Isolation and culture of PDLSCs
Human PDL tissue samples were obtained from third molars or teeth extracted for orthodontic reason. Written informed consent was provided by all donors (n = 5, aged 18 to 24 years), and the subsequent studies were approved by the Ethics Committee of the School of Stomatology, Fourth Military Medical University, Xi’an, Shannxi, China. In summary, freshly extracted teeth were washed using cold phosphate-buffered saline (PBS; Corning, New York, USA), and gingival tissue was excluded. PDL tissues were scraped from the middle third of the root surface and cut into small pieces (approximately 1 mm3). Next, the periodontal samples were digested by type I collagenase (Sigma-Aldrich, St. Louis, USA) for 45 min in dark. After digestion, the PDL tissues were resuspended in complete Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Sijiqing, Hangzhou, China), 1% penicillin (Invitrogen, Carlsbad, CA, USA) and streptomycin (Invitrogen). The PDL tissues were finally transferred to a 6-well culture plate and cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The medium was refreshed every 2 days. Cells from passages 3 to 5 were used in the following experiments.
Flow cytometry analysis
Flow cytometry analysis was conducted to identify the immunophenotypes of PDLSCs. PDLSCs were collected and then transferred into sterile Eppendorf tubes (Invitrogen) at 5×105 cells per tube. The cells were incubated with FITC-conjugated monoclonal antibodies against human CD31, CD35, CD45, CD90, CD105, and CD146 (all from eBioscience, San Diego, USA). A cell suspension without antibodies served as the blank control. After labeling for 1 h in dark, the PDLSCs were washed and resuspended with PBS. These procedures were performed on ice. The immunophenotypes of the cells were assessed with a Beckman Coulter Epics AL cytometer (Beckman Counter, Fullerton, USA).
Colony forming unit (CFU) assay
To assess colony-forming efficiency of PDLSCs, cells in passage 4 were seeded in 100-mm-diameter culture dishes (Invitrogen) at 1×103 cells/dish. The medium was refreshed every 2 days. After 14 days of cultivation, the cells were fixed using 4% paraformaldehyde (Invitrogen) for 30 min and then stained with 0.1% toluidine blue (Sigma-Aldrich) for 20 min. Cell colonies were photographed with an inverted microscope (Olympus Optical, Tokyo, Japan). Cell aggregates containing more than 50 cells were recognized as colonies.
Cell Counting Kit-8 (CCK-8) assay
Proliferation ability of PDLSCs was measured with Cell Counting Kit-8 assay. PDLSCs were seeded in 96-well culture plates at a density of 1×103. After 24 h of cell adhesion, 200 μL medium with 20 μL CCK-8 reagent (Invitrogen) was added to the test wells. The plate was then incubated at 37 °C for 2 h. After incubation, absorbance at 450 nm was detected with a microplate reader (TECAN, Männedorf, Switzerland) to measure the proliferation capacity of PDLSCs. During the 8-day culture, all procedures mentioned were performed at proscribed time points every day.
Osteogenic differentiation assay
For osteogenic induction, PDLSCs were seeded in 6-well plates at a density of 1 × 105 until 70~80% confluence. Cells were then cultured with osteo-inductive medium: complete DMEM or high glucose (25 mmol/L) DMEM supplemented with 10% (v/v) FBS, 1% penicillin and streptomycin, 50 μg/ml vitamin C, 10 nM dexamethasone and 10 mM β-glycerophosphate. The medium was refreshed every 2 days.
Alkaline phosphatase (ALP) staining was performed at 7 days after osteogenic induction. PDLSCs were stained using a BCIP/NBT ALP Color Development kit (Biotime, Shanghai, China) and then observed and photographed with an inverted microscope. ALP activity measurement was performed using an ALP assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Alizarin red staining was conducted at 21 days after osteogenic induction. PDLSCs were fixed using 4% paraformaldehyde (Invitrogen) for 30 min. Calcium deposits were stained and then observed and photographed with an inverted microscope. The stained areas were then dissolved in 6% cetylpyridine (Sigma-Aldrich) for 15 min, and the absorbance at 560 nm was assessed using a microplate reader for quantitative assay.
Adipogenic differentiation assay
For adipogenic induction, PDLSCs were seeded in 6-well plates at a density of 1 × 105 until 80~90% confluence. Cells were then cultured with adipo-inductive medium (Cyagen, Guangzhou, China). The medium was refreshed every 2 days.
Oil red O staining was performed at 21 days after adipogenic induction. PDLSCs were fixed using 4% paraformaldehyde (Invitrogen) for 30 min. Lipid droplets were stained and then observed and photographed with an inverted microscope. The stained areas were then dissolved in isopropanol (Sigma-Aldrich) for 15 min, and absorbance at 560 nm was measured using a microplate reader for quantitative assay.
Chondrogenic differentiation assay
For chondrogenic induction, PDLSCs were collected in 15-mL centrifuge tubes (Invitrogen) at a density of 2×105 cells/tube. The cells were washed with PBS and then resuspended in chondro-inductive medium (Cyagen, Guangzhou, China). After 24 h, cell spheres were visible at the bottom of the tubes. The medium was refreshed every 2 days.
Alcian blue staining was performed at 21 days after chondrogenic induction to measure the chondrogenic differentiation ability of PDLSCs.
