Animal Care Committee at the Ninth People’s Hospital in Shanghai Jiao Tong University School of Medicine (Shanghai, China) approved animal experiments.
Human DPSC isolation and identification
Cells were isolated from dental pulp. Briefly, we removed tissue and immersed it in a digestive solution (4.0 mg/mL dispase and 3.0 mg/mL: type I collagenase) for 1 h at 37°C. It was filtered using 70-μm cell strainers to get an hDPSC suspension. The cells were plated in T25 flasks and cultured them in culture medium DMEM/F12 with fetal bovine serum of 10% and 1% penicillin/streptomycin at 37°C and 5% CO2.
For cell phenotypic analysis using surface proteins, we harvested DPSCs using 5 mM ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS). We then incubated cells with FITC-conjugated antibodies against human CD44, CD45, CD34, CD29, CD90, CD73, and CD105. Isotype antibodies that matched would serve as controls (Becton Dickinson, San Jose, CA). We washed cells with cold PBS containing 2% fetal calf serum. We acquired 1000 labeled cells and analyzed them through an immunofluorescence microscope (Becton Dickinson).
Plasmid transfection and construction
Overexpressed pcDNA3.1-SOX11 and pcDNA3.1-circ-FURIN plasmids were purchased from GeneChem Co., Ltd. (Shanghai, China). The siRNA against circ-FURIN (si-circ-FURIN), miR-125 mimic, and miR-125 inhibitor were also purchased from GeneChem Co., Ltd. Lipofectamine® 3000 (Invitrogen Life Technologies, USA) was used for cell transfections following standard manufacturer instructions.
Strand-specific high-throughput RNA-Seq library construction
Total RNA was retrieved from osteogenic DPSC differentiation after 0 or 14 days through TRIzol reagent (Invitrogen, Carlsbad, CA, USA) by using ~3 μg of RNA from individual samples with VAHTS Total RNA-seq (H/M/R) Library Prep Kit from Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) to erase ribosomal RNA and retain other mRNA’s, like non-coding RNAs. RNAs were exposed to 40 U of RNase R (Epicenter) at 37°C for 3 h after TRIzol purification. KAPA Stranded RNA-Seq Library Prep Kits (Roche, Basel, Switzerland), which were subjected to deep sequencing via Illumina HiSeq 4000 at Aksomics, Inc. (Shanghai, China), was used to prepare RNA-seq library
Total RNA isolation and quantitative reverse transcription (RT-q)PCR
Total RNA was isolated from tumor cells, tissues, and serum through TRIzol reagent (Invitrogen) abiding by procedures. RNA sample purity and concentration were detected using a spectrophotometer via measuring absorbance values at 260, 230, and 280 nm through NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA). The OD260/OD280 ratios in [1.8, 2.1] and OD260/OD230 ratios >1.8 were considered excellent. We calculated relative expression using 2-ΔΔCt approach. RT-qPCR amplification was performed via the aftermentioned primers: circ-FURIN: forward: 5′-GTTAGAGGTTTTAAAGTG-3′; reverse: 5′-CATCCAGCTCCAGAGCACCTGG-3′; osterix (OSX): forward: 5′-ACTGCCCCACCCCTTAGACA-3′; reverse: 5′-GAGGTGCACCCCCAAACCAA-3′; SOX11: forward: 5′-AGCAAGAAATGCGGCAAGC-3′; reverse: 5′-ATCCAGAAACACGCACTTGAC-3′; runt-related transcription factor 2 (RUNX2): forward: 5′-TGGTTACTGTCATGGCGGGTA-3′; reverse: 5′-TCTCAGATCGTTGAACCTTGCTA-3′; osteocalcin (OCN): forward: 5′-AGCCACCGAGACACCATGAGA-3′; reverse: 5′-GGCTGCACCTTTGCTGGACT-3′; alkaline phosphatase (ALP): forward: 5′-GAACGTGGTCACCTCCATCCT-3′; reverse: 5′-TCTCGTGGTCACAATGC-3′; miR-125: forward: 5′-GGGTCCGAGGTATTCGCACT-3′; reverse: 5′-TCCCTGAGACCCTTTAACCTGTG-3′; U6: forward: 5′-CTCGCTTCGGCAGCACA-3′; reverse: 5′-AACGCTTCATTTGCGT-3′; and GAPDH: forward: 5′-AATCCCATCACCATCTTCC-3′; reverse: 5′-CATCACGCCACAGTTTCC-3′. We normalized circ-FURIN, ALP, SOX11, OCN, OSX, and RUNX2 expression levels to GAPDH, while we normalized miR-125 expression level to U6.
Dual-luciferase reporter assays
Binding sites for circ-FURIN and SOX11 3′-UTR, i.e., circ-FURIN-WT, circ-FURIN-Mut, SOX11-3′UTR-WT, and SOX11-3′UTR-Mut, were placed into pGL3 promoter vector (Realgene, Nanjing, China) for dual-luciferase reporter assay. Cells were plated into 24-well plates and transfected with 80 ng plasmid, 50 nM miR-125 mimics, 5 ng Renilla luciferase vector pRL-SV40, along with NC reagents through Lipofectamine 2000 (Invitrogen). Cells were gathered and detected two days followed by transfection through Dual-Luciferase Assay (Promega, Madison, WI, USA) according to protocols. All experiments were independently repeated in triplicate.
We used NBT/BCIP staining kit (CoWin Biotech, Beijing, China) for ALP staining following instructions. DPSCs were seeded in 24-well plates to be cultured in osteogenic medium for 1~2 w. Cellswere fixed with 4% PFA for half of an hour and incubated them in a staining reagent in dark for twenty minutes.
In order for detection of extracellular matrix calcium deposition, we seeded DPSCs in 24-well tissue plates and cultivated them for one to two weeks in osteogenic medium. Then, 0.1% Alizarin Red S (ARS, Sigma-Aldrich, Saint Louis, MO, USA) solution at pH 4.2 was used to stain nodules that calcified followed by fixing DPSCs.
In vivo heterotopic bone formation assay
DPSCs were induced in osteogenic medium for one week before in vivo investigation. Cells were resuspended to be incubated with 7 mm × 5 mm × 2 mm Bio-Oss Collagen (Geistlich, GEWO GmbH, Baden-Baden, Germany) scaffolds for one hour at 37°C after centrifugation at 150 g for 5 min. Cells were subcutaneously implanted on backs of five-week-old BALB/c homozygous nude (nu/nu) mice (five mice per group) as previously described . The implants were harvested after eight weeks.
Hematoxylin and eosin (H&E) and Masson’s trichrome staining and immunohistochemical analyses
Specimens in 10% EDTA (pH 7.4) was decalcified for 1 min after embedding in paraffin and dehydration. We cut sections (5 μm) and stained them with H&E as well as Masson’s trichrome. We performed immunohistochemical analysis following previously described procedure . Specimens were maintained in 5% normal goat serum for half of an hour and incubated with primary antibody against OCN (Santa Cruz Biotechnology) at 4°C overnight. Our team processed sections via ABC detection kit (Vector Laboratories, Burlingame, CA) and visualized with Olympus microscope (Olympus Co., Tokyo, Japan).
Our team expressed results as mean ± standard deviation (SD). GraphPad Prism software (GraphPad, La Jolla, CA, USA) was used to compute significance. P-value ≤0.05 inferred statistical significance.