Animals
C57BL/6J mice and BALB/c nude mice were purchased from Vital River Laboratory Animals Technology (Beijing, China). All animal experiment protocols (2018029) were approved by the Institutional Animal Care and Use Committee of China Medical University.
Antibodies and reagents
Anti-CD9, CD63, Alix, calnexin, CD31, CD34, CD45, CD73, CD90 and CD105 antibodies were purchased from Abcam (Cambridge, UK). Anti-Ki-67, RhoA, Rac1, Cdc42 and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, USA). Alexa Fluor 488 and Alexa Fluor 568 secondary antibodies were purchased from Proteintech (Rosemont, IL, USA). Goat anti-rabbit/anti-mouse IgG IRDyel 800cw secondary antibodies were purchased from Abbkine (Redlands, CA, USA). PKH-26 and PKH-67 kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). LipofectamineTM RNAiMAX, FMTM4-64FX and ActinGreenTM 488 were purchased from Thermo Fisher (Eugene, Dregon, USA). SiCdc42, Cdc42-EGFP and Cdc42-mCherry fusion protein expression plasmids were purchased from GenePharma (Suzhou, China). Cdc42 inhibitor ML141 was purchased from MedChemExpress (Monmouth Junction, USA).
SCAP isolation and characterization
Human third molars with immature roots were obtained from healthy donors with an age range from 12 to 15 years in the clinic at the School of Stomatology affiliated with China Medical University. Informed consent was obtained from all patients and their parents. The apical papilla was gently separated and digested with dispase II (Boehringer Ingelheim, Mannheim, Germany) and collagenase type I (Worthington Biochemical Co., Lakewood, CO, USA). Single-cell suspensions were seeded and cultured in alpha-minimum essential medium (α-MEM, HyClone, Logan, UT, USA) supplemented with 15% (v/v) fetal bovine serum (FBS, MRC, Uruguay), 1% (v/v) penicillin-streptomycin solution (HyClone), 2 mM L-glutamine (Biosource/Invitrogen, USA), and 0.1 mM L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), and incubated at 37°C with 5% CO2. The expression of MSC surface markers, including CD31, CD34, CD45, CD73, CD90, and CD105 was detected by flow cytometry. The multipotent differentiation potential of SCAP, including osteogenesis and adipogenesis, were evaluated using osteogenic and adipogenic differentiation media for four weeks. Alizarin red S and Oil red O staining were used to detect the formation of mineralized nodules and lipid droplets, respectively.
SCAP-Exo isolation and identification
SCAP were cultured in exosome-free medium for 48 h. The culture supernatant was collected and centrifuged at 4°C in three different speeds: 3,000 × g for 20 min, 20,000 × g for 30 min, and 120,000 × g for 2 h in an ultracentrifuge (Beckman Optima L-100XP, USA). Exosomes were resuspended in sterile PBS and stored at -80°C. SCAP-Exo were observed by transmission electron microscopy (TEM) (H-800, Hitachi, Japan). A nanoparticle tracer analyzer (ZetaView, Germany) was used to measure the size of the particles. Exosomal surface markers, including CD9, CD63, and Alix were detected by western blotting. Cdc42 siRNA and Cdc42 inhibitor ML141 (20 μM) were used to treat SCAP (whole cells). The exosome-free medium was changed and the cells were cultured for 48 h. The culture supernatant was collected and centrifuged to isolate SCAPsiCdc42-Exo and SCAPML141-Exo.
Quantification of exosomes
Twenty microliters of SCAP-Exo were added to 50 μL protein lysate and lysed on ice for 1 h. BCA protein assay kit was used to generate the standard curve of protein concentration and was subsequently used to measure the concentration of SCAP-Exo.
SCAP-Exo treatment in wound healing of CSD in the palatal gingiva
The wound model was identical to a previous study [20]. Full-thickness circular gingival wounds (soft tissue defects) with a diameter of 2.0 mm were made in the palates of C57BL/6J mice using a biopsy punch (n = 5). SCAP-Exo or SCAPsiCdc42-Exo was suspended in PBS at a concentration of 1 μg/μL. Forty microliters SCAP-Exo, SCAPsiCdc42-Exo, or PBS (as control) were injected submucosally into four symmetrical sites around the wounds after they were created, according to previous protocol [21], so that 40 μg exosomes were applied locally on each wound. Mice were sacrificed 7 days post-operation, fixed in 4% paraformaldehyde, and decalcified with 10% ethylenediaminetetraacetic acid solution. The samples were embedded in paraffin, sectioned and stained with haematoxylin and eosin (H&E). In addition, the samples were embedded in optimal cutting temperature compound and sectioned. The frozen sections were stained with immunofluorescent CD31, an endothelial marker of micro-vessels, and the CD31 positive area was analysed using the Image J software (1.50i, National Institutes of Health, Bethesda, MD, USA).
In vivo tracking experiment
PKH-26-labelled SCAP-Exo or PBS (as control) was injected submucosally into four symmetrical sites around the wounds once after the wounds were created. Mice were sacrificed after 7 days post operation. Frozen sections and fluorescence images were used to observe the fate of SCAP-Exo.
Real-time live-cell imaging (RT-LCI)
Human umbilical vein endothelial cells (HUVECs) were seeded into a 35-mm dish (81158, Ibidi, Germany) and stained with FMTM 4-64FX. SCAP-Exo were labelled with PKH-67 and added to the HUVECs. The process of HUVECs taking up SCAP-Exo was observed under the laser confocal microscope (ECLIPSE Ti2, Nikon, Japan) for 30 min continuously.
Western blot analysis
The protein concentration was detected using a BCA protein assay kit. Proteins (20 μg) were loaded onto a 12% sodium dodecyl sulphate-polyacrylamide gel for electrophoresis, and then transferred to polyvinylidene difluoride membranes. The membranes were exposed to the appropriate primary and secondary antibodies. Finally, the bands were revealed using an Odyssey CLx instrument (LI-COR, Lincoln, NE, USA). The density of the bands was measured with Image J to quantify protein expression.
Tube formation assay
Matrigel (50 μL) (#356234, BD Biosciences, San Jose, CA) was precoated in each well of a 96-well plate and polymerized at 37 °C. HUVECs and SCAP-Exo-pretreated HUVECs were seeded at a density of 1.5 × 104 cells/well and cultured for 8 h. Photos of tube formation were taken by a stereoscopic microscope (ECLIPSE TE2000-S, Nikon, Japan). The indexes of tube formation were analysed using Image J.
Matrigel plug assay
Matrigel (200 μL) (#356231, BD Biosciences, San Jose, CA) was mixed with SCAP-Exo, SCAPsiCdc42-Exo, SCAPML141-Exo or PBS on ice. The mixtures were injected subcutaneously into the dorsum of BALB/c nude mice (n = 5). After 14 days, the matrigel plugs were extracted. H&E staining was used and the number of vessels in the matrigel was counted.
Cell proliferation assay
Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) and Ki-67 staining assay. HUVECs were seeded into 96-well plates at a density of 2,000 cells/well, and cultured with SCAP-Exo. The plates were incubated for 24, 48, and 72 h. CCK-8 solution (Dojindo, Kumamoto, Japan) was added and incubated in the dark. The absorbance of each well was measured at 450 nm using a microplate reader (Tecan, Salzburg, Austria). In addition, HUVECs (2 × 104/well) were seeded on glass coverslips placed inside a 12-well plate and cultured to the logarithmic phase. Thereafter, cells were fixed and stained with Ki-67 immunofluorescent antibody. The number of Ki-67-positive cells was indicated as a percentage of the total cell number.
Cell migration assay
Cell migration was measured using the transwell cell migration and scratch wound healing assay. HUVECs were seeded into the upper transwell insert of a 24-well plate at a density of 1 × 104 cells/well. SCAP-Exo were added into the lower chamber and incubated for 24 h. Thereafter, cells in the transwell chamber were removed. After staining with crystal violet, the number of cells migrating below the transwell layer was counted. Moreover, HUVECs (5 × 105/well) were seeded into a 6-well plate and a scratch in the cells was made with a 200 μL sterile tip. The serum-free medium containing SCAP-Exo was then replaced. After 0, 12, and 24 h, the boundaries of the scratches were recorded and the wound closure rates were measured and calculated using Image J.
Pull-Down assay
RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit (Cytoskeleton, Denver, CO, USA) was used following the manufacturer’s instructions. Briefly, the equivalent protein amounts of lysate were added to a pre-determined amount of rhotekin-RBD (for RhoA activation) or PAK-PBD beads (for Rac1 and Cdc42 activation) and incubated at 4 °C on a rotator for 1 h. Next, the beads were centrifuged and washed. The bead pellets were resuspended with 20 μL loading buffer and boiled. The samples were then analysed by western blot.
F-actin immunofluorescence staining
HUVECs were fixed for 30 min and stained with ActinGreenTM 488 at 4 °C for 30 min. Pseudopodia formation was observed by fluorescence microscopy (ECLIPSE 80i, Nikon, Japan). We counted the number of filopodia and used Image J software to quantitatively analyze the length of filopodia.
Plasmid transfection and fluorescence co-localization
The Cdc42-EGFP or Cdc42-mCherry fusion protein expression plasmids were transfected into SCAP to extract SCAPCdc42-EGFP-Exo or SCAPCdc42-mCherry-Exo. SCAPCdc42-EGFP-Exo was added to HUVECs and passaged to the 6th passage. SCAPCdc42-mCherry-Exo was added to HUVECs overnight. Cells were incubated with Cdc42 primary antibody and fluorescent secondary antibody. The co-localization of Cdc42 and Cdc42-mCherry was observed by confocal microscopy (ECLIPSE Ti2, Nikon).
Statistical analysis
All data were recorded as the mean ± standard deviation (SD). Comparisons between two groups were analysed using an independent two-tailed Student’s t test, and comparisons between more than two groups were analysed using one-way analysis of variance (ANOVA) with SPSS 20.0 (SPSS Inc., Chicago, IL, USA). A value of P < 0.05 was statistically significant.