Isolation of dental pulp stem cells
Human dental pulp stem cells were isolated from extracted teeth from healthy young (16-39-year-old) and elderly (40-76-year-old) patients (hereafter referred to as “young DPSCs” and “elderly DPSCs”, respectively)—after obtaining informed consents—at Tokyo Medical University Hospital. This study was approved by the institutional ethics committee of the Faculty of Medicine, Tokyo Medical University, Japan (approval no. 3486). Dental pulp was extracted and minced into small pieces, followed by enzymatic digestion using 3 mg/mL collagenase type I (Sigma-Aldrich) for 45 min at 37 °C; single-cell suspensions were obtained by passing the cells through a 70 µm cell strainer. The isolated cells were plated at a density of 1 × 105 cells on 100 mm dishes and cultured in alpha-modified Eagle’s medium (αMEM; Gibco/BRL) supplemented with 15 % fetal bovine serum (FBS; Biowest) and 1 % penicillin/streptomycin (P/S; Wako Pure Chemical Industries). Cells were passaged at 70 % confluence. Young and elderly DPSCs in passage 3 (P3) or P4 were used for experiments. Dental pulp stem cells were cultured in 12-well plates containing osteogenic differentiation media with and without TH. We used αMEM supplemented with 10 % FBS, 1 % P/S, and 10 nM dexamethasone (Wako Pure Chemical Industries) for osteogenic induction (osteogenic medium [OM]). We added TH (Takeda Chemical Industries) at the optimal concentration of 10-6 M to the OM, as described previously .
Dental pulp stem cells (1 × 105 cells) were cultured in 100 mm dishes at a density of 1 × 105 cells until they reached 70 % confluence. To calculate the initial percentage of DPSCs from young and elderly patients, the number of cells initially plated and the number of cells after detachment at 70 % confluence were counted. Cell proliferation of young and elderly DPSCs was compared by collecting and counting cells at P1, P2, and P3.
Flow cytometry analysis
A single-cell suspension was obtained by detaching the cells using 0.25 % trypsin and washing them with phosphate-buffered saline (PBS). Cells were fixed with 10 % FBS for 10 min at 37 ℃ and then incubated with the following antibodies for 90 min at 4 ℃: fluorescein isothiocyanate (FITC)-conjugated anti-human CD14, phycoerythrin (PE)-conjugated human CD29, FITC-conjugated human CD34, FITC-conjugated human CD44, PE- conjugated human CD73, FITC-conjugated human CD81, PE- conjugated human CD90, FITC-conjugated human CD105, and FITC-conjugated human CD146 (BioLegend). Cells were washed with PBS and then fixed in 4 % paraformaldehyde (PFA) for 10 min at 4 ℃. Non-labelled cells were used as negative controls. Cells were analyzed using a flow cytometer (BD Biosciences), and data analysis was performed using FlowJo software (FlowJo; FlowJo, LLC).
Alizarin red S staining
Young and elderly DPSCs were cultured in OM with or without TH for 14 days and stained with alizarin red S (Sigma-Aldrich), as described previously. In brief, cells were fixed with 10 % formaldehyde in PBS for 10 min at 4 ℃, followed by two washes with distilled water. Next, the cells were stained in 1 % alizarin red S solution for 15 min. After staining, they were washed twice with distilled water.
Alkaline phosphatase staining
Young and elderly DPSCs were stained with alkaline phosphatase (ALP) as described previously. In brief, the cells were fixed in 70 % ethanol after they were rinsed with PBS, followed by staining for 10 min with 0.01 % naphthol AS-MX phosphate (Sigma-Aldrich), using 1 % N,N-dimethyl formamide (Wako Pure Chemical Industries) as the substrate and 0.06 % Fast BB salt (Sigma-Aldrich) as a coupler.
Reverse transcription-polymerase chain reaction analysis
Total RNA from young and elderly DPSCs cultured in regular medium (RM Dulbecco’s Modified Eagle Medium [DMEM, GIBCO/BRL] supplemented with 10% FBS and 1% P/S), OM, and OM with TH was isolated using TRIzol (Invitrogen), and reverse transcription was performed using QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer’s instructions. Real-time polymerase chain reaction of Runx2, an osteoprogenitor maker, Alp and Colla1, early markers of osteoblast differentiation, and osteocalcin, a mature osteoblast marker, was performed in a Lightcycler 96 (Roche Diagnostics) using THUNDERBIRD SYBR qPCR Mix (Toyobo), as described previously. GAPDH was used as an endogenous control. The primer sequences used in this study are presented in Table 1.
Transplantation of young and elderly dental pulp stem cell sheets into a mouse calvarial defect model
A mouse calvarial defect model was used for animal experiments, as described previously, following the guidelines of Animal Care and Use committee of the Faculty of Medicine, Tokyo Medical University. In brief, young and elderly DPSC sheets were cultured on temperature-responsive dishes in OM with TH treatment for 14 days, and each sheet was transplanted into the calvarial defects (3.5 mm in diameter) created in the right parietal bone using biopsy punches (Kai Corporation). Four mice were used in each experimental group. Eight weeks after transplantation, mice were sacrificed, and calvaria were harvested.
The calvarial defects were examined by micro-computed tomography (micro-CT; SMX-90CT; Shimadzu) The scanning conditions were as follows: 90 kV, 110 µA, and a field of view (XY) of 10 mm; the resolution of one CT slice was 512 × 512 pixels. The data were reconstructed and analyzed using morphometric software TRI/3D-BON (Ratoc System Engineering). Bone volume (BV), bone mineral content (BMC), and bone mineral density (BMD) of a 5 × 5 × 3 mm3 cuboid area located in the center of the initial defect area were calculated.
Each experiment was replicated at least three times. Statistical analysis was performed using SPSS 24.0 software (IBM); all data are expressed as mean ± SD. Statistical significance was evaluated using one-way ANOVA, and p values were calculated with Student t test. P < 0.05 or P < 0.01 was considered to indicate statistically significant differences between two groups.