DPSCs extraction and cell culture
Primarily healthy third molars were collected from young people aged 18-25 years after informed consent at Oral and Maxillofacial Surgery of the Jiangsu Provincial Stomatological Hospital. The collection process obeyed the ethical approval of Nanjing Medical University. Tooth was removed and then placed in PBS buffer containing 100 U / mL penicillin. After washing with a sterile saline solution, the surface adhered gingival tissues and blood clots were removed. Dental pulp was gently harvested in fresh culture medium And digested in 4 mg / mL trypsin (Gibco, Life Technologies, Grand Island, NY) containing 3 mg / mL collagenase type I (Gibco, Life Technologies) at 37°C. 30 min later, isolated cells were inoculated in 6-cm culture dishes with α-MEM (Gibco, Life Technologies) containing 10% fetal bovine serum (FBS, Gibco, Life Technologies), 100 μg/mL streptomycin and 100 U / mL penicillin in a 5% CO2 incubator at 37°C. On the third day, solution was changed and medium was replaced every two days since after. Cell passage at a ratio of 1:3 was conducted at 70-80% confluence, and third - fifth generation cells were utilized for subsequent experiments. Isolated DPSCs were induced for osteogenesis at 50–60% confluence in osteogenesis medium (OM,Human Dental Pulp Stem Cell Osteogenic Differentiation Basal Medium, Cyagen Biosciences Inc, USA): standard GM containing 100 μM ascorbic acid, 2mM 2‐glycerophosphate, and 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA). OM was replaced every 2 days.
DPSCs characterization
STRO-1 is a protein-tagged gene of MSCs, which is the first isolated monoclonal antibody to identify MSCs. Cultured cells (3d) were subjected to immunofluorescence staining with an antibody of STRO-1 (1:200, Novus Biologicals, Littleton, CO, USA), followed by determination of positive expression of STRO-1. Meanwhile, cells were incubated with CD34-FITC, CD45-PerCP, CD90-PE, CD105-APC and CD73-PE (Miltenyi, Bergisch Gladbach, Germany), and subjected to FCM analysis (BD Biosciences, CA, USA).
Tri-lineage differentiation of DPSCs
Mineralized nodule formation of DPSCs was determined by ARS staining, as described previously25. Osteogenesis ability of third generation DPSCs was examined by induction in OM for 14 days according to the instructions. Then, DPSCs were reacted in 4% paraformaldehyde for 15 minutes and dyed with ARS (pH=4.2, Sigma, Aldrich) for 10 min. ARS was diluted in 10% cetylpyridinium chloride (CPC) to calculate the number of calcified nodules. OD value was determined at 570 nm.
DPSCs were incubated in adipogenic differentiation medium (adult fat adipose‐derived stem cell adipogenic differentiation medium, Cyagen Biosciences Inc, USA). When the cell fusion reached 80%, the adipogenic induction group was added with 2 ml OriCell adipogenic differentiation medium A solution. After 3 days, OriCell adipogenic differentiation medium B solution was replaced. After 24 h, change the A solution to culture. After 25 days, Oil Red O staining was conducted to assess adipogenic differentiation in fixed DPSCs.
Three-dimensional pellet culture of DPSCs (2.5×105 cells) in a 15 ml sterile tube was conducted for 25-day chondrogenic differentiation. Induction medium was changed every two days with the lids of the tube loosened. Pellets fixed and embedded in OCT compounds in 5 µm thickness (Sakura Finetek Co., Ltd., Tokyo, Japan) were dyed with Alcian Blue.
Cell transfection
Three circSIPA1L1 siRNAs (100 nM) were designed by Ribobio (Ribobio, China) and their sequences were as follows: siRNA-1: CTGGATGAACAAGGGAGAA; siRNA-2: ATGAACAAGGGAGAAAGCA; siRNA-3: AGGGAGAAAGCATGGGATT (Figure 1E),the si-NC group were transfected with randomized sequence of siRNA, the transfection efficacy was tested by RT-PCR and at last, circSIPA1L1 siRNA-1 and siRNA-3 were selected (Figure 1F).Meanwhile, miR-617 mimic (50 nM), miR-617 mimic NC (50 nM), miR-617 inhibitor (100 nM) and miR-617 inhibitor NC (100 nM) were purchased from Ribobio as well. To overexpress the circSIPA1L1, we designed an overexpression plasmid of circSIPA1L1 and the NC group were transfected with an empty vector, after transfection using Lipofectamine 2000 (Invitrogen, USA) for 48-96 h, the transfection efficacy was tested by RT-PCR Complete medium was replaced at 6 h.
Flow cytometry
DPSCs were cultured for three days, then collected by trypsin (Beyotime, Haimen, China) and fixed in alcohol overnight at 4 °C in dark. After PBS wash, samples were subjected to FACScan flow cytometer (BD Biosciences, San Jose, CA) and independently analyzed for three times.
