2.1 Cell culture
SCAPs were isolated from dental apical papilla tissue. Dental apical papillae were isolated and transferred into phosphate-buffered saline (PBS; Gibco) containing penicillin-streptomycin (Pen-Strep; Gibco) and trimmed to small pieces under sterile conditions. The samples containing stem cells were transferred into a flask containing α-modified Eagle’s minimum essential medium (α-MEM; Gibco), 10% fetal bovine serum (FBS; BI) and 2% Pen-Strep.
2.2 Characterization of SCAPs
2.2.1 Immunofluorescence staining (IF)
SCAPs were cultured for 3 days. Briefly, cells were incubated with primary antibody STRO-1 (eBioscience) and Cy3-conjugated secondary antibodies. After counterstained with 4,6-diamidino-2-phenylindole (DAPI), cells were observed under a fluorescence microscope (Leica, Germany).
2.2.2 Flow Cytometry Analysis
For surface markers characterization, cells were stained with fluorescent monoclonal