlowJo (BD, NY, USA).
RNA extraction and qRT-PCR
After culture for 4 days, total RNA was extracted with TRIzol reagent (TransGen, Beijing, China) and a RNA kit (TransGen, Beijing, China). cDNA was synthesized with a First-Strand cDNA Synthesis Kit (TransGen, Beijing, China). The qPCR was carried on the 7500 Real Time PCR system (Thermo Fisher, MA, USA), using a green qPCR supermix (TranGen, Beijing, China). Results were analyzed and normalized to the housekeeping gene GAPDH using the ΔΔCt method. All primers (Sangon, Shanghai, China) were listed in Additional file 3: Tab. S1.
After culture for 4 days, total RNA was extracted with TRIzol reagent (TransGen, Beijing, China), RNA sequencing was performed in collaboration with BGI-shenzhen following the standard operational procedure (https://www.genomics.cn/). The expression of human genes was performed by transforming mapped transcript reads to TPM. The significant levels were corrected by Q value with a threshold (Q value < 0.05) by Bonferroni. Differential expression analysis was performed using the DESeq2 (v1.4.5), Dr. Tom (BGI, Shenzhen, China) and R language (v4.0.3) ComplexHeatmap package (v2.4.3) with Q value < 0.05 and fold change > 1.5. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed by Dr. Tom (BGI, Shenzhen, China) and R language TopGO package (v2.42.0) and ClusterProfiler package (v3.18.1).
Western blot analysis
After culture for 4 days, cells were lysed in RIPA lysis buffer (Solarbio, Beijing, China) and 1 mM PMSF (Solarbio, Beijing, China) on ice. The concentration of protein was metered using the BCA Protein Assay Kit (Thermo Scientific, MA, USA) and heat denaturation of protein in protein sample loading buffer (EpiZyme, Shanghai, China) at 100℃ for 10 min. Equal quantities of protein were separated on 10% SDS-PAGE gels by electrophoresis, transferred onto 0.45 um polyvinylidene-fluoride membranes (PVDF) (Immobilon, MA, USA). Membranes were blocked with 5% (w/v) non-fat milk in TBST buffer pH 7.5 for 1 hour at room temperature, incubated with the first antibody. Anti-GAPDH, PI3K p85, PI3K p110, AKT, phosphorylated-PI3K p85 and phosphorylated-AKT were used as primary antibodies. All antibodies were listed in Additional file 4: Tab. S2. After washed with TBST 10 minutes for 3 times and followed by incubation with secondary antibodies at room temperature for 1 hour. All uncropped blots were visualized by ECL reagents (TransGen, Beijing, China).
Rat tooth extraction model
The experimental protocol was approved by the Medical Ethics Committees of Stemmatological Hospital, Tianjin Medical University. The Unit for Laboratory Animal Medicine facilitated animal care at the Tianjin Hospital of ITCWM. This study conformed with the ARRIVE guidelines 2.0. Isolation and characterization of rat DFSCs were performed according to our previous study (29) and NAC treatment was the same as before. Under anesthesia by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg), the right maxillary first molars were extracted on 20 Sprague-Dawley rats (male, 6-week-old) purchased from Beijing SPF Biotechnology. The sockets were formed using an 1# small ball drill to remove residue roots and bone pieces as much as possible, absorbable gelatin sponges (Xiang’en, Jiangxi, China) absorbed with PBS vehicle (15 µL), 5 mM NAC solution (15 µL), rat DFSC suspension (106 cells, 15 µL) or 5 mM NAC-treated rat DFSC suspension (106 cells, 15 µL) were placed into tooth extraction sockets depending on the randomized allocation of the animals, the sockets were finally sealed with tissue adhesive (n-butyl cyanoacrylate; 3M, MN, USA) (Fig. 5A). A few animals with unsuccessful extraction were excluded. Only the experimenter who filled the sponges was aware of the group allocation. Based on different fillers, rats were divided into four groups (n = 4/group): CON (PBS vehicle), NAC (5 mM), CELL (rat DFSCs), NAC + CELL (5-mM-NAC-treated rat DFSCs). Four groups were always housed separately for the quality assurance of the treatment with the same housing condition and no diet modifications. Rats were humanely sacrificed by an overdose of anesthetics on postoperative day 7.
Right maxillary samples were fixed with 4% paraformaldehyde (Servicebio, Hubei, China) for 2 days at 4°C, decalcified with EDTA solution (Servicebio, Hubei, China) for 21 days at room temperature, embedded in paraffin (Leica, Wetzlar, Germany) and cut into 4-µm-thick slices using a HistoCore AUTOCUT (Leica, Wetzlar, Germany). Extraction socket sections were stained with haematoxylin-eosin (HE) and masson trichrome following the manufacturer’s instructions (Servicebio, Hubei, China). Optical microscope (Nikon, Tokyo, Japan) was used to scan sections.
Micro-computed tomography assessment
Right maxillary samples were fixed with 4% paraformaldehyde (Servicebio, Hubei, China) for 2 days at 4°C, transferred to 70% alcohol, scanned using a SkyScan1276 system (BRUKER, MA, USA) at 10-µm scaled image pixel size with an energy level of 55 kV. The scans were reconstructed to produce 3-dimensional images by CTvox (BRUKER, MA, USA) and 2-dimensional section images by DataViewer (BRUKER, MA, USA). The extraction socket of the first molar was isolated by manual contouring and analyzed with regard to bone volume/tissue volume, bone mineral density and trabecular parameter (trabecular thickness, trabecular number, trabecular separation) using CTan software (BRUKER, MA, USA).
Data represented mean ± standard error (SEM) of independent samples. Statistical analysis was performed by Student’s t-test using GraphPad Prism 8 (GraphPad Software, CA, USA). Statistically significant differences between groups were determined by p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).