Cell proliferation assay
Proliferative potential of DPSCs was determined by the Cell Counting Kit-8 (CCK-8) (Dojindo, Tokyo, Japan) assay and EdU incorporation assay. For CCK-8 assay, 3 × 103 DPSCs were inoculated in each well of a 96-well plate. After 24 hours of culture, the medium were replaced with osteogenesis medium. After 1, 3, 5, 7, and 9 days of culture, DPSCs were treated with CCK‐8 regents at 37°C for 2 h and the optical density (OD) at 450 nm was measured by a microplate reader.
For EdU incorporation assay, 5 × 103 DPSCs were treated with 50 mM 5 ethynyl-20-deoxyuridine (EdU, Ribobio) at 37 °C for 6 h. After 30-min fixation in 4% paraformaldehyde (PFA), DPSCs was treated with 2 mg / ml glycine for 10 min, 0.5% Triton X-100 and 1 × Apollo reaction mixture for 30 min. Subsequently, DPSCs were treated with 1×Hoechst-33342 solution in dark for 30 min at room temperature (RT) and images were captured by fluorescence microscopy.
ALP staining
At day 7 of osteogenesis, DPSCs were fixed in 4% PFA for 15 min and washed with PBS for three times. ALP staining was performed using the NBT / BCIP staining kit (Beyotime, China) and images were captured using a microscope (Olympus, Japan).
Western blot
RIPA lysis buffer (Beyotime, China) was used to isolate cellular protein, which was further loaded onto 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, MA, USA). After 2-h blockage in 5% skim milk, the membrane was incubated with diluted OSX, RUNX2, ALP (Abcam, UK), Smad3 and GAPDH (Cell Signaling Technology) at 1:1000 overnight at 4 °C. After TBST wash for three times, the membrane was reacted with the corresponding secondary antibody for 1 h at RT. Grey value was analyzed by ImageJ software.
Reverse transcription polymerase chain reaction (RT-PCR)
RNAs extracted from DPSCs using TRIzol (Invitrogen, USA) underwent reverse transcription by PrimeScript RT kit (TaKaRa, Otsu, Japan). RT-PCR was performed on an ABI 7300 real-time PCR system with Universal ChamQTM SYBR Green quantitative PCR Master Mix (Vazyme, Nanjing, China). GAPDH and U6 were the internal references for mRNA and miRNA, respectively. Bulge-Loop miRNA qPCR Primer kit (RiboBio) was used for measuring miRNA-617 expression. Primer sequences for ALP, OSX, RUNX2 and GAPDH were depicted in Table 1. Expression levels were calculated by the 2−ΔΔCt method as previously reported 25.
Immunofluorescence staining
After PBS washing for three times, DPSCs were subjected to 30-min incubation in 4% paraformaldehyde, 15-min incubation in 0.1% Triton X-100 (Beyotime) and 2-h blockage in normal goat serum (DCS / BioGenex, Hamburg, Germany) at RT. After treatment with primary and T fluorescent dye-labeled designated secondary antibody at appointed time points, nuclei were counterstained with DAPI (Beyotime). Immunofluorescence images were observed under a fluorescent inverted microscope (Olympus, Shanghai, China).
Dual-luciferase reporter assay
Dual-luciferase reporter assay was conducted as described previously 25. In brief, HEK293T cells seeded in 24-well plates (5×105 cells/well) were co-transfected with Firefly Luciferase reporter vector (800 ng), Renilla Luciferase reporter vector (5 ng wild-type or mutant-type, GeneChem, Shanghai, China) and 50 nM miR-617 mimics or negative control using Lipofectamine 2000. Luciferase activity measured by the Dual-Luciferase Reporter Assay System (Promega) was finally calculated as Firefly luciferase activity normalized to that of Renilla.
Animal procedures
Animal procedures followed institutional guidelines and got approval of the Ethics Committee of Nanjing Medical University. Fifteen 5-week homozygous nude mice were provided by the Animal Center of Nanjing Medical University. Mice were habituated for 1 week with 3-4 per cage. They were randomly assigned into three groups (n=5 per group) and subcutaneously transplanted with DPSCs transfected with si-NC, si-circSIPA1L1-1 or si-circSIPA1L1-3, respectively. Specifically, transfected DPSCs underwent osteogenesis for 2 weeks, followed by treatment with Bio-Oss collagen (Geistlich, Germany) scaffold for 12 h at 37 °C. Make two longitudinal incisions on the back of the nude mouse, and bluntly separate to form dorsal subcutaneous pocket, where two implants were inserted. Eight weeks later, the implant was removed and fixed in 4% PFA.
Histology
Sections were decalcified in 10% EDTA (pH 7.4) for 4 weeks with EDTA solution replacement every other day, dehydrated and paraffin embedded. Subsequently, sections were sagittaly sectioned, deparaffinized and visualized by hematoxylin and eosin (H&E) or Masson's trichrome staining. Images were captured using a microscope.
Statistical processing
Statistical Package for Social Sciences (SPSS) software 16.0 was used for statistical analyses. One-way analysis of variance (ANOVA) and Student's t-test were used for comparing differences. A two-tailed P < 0.05 considered as statistically significant. Data were expressed as mean ± SD of from at least three independent experiments.