Cell Isolation and Aggregate Culture
Healthy human impacted third molars or extracted teeth for orthodontic reasons were collected from individuals aged 18-40 years. The standard of healthy periodontal tissues: no bleeding on probing, probing depth≤4mm, alveolar bone loss≤3mm. PDLSCs were isolated and cultured as previously described . Briefly, the periodontal ligament was gently separated from the middle part of the root surface and enzymatically digested with type I collagenase (3mg/ml, Sigma-Aldrich, USA) for 2h at 37℃ (suspended every 15 mins). Cells were then plated in 6-well plates in alpha modification of Eagle’s medium (a-MEM; Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA), 0.292 mg/ml L-glutamine (Invitrogen, USA), 100 units/ml penicillin (Invitrogen) and 100 mg/ml streptomycin (Invitrogen) at 37 °C in 5% CO2, cultured for 2 weeks and the medium was changed every 3 days. To further purify the stem cells, single-cell-derived colonies were obtained using the limiting dilution technique.
Normal exfoliated human deciduous incisors were collected from 7-8-year-old children. SHED was isolated and cultivated in accordance with the protocol previously described , that is, the pulp was separated from remnant crown and the digested with 3mg/ml type I collagenase (Sigma) and 4 mg/mL dispase (Sigma) for 1h at 37℃. The single suspensions were seeded into 6-well plates with regular cell culture medium mentioned above. SHED used were at passage 2-5, and the same passage were used for each experiment.
PDLSCs aggregates were prepared as described previously . 1-2×105 PDLSCs per well were seeded in 6-well-plate with basal medium and changed for aggregate induction medium with SHED aggregate-derived exosomes at different concentrations when reaching 100% confluence. The induction medium was refreshed every 3-4 days. The aggregate was harvested after 10 days for the following experiments.
Exosomes Isolation and Characterization
SHED aggregates were prepared as described previously . Then the SHED aggregate or SHED were cultured in the medium with exosome-deleted FBS for 48h. Exosomes-deleted FBS was prepared by ultracentrifuging for 16h at 100000g before use. Finally, the supernatants were collected and several centrifugates were performed for exosomes isolation as described previously . Briefly, the supernatants were centrifuged at 300g for 10 min, 2000g for 10 min, and 16000g for 30min to remove dead cells and cellular debris, respectively. Then, the supernatants were ultra-centrifuged at 150000g for 70min, and additionally washed with PBS for another 70min at 150000g. All procedures were performed at 4℃. The final pellets were resuspended in sterile PBS and stored at -80℃ for following experiments.
Western blotting was performed to detected exosome markers, including CD63 (ab217345, Abcam, USA), CD81 (sc-166029, Santa Cruz-Biotechnology, USA), CD9 (ab92726, Abcam). Cell extract was considered as control. Transmission electron microscopy (TEM) (Thermo Fisher, USA) was conducted to observe the morphology of exosomes. The exosomes were dropped onto carbon-coated copper grids and kept for 3-4 min, then dried with filter paper and stained with 1%phosphotungstic acid for 10s, finally washed with distilled water for 30s. Nanoparticle tracking analysis (NTA) was utilized to identify the particle size and distribution.
Exosomes Uptake Assay
Enriched exosomes were labeled with PKH67 (Sigma) according to the manufacturer’s protocol. PDLSCs were seeded in 24-well plates with climbing, and incubated with the labeled exosomes for 4 and 24h, respectively. Hoechst (33342, Sigma) was used for staining the nuclei. The uptake of exosomes was visualized by confocal fluorescence microscope (Nikon, Japan).
Tube Formation Assay
In vitro angiogenesis was determined by tube formation assay in Matrigel. PDLSCs (4×104) were seeded onto Matrigel (Sigma)-coated 96-well plate and cultured in FBS-free medium in the presence of exosomes at the indicated concentrations (0, 5, 10, 15, 30, and 60mg/ml) for 6h. The images of tube formation were acquired by inverted phase contrast microscope (Leica, Germany). The total tube length, number of junctions and number of nodes were calculated by randomly selecting five fields per well using ImageJ 1.53c (National Institutes of Health, USA). The same experimental approach was used in PDLSCs after transfection with mimics and inhibitor.
Alkaline phosphatase (ALP) staining and Alizarin Red S (ARS) Staining
PDLSCs were seeded in 12-well plate and cultured until the cells reached 80% confluence, then incubated with exosomes at different concentrations (0, 15, 30, and 60mg/ml) for 48h and further induced with osteogenic medium (basal medium supplemented with 50 mg/mL L-ascorbic-2-phosphate (MP Biomedicals, USA), 0.1 mM dexamethasone, and 5 mM b-glycerophosphate (Sigma)) for 7 days or 28 days for ALP staining or ARS staining, respectively. For ALP staining, the cells were rinsed with PBS, then nitro blue tetrazolium (NBT) and 5-bromo-4-chroro-3-indolyl phosphate (BCIP) (Beyotime, China) were added for the staining. 30 min later, staining agent was discarded and cells were rinsed two times with PBS. For ARS staining, the cells were fixed with 60% isopropyl alcohol and stained with 2% ARS (Kermel, China). After rinsing with distilled water, stained calcium nodules were identified under microscope. Finally, the mineralized nodules were dissolved with hexadecyl pyridinium chloride, and absorbance was quantitatively measured at 570 nm for statistical analysis.
