Isolation and culture of PDLSCs
PDLSCs were cultured from three females (aged 20 to 28 years), as described in previous studies. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Teaque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Inc., Marlborough, MA, USA) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B; Nacalai Teaque, Inc.) at 37 °C under 5% CO2. Passage-zero PDLSCs were seeded and cultured until Passage 4 to 7 for use in experiments. Characterization of PDLSCs was performed as previously described, including analysis by flow cytometry using a FACSVerse (BD Pharmingen, NY, USA) with antibodies (obtained from BioLegend, Inc. San Diego, CA, USA) against human cluster of differentiation 34 (CD34), CD45, CD73, CD90, CD105, and stromal cell surface marker 1 (STRO-1).
For growth at various glucose concentrations, PDLSCs were cultured in DMEM supplemented with 10% FBS and glucose at one of the five following concentrations: 100 mg/dL (physiological control), 75 mg/dL, 50 mg/dL, 25 mg/dL and 0 mg/dL.
PDLSCs were cultured with antibodies against human CD34, CD45, CD73, CD90, CD105, and STRO-1 (Santa Cruz Biotechnology, Inc. Dallas, Texas, USA). Fluorescent immunostaining was performed using Alexa Fluor 488® (Thermo Fisher Scientific, Inc., Waltham, MA, USA) as the secondary antibody, and the nuclei were counterstained using 4’,6-diamidino-2-phenylindole (DAPI) (Dojindo Molecular Technologies, Inc. Kumamoto, Japan). Images of stained cells were obtained using an all-in-one fluorescence microscope (BZ-9000, Keyence Corp., Osaka, Japan).
Cell proliferation assay
PDLSCs were cultured in 96-well plates at 2×10⁴ cells/mL in glucose-supplemented culture medium (at 100, 75, 50, 25, or 0 mg/dL) for 1, 3, and 7 days. The number of viable cells at each timepoint was determined using a cell proliferation assay kit (Cell Count Reagent SF, Nacalai Teaque, Inc.) according to the manufacturer’s protocol; results were determined by measuring absorbance at 450 nm with a spectrophotometer (SpectraMax iD3, Molecular Devices Inc., San Jose, CA, USA).
Staining of live cells
PDLSCs were cultured in 24-well plates at 2×10³ cells/mL in glucose-supplemented culture medium (at 100, 75, 50, 25, or 0 mg/dL) for 1, 3, and 7 days. At each time point, live cells were stained for 15 min at 37 °C with 4 μM Calcein-AM solution (Dojindo Molecular Technologies, Inc.). Images were obtained with an all-in-one fluorescence microscope (BZ-9000, Keyence Corp.) and the percentage of positive staining area in images was determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Cell migration assay
Cell migration was assessed using a wound-repair assay kit (Ibidi GmbH, Inc., Martinsried, Germany). Briefly, PDLSCs were cultured in a cell culture insert at 5×10⁵ cells/mL. The culture insert was removed after the cells reached confluence, leaving a 500-μm wide “wound” in the confluent monolayer, and the medium was replaced with serum-free low-glucose medium. The images of each wound at 0, 12 and 24 h (as the cells migrated to fill in the intervening space) were photographed with a digital microscope camera (Olympus LS, Olympus Corp., Tokyo, Japan). The percentage of healed-wound area was determined by using ImageJ software.
Alkaline phosphatase (ALP) staining and activity
PDLSCs were cultured in 24-well plates at 4×10⁴ cells/mL in glucose-supplemented osteogenic medium (at 100, 75, 50, 25, 0 mg/dL) and incubated for 7 and 14 days. At each time point, PDLSCs were stained using an ALP staining kit (Sigma-Aldrich, Inc. St. Louis, MO) in accordance with the manufacturer’s protocol. At the same time points, PDLSCs were treated with 0.2% Triton X-100 (Sigma-Aldrich, Inc.) and ALP activity was measured following addition of 1-step PNPP (Pierce Biotechnology, Inc., Rockford, IL, USA.); product formation data were determined by measuring absorbance at 405 nm with a spectrophotometer (SpectraMax iD3, Molecular Devices Inc.), and the values were normalized to the DNA content measured by Pico Green dsDNA Assay Kit (Invitrogen, Inc. Paisley, UK.) in the respective sample.
Extracellular calcium deposition and Alizarin red S (ARS) staining
PDLSCs were cultured in 24-well plates at 4×10⁴ cells/mL as for the ALP staining assay. After the osteogenic medium was removed, cells were washed with PBS and extracellular calcium was dissolved with 10% formic acid. The deposition of extracellular calcium was quantified using the Calcium E-test kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer’s protocol. For the ARS assay, PDLSCs were cultured for 7 and 14 days with low-glucose osteogenic medium, then stained for 3 minutes at room temperature with a solution of 1% ARS (Wako Pure Chemical Industries, Ltd.) and washed three times with PBS.
Real-time polymerase chain reaction (Real-time PCR)
Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen, Inc., Venlo, the Netherlands) according to the manufacturer’s instructions, and 10 μL total RNA from each sample was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (TAKARA Bio, Inc., Shiga, Japan) on the StepOnePlus Real-time PCR System (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. PCR was performed to determine the expression level of Runt-related transcription factor 2 (Runx2), and gene expression was normalized to that of the housekeeping gene encoding glyceraldehyde phosphate dehydrogenase (GAPDH).
Measurement of osteocalcin (OCN)
PDLSCs were cultured in low-glucose osteogenic medium for 14 days. The spent culture medium then was collected and the OCN was quantified using an OCN detection kit (TAKARA Bio, Inc.) according to the manufacturer’s protocol.
Western blot analysis
Total protein was extracted using radio-immunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, Inc.) and a phosphatase cocktail (Nacalai Teaque, Inc.). Protein samples were separated on a 12.5% gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were blocked for 60 min at room temperature using Blocking One (Nacalai Teaque, Inc.) and then incubated overnight at 4 °C with primary antibodies against Akt, p-Akt (S473), HIF-1α, ERK1/2, p-ERK1/2, or β-actin (Cell Signaling Technology, Inc., Danvers, Massachusetts, USA). Detection of primary antibodies was performed by incubation for 1 hour at room temperature with horseradish peroxidase (HRP) -conjugated secondary antibodies (Invitrogen, Inc.). Immunoreactive bands were visualized with a chemiluminescence kit (Nacalai Teaque, Inc.) and signals were detected with the ChemiDoc MP system (Bio-Rad Laboratories, Inc.). Densitometric analysis was performed using ImageJ software.
After achieving confluence, PDLSCs were stimulated by osteogenic medium for 7 and 14 days, and the supernatants at each time point were collected for analysis. Lactate secretion into the medium was measured using a Lactate Assay Kit-WST (Dojindo Molecular Technologies, Inc.) according to the manufacturer’s instructions.
Data are presented as mean ± SD. Parametric data from three or more groups were analyzed using a two-tailed One-way Analysis of Variance (ANOVA) with post hoc Tukey’s test. Values of p <0.05 were considered significant.