All DPSCs in our experiments derived from human patients. Cells were isolated from the extracted third molars of human dental patients between 18 and 22 years old. The process were reviewed and approved by the institutional Review Board of the Hospital of Stomatology of Guangzhou Medical University, and a signed informed consent document was obtained from each patient. hDPSCs were prepared using explants and the trypsin digestion technique. Cells were cultured and expanded in alpha minimum essential medium (α-MEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin solution. Cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Fresh culture media was replenished every 3 days, Third-passage cells were harvested for the subsequent experiments.
Flow cytometry assay
Cultured cells in their 3rd passage were identified based on the surface antigens of hDPSCs using a flow cytometry method. Cells were trypsinized and incubated in PBS containing 0.1% FBS for 45 min with fluorescein-conjugated monoclonal antibodies against CD34, CD45, CD73, CD90, CD146 and CD166 (BD Biosciences, San Jose, CA, USA). Flow cytometry analysis was performed using a flow cytometer (FACSCalibur, BD Biosciences).
After odotoblastic induction for 3 days, the differentiated hDPSCs in the culture dishes were isolated and hDPSCs cultured in normal culture medium were observed as the control. Total RNA was isolated with TRIzol (Invitrogen) according to the manufacturer’s instructions. A LncRNA microarray (KangChen Bio-tech, Shanghai, China) using induced group and non-induced group was performed. Differentially expressed lncRNAs were identified based on fold change (>2), as well as P< 0.05.
Odontogenic differentiation induction and treatment of hDPSCs
hDPSCs (passages 4~6) were seeded in 6-well plates at a density of 1×105 cells each well. To induce odontogenic differentiation of hDPSCs, cells were cultured with odontogenic medium (10% FBS, 10-8 mol/L dexamethasone, 10 mmol/L β-glycerophosphate and 50 mg/mL ascorbic acid in α-MEM) for 14 days. Control samples were cultured in 10% FBS in α-MEM with no supplements. For functional experiments, recombinant human protein BMP-2 (rhBMP-2; Gibco, USA) and FGF9 (rhFGF9; Peprotect, USA) were purchased and used to stimulate hDPSCs. hDPSCs were cultured with odontogenic medium consisting of rhBMP-2 (20 ng/mL) and rhFGF9 (20 ng/mL) separately. Fresh medium was changed every 3 days.
ALP assay and Alizarin Red S staining
Cells lysates were harvested after odontoblatic differentiation, and ALP activity was evaluated and analyzed following the manufacturer’s instructions (ALP Diagnostics Kit, Yeasen, Shanghai, China). hDPSCs were fixed in 4% paraformaldehyde, and mineral nodules were detected by Alizarin Red S staining (BestBio, Shanghai, China).
RNA preparation and qRT-PCR
Total RNA was isolated from cultured cells with TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s protocol. Reverse transcription was performed with PrimeScript RT Master Mix (TaKaRa, Japan) for lncRNA and mRNA. For miRNA examination, cDNA was synthesized using a miRNA First Strand cDNA Synthesis Kit (Sangon Biotech, China). RNA expression was measured by quantitative real-time PCR analysis using a CFX96 Real-Time PCR instrument (Life Technology, USA) and SYBR Green reagent (TaKaRa, Japan). GAPDH was used as an internal control for lncRNA and mRNA. The expression of miRNA was normalized to U6. Primers were synthesized by Generay Technologies (Shanghai, China), and all sequences are available in Additional file 1: Table 1. The 2-ΔΔCT method was used to calculate relative expression levels.
Western blot analysis
Cells were lysed in RIPA Buffer (Beyotime, China) combined with a cocktail of protease inhibitors (Thermo Scientific, Rockford, IL). Equal amounts of proteins (25 μg) of different groups were separated by 10% SDS-PAGE and transferred to 0.45-μm PVDF membranes (Millipore, USA). The membrane was first blocked with 5% BSA for 1 h at room temperature and incubated at 4 °C overnight with primary antibodies: GAPDH (1:1000; Abcam, Cambridge, UK), RUNX2(1:1000; Cell Signaling Technology, Danvers, MA), DSPP (1:1000; Abcam, Cambridge, UK) and DMP-1 (1:1000; Abcam, Cambridge, UK). After washed with Tris-buffer saline containing 0.05% Tween 20 (TBST) for three times and 5 min each, the membranes were incubated with secondary antibodies labelled with horseradish peroxidase (1:3000; Cell Signaling Technology, Danvers, MA) at room temperature for 1 h. Blots were visualized using ECL Western Blotting Substrate.