Selection of the optimal concentration of metformin
To select the optimal concentration of metformin (Sigma-Aldrich), the toxic and protective effects of metformin were assessed by CCK-8 assay. For toxic effect, PDLSCs were seeded in 96-well culture plates at a density of 5×103 with different concentrations of metformin (0, 10, 100, 500 and 1000 µM). Cell viability was measured at 6, 12, 24, 48 and 72 h later. For protective effect, PDLSCs were seeded in 96-well culture plates at a density of 1×103 with the aforementioned concentrations of metformin. Cell viability was measured daily during the 6-day culture period. Accordingly, concentrations of metformin that induced cell toxicity were excluded. Ultimately, a concentration that conferred the maximal protective effect was selected.
RNA-seq analysis was performed to detect differentially expressed genes in PDLSCs cultured in different osteo-inductive mediums. In brief, total RNA was extracted using a TRIzol reagent kit (Invitrogen). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA) and verified using RNase-free agarose gel electrophoresis. Then, eukaryotic mRNA was enriched with Oligo (dT) beads, and total RNA was fragmented into short fragments using fragmentation buffer and reversely transcribed into cDNA with random primers. Second-strand cDNA was synthesized by DNA polymerase I, RNase H, dNTP and buffer. The cDNA fragments were purified with a QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), and then, the ends were repaired, and poly(A) was added. The fragments were ligated to Illumina sequencing adapters. Desired genes were selected and compared to determine changes in the expression levels of relevant genes. After purification, cDNA fragments were sequenced using an Illumina Novasqr6000 by Gene Denovo Biotechnology CO (Guangzhou, China).
Lentivirus transfection was performed to stably upregulate the expression of natriuretic peptide receptor 3 (NPR3) in PDLSCs. In summary, PDLSCs were cultured until 40~50% confluence and then transfected with lentivirus containing NPR3 sequence in the presence of polybrene (1 μL/mL). The medium was replaced 24 h after transfection. Cells transfected with lentivirus lacking NPR3 sequence were used as negative controls. Lentiviruses containing or lacking NPR3 sequence were designed and synthesized by Gene Pharma (Shanghai, China).
The efficiency of lentivirus-mediated upregulation of NPR3 expression was confirmed by qRT-PCR, Western blot analysis and Immunofluorescence staining.
Enzyme-linked immunosorbent assay (ELISA)
After 7 days of osteogenic induction, the culture mediums were collected and centrifuged to remove the cells and debris. The concentrations of C-type natriuretic peptide (CNP) secreted into the cell culture supernates were detected with a ELISA kit (Rebiosci, Shanghai, China).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Quantitative real-time polymarese chain reaction (qRT-PCR) was conducted to measure mRNA expression levels following the manufacturer’ s instructions. In brief, Total RNA was extracted from cells with TRIzol reagent (Invitrogen). Extracted total RNA was reverse transcribed to cDNA using Evo M-MLV RT Premix (Takala, Shiga, Japan). Quantitative real-time PCR was performed using the SYBR Green Premix Pro Taq HS qPCR kit (Tli RNaseH Plus; TaKaRa), and the results were analyzed with a CFX96 Real-time RT-PCR system (Bio-Rad, Hercules, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize the expression levels. The expression of target genes was calculated using the 2-ΔΔCT method. The primer sequences used for qRT-PCR are listed in Table 1.
Western blot analysis
Western blot analysis was performed to measure protein expression levels in PDLSCs. Briefly, Prepared cells were first lysed in RIPA lysis buffer (Biotime) supplemented with protease and phosphatase inhibitors (Biotime). A bicinchoninic acid (BCA) assay kit was then used to measure protein concentrations. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Biotime) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk at room temperature for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000; goat anti-rabbit IgG, CST, #7074; goat anti-mouse IgG, CST, #7076) at room temperature for 2 h. Subsequently, the blots were visualized using enhanced chemiluminescence substrate (ECL kit, Millipore), and protein bands were analyzed with Image J software. GAPDH was used as the housekeeping gene for internal normalization. The following primary bodies were used for Western blotting: GAPDH (1:3000; Affinity, AF7021), ALP (1:10000; Abcam, ab108337), RUNX2 (1:1000; CST, #12556), BMP2 (1:1000; Abcam, ab214821), OCN (1:1000; Santa cruz; Sc-390877), NPR3 (1:1000; Abcam, ab177954), p38 MAPK (1:1000; CST, #9212), p-p38 MAPK (1:1000; CST, #4511), JNK (1:1000; CST, #9252), p-JNK (1:1000; CST, #4668), ERK1/2 (1:1000; CST, #9102) and p-ERK1/2 (1:1000; CST, #9101).
Immunofluorescence staining was performed to visualize NPR3 in PDLSCs. Briefly, cells were fixed with 4% paraformaldehyde for 30 min, followed by treatment with 0.5% Triton X-100 and 2% bovine serum albumin (BSA; Sigma-Aldrich). Then, the cells were incubated with primary antibodies against NPR3 (1:100; Abcam, ab97389) over night. The secondary antibodies for NPR3 were Alexa Fluor 488 AffiniPure donkey antirabbit IgG (1:100; Yeasen Biotech Company, Shanghai, China). Finally, the cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Abcam, ab104139), and the fluorescence images were acquired using a Beckman Coulter Epics XL cytometer (Beckman Coulter).
All data are presented as the mean ± standard deviation of at least three independent experiments. Statistical analysis was performed using GraphPad Prism 8 software. One-way analysis of variance followed by Tukey’s multiple comparisons tests, Sidak’s multiple comparisons tests or Dunnett's multiple comparisons was used for comparing more than two groups, and unpaired two-tailed Student’s t test was used for comparisons of two unpaired groups. Statistical significance was expressed as P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***).