Scanning Electron Microscopy (SEM)
The cell aggregates from each group were washed with PBS three times, fixed with 2.5% glutaraldehyde at 4℃, dehydrated and dried in a critical-point dryer. Finally, the specimens were observed under SEM (Hitachi S-4300; EIKO Engineering, Tokyo, Japan).
Total proteins were isolated from cell aggregates. Extracellular matrix (ECM)- and angiogenesis-related proteins were analyzed by western blot as previous described . Primary antibodies employed in this study included fibronectin (ab2413, Abcam), integrinβ1 (ab52971, Abcam), collagen type I (COL-I) (ab34710, Abcam), angiogenin (ANG) (ab10600, Abcam), platelet derived growth factor (PDGF) (3174, Cell Signaling Technology), phosphate-SMAD1/5 (#9516, Cell signaling Technology), transforming growth factor-β receptor II (TGFβRII) (sc-400, Santa Cruz Biotechnology), phosphate-SMAD2/3 (sc-11769, Santa Cruz Biotechnology) and GAPDH (30201ES20, YEASEN). Secondary antibody was HRP conjugated to antibodies to rabbit and mouse.
Small RNA sequencing
Small RNA sequencing was performed by BGISEQ-2000 and the sequencing libraries were constructed in BGI online platform (Shenzhen, China). The miRNAs expression with significant differences between the SHED- and SHED aggregate- derived exosomes was shown by the heatmap.
Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted with TRIzol reagent (Invitrogen) and converted to cDNA using Mir-XTM miRNA First-Stand Synthesis Kit (Takara, Japan). Then RT-PCR was conducted with SYBR® Premix Ex TaqTM II (Takara) using the quantitative PCR System (Bio-Rad, USA). The expression of miR-222 was normalized to that of U6 snRNA. The miR-222 primer was AGCUACAUCUGGCUACUGGGU (RiboBio, China). The forward and reverser primer of U6 were included in the Mir-XTM miRNA First-Stand Synthesis Kit (Takara).
miRNA mimics or inhibitors were transfected into PDLSCs using riboFECTTM CP (RiboBio, China) according to the manufacturer’s protocol. The miRNA mimics and inhibitor used were from Sangon Biotech (Shanghai, China).
Periodontal Bone Defect Model
The use of Sprague-Dawley (SD) male rats for research was approved by the IRB of FMMU. The surgical procedures were based on the guidelines of the Animal Care Committee of FMMU. The periodontal defect model was created as previously described . A periodontal bone defect of approximately 3×2×1 mm3 was created at the buccal alveolar bone of left mandibular molars. A total of 15 SD rats (8 weeks old) from the FMMU Animal Center were randomly divided into 3 groups: (1) a control group without cell and β-tricalcium phosphate (β-TCP) (provided by University of Extremadura, Spain) implantation (control, n=5); (2) a group treated with PDLSCs aggregate wrapping β-TCP (PDLSCA, n=5); and (3) a group treated with combination of PDLSCs aggregate and SA-Exo, which wrapping β-TCP (PDLSCA+SA-Exo, n=5). After 6 weeks, the rats’ mandible samples were harvested and fixed with 4% paraformaldehyde for 48h.
The mandible samples were scanned using a Micron X-ray 3D Imaging System (YXLON, Germany) with 10mm resolution. Three-dimensional (3D) images were reconstructed and analyzed by the VG Studio 3.4 (VG, Germany). The ratio of new bone volume to tissue volume (BV/TV) was calculated.
After micro-CT analysis, the mandibles were decalcified with 17% ethylenediaminetetraacetic acid (EDTA) (MP Biomedicals) for 1 month and embedded in paraffin. Paraffin sections (3um thick) were stained using hematoxylin and eosin (HE) as described . Photographs were taken using a microscope (OLYMPUS, Japan). The percentage of new bone area in the total area was evaluated quantitatively from 3 randomly-selected sections by ImageJ 1.53c.
Masson’s Trichrome Staining
The paraffin sections were stained with Masson’s trichrome staining (Baso Diagnostic Inc., China) according to the manufacturer’s instructions. Photographs were taken using a microscope (OLYMPUS).
The sections were deparaffinized and rehydrated, then subjected to antigen retrieval in boiled sodium citrate buffer solution (pH 6.0) for 10 minutes and cooled to room temperature. Slides were permeabilized with 1% TritonX-100 for 10 minutes and then blocked for 1h in blocking buffer (goat serum). After blocking, samples were incubated with primary antibody overnight at 4°C and then incubated with the related fluorescence secondary antibodies for 1 hour at room temperature. Hoechst (MP Biomedicals) was used for counterstaining the nuclei. The immunofluorescent images were obtained using confocal fluorescence microscope (Nikon). Antibodies against fibronectin (ab2413, Abcam) and CD31 (ab212712, Abcam) were used in this study.
All results were presented as the mean ± standard deviation (SD) of at least three independently experiments. Two-group comparisons were analyzed by Student’s t tests. Comparisons among three or four groups were evaluated by one-way ANOVA followed by LSD post hoc test. P value less than 0.05 was reflected to specify statistical significance.