Recombinant lentiviruses targeting H19 (shH19-1 and shH19-2) and Lenti-shNC were purchased from GenePharma Company (Shanghai, China). hDPSCs were transfected by lentiviruses exposure in 1 mL α-MEM supplemented with 10% FBS and 5μg /mL polybrene for 24 h. H19 overexpression plasmid pcDNA3.1-H19, miR-140-5p mimics and and scramble control (NC) were chemically synthesized by GenePharma. When hDPSCs were 70%-80% confluent, pcDNA-H19 and miRNA mimic transfection was performed using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. qRT-PCR analysis was used to detect H19 and miR-140-5p expression levels to validate the transfection efficiencies. The cells were cultured in mineralizing medium for odontoblastic differentiation 48 h after transfection.
Dual luciferase reporter assay
The luciferase reporter vector psiCHECK-2 containing full-length of H19 sequence, the 3’ untranslated region (UTR) sequence of BMP-2/FGF9 and their relevant mutant types were synthesized by Synbio, China. HEK293T cells were seeded into 24-well plates at a density of 5×104 cells per well the day before transfection. Cells were transiently co-transfected with corresponding psiCHECK-2 vector and miR-NC/miR-140-5p mimics using Lipofectamine 3000. Renilla and Firefly luciferase activity were measured separately 48 h after transfection using a dual-luciferase reporter assay system (Promega, USA) following the manufacturer′s instructions. These experiments were repeated three times.
The Human Enzyme-linked immunosorbent assay (ELISA) kits were obtained from CLOUD-CLONE CORP (Wuhan, China). The culture supernatants were collected on day 2 after odontogenic induction treatment. The secreted levels of BMP-2 and FGF9 were quantified using corresponding ELISA kits, according to the manufacturer’s protocol.
In vivo odontogenesis assay
BALB/c nude mice (5 weeks old, five mice were included in each group) were purchased from the Experimental Animal Center, Guangzhou University of Chinese Medicine. All the animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine and were performed in accordance with established guidelines. hDPSCs with H19 overexpression and negative controls induced under odontoblastic medium for 1 week were harvested and incubated with Bio-Oss Collagen (Geistlich, Germany) scaffolds for 1 h at 37 °C. The hDPSCs-loaded scaffolds were implanted subcutaneously into the nude mice. Implants were harvested 6 weeks after implantation and fixed in 4% PFA.
Analyses of bone formation in vivo
Micro-CT analysis was performed using a high-resolution Micro-CT (Bruker, Karlsruhe, Germany) set as 10μm of the voxel resolution of the scanned volumes, 500 ms of the 360 rotational steps per time. Micro-CT image slices were reconstructed and the ratio of new bone volume to existing tissue volume (BV/TV) were calculated by Micro-CT image analysis software. For histological examination, the specimens were decalcified in 10% EDTA (pH 7.4) for 30 days. After dehydrated and embedded with paraffin, tissue samples of 5 μm thick paraffin section were stained with hematoxylin and eosin (H&E) and Masson’s trichrome. Immunohistochemical staining was conducted using HRP/DAB (ABC) detection IHC kit and human-specific primary antibodies, including DMP-1 (1:100). All products for IHC were from Abcam. Histological staining was captured under the microscope (Leica Microsystems, Germany) with an increase of ×100.
Statistical analyses were performed using GraphPad Prism7.0 (GraphPad Prism, Inc., La Jolla, CA, USA). One-way analysis of variance (ANOVA) and Student’s t test (two-tailed) were used to evaluate the statistical significance. All data are shown as the means ± SD from three independent experiments. Statistical significance was defined as P < 0.05.