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, anti-CD63, anti-Alix, anti-calnexin, anti-CD31, anti-CD34, anti-CD45, anti-CD73, anti-CD90, anti-CD105, anti-Collagen Ⅰ and anti-Fibronectin antibodies were purchased from Abcam (Cambridge, UK). Anti-Ki-67, anti-RhoA, anti-Rac1, anti-Cdc42 and anti-β-actin antibodies were purchased from Cell Signaling Technology (Danvers, USA). Alexa Fluor 488- and Alexa Fluor 568- conjugated secondary antibodies were purchased from Proteintech (Rosemont, IL, USA). IRDye 800cw-conjugated goat anti-rabbit/anti-mouse IgG 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, Oregon, USA). SiCdc42, Cdc42-EGFP and Cdc42-mCherry fusion protein expression plasmids were purchased from GenePharma (Suzhou, China). The 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 aged 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) foetal 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 the potential for osteogenesis and adipogenesis, was 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 an ultracentrifuge at three different speeds: 3,000 × g for 20 min, 20,000 × g for 30 min, and 120,000 × g for 2 h (Beckman Optima L-100XP, USA). The 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 analyser (ZetaView, Germany) was used to measure the sizes of the particles. Exosomal surface markers, including CD9, CD63, and Alix, were detected by Western blotting. Cdc42 siRNA and the 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 microlitres of SCAP-Exo were added to 50 μL of protein lysate and lysed on ice for 1 h. A BCA protein assay kit was used to generate a standard curve of the 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 that in 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 microlitres SCAP-Exo, SCAPsiCdc42-Exo, or PBS (as control) was injected submucosally into four symmetrical sites around the wounds after they were created, according to a previous protocol [21], so that 40 μg of exosomes were applied locally on each wound. The mice were sacrificed 7 days post-operation. Tissue samples were 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. In order to characterize the newly healed gingival tissues, the frozen sections were stained with immunofluorescent Collagen I and Fibronectin which are the matrix markers. The frozen sections were also stained with immunofluorescent CD31, an endothelial marker of micro-vessels, and the CD31-positive area was analysed using the ImageJ software (1.50i, National Institutes of Health, Bethesda, MD, USA).
In vivo tracking experiment
PKH-26-labelled SCAP-Exo or PBS (as a control) was injected submucosally into four symmetrical sites around the wounds once after the wounds were created. The mice were sacrificed 7 days post operation. Frozen sections and fluorescence images were used to observe the fates 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 SCAP-Exo uptake by HUVECs was observed under a laser confocal microscope (ECLIPSE Ti2, Nikon, Japan) for 30 min continuously.
Western blot analysis
Protein concentrations were detected using a BCA protein assay kit. Proteins (20 μg) were loaded onto a 12% sodium dodecyl sulphate-polyacrylamide gels 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 each band was measured with ImageJ to quantify protein expression.
Tube formation assay
Matrigel (50 μL) (#356234, BD Biosciences, San Jose, CA) was used to precoat 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. Photographs of tube formation were taken with a stereoscopic microscope (ECLIPSE TE2000-S, Nikon, Japan). The indexes of tube formation were analysed using ImageJ.
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 backs of BALB/c nude mice (n = 5). After 14 days, the Matrigel plugs were extracted. H&E staining was used and the vessels in the Matrigel were counted.
Cell proliferation assay
Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) and Ki-67 staining assays. HUVECs were seeded into 96-well plates at a density of 2,000 cells/wel and cultured with SCAP-Exo. The plates were incubated for 24, 48, and 72 h. CCK-8 solution (Dojindo, Kumamoto, Japan) was added, and the plates were 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, the cells were fixed and stained with an immunofluorescent Ki-67 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 transwell cell migration and scratch wound healing assays. 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 to the lower chamber and incubated for 24 h. Thereafter, the cells in the Transwell chamber were removed. After staining with crystal violet, the cells that migrated below the Transwell layer were 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 medium was then replaced with serum-free medium containing SCAP-Exo. After 0, 12, and 24 h, the boundaries of the scratches were recorded and the wound closure rates were calculated using ImageJ.
Pull-down assay
A RhoA/Rac1/Cdc42 Activation Assay Combo Biochem Kit (Cytoskeleton, Denver, CO, USA) was used following the manufacturer’s instructions. Briefly, 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 in 20 μL of loading buffer and boiled. The samples were then analysed by Western blot assay.
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 filopodia and used ImageJ software to quantitatively analyse the lengths of filopodia.
Plasmid transfection and fluorescence co-localization
Cdc42-EGFP or Cdc42-mCherry fusion protein expression plasmids were transfected into SCAP, and SCAPCdc42-EGFP-Exo or SCAPCdc42-mCherry-Exo was extracted. SCAPCdc42-EGFP-Exo was added to HUVECs, and the cells were passaged to the 6th passage. SCAPCdc42-mCherry-Exo was added to HUVECs, and the cells were incubated overnight. The cells were then 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 among more than two groups were performed using one-way analysis of variance (ANOVA) with SPSS 20.0 (SPSS Inc., Chicago, IL, USA). A value of P < 0.05 was considered to indicate statistically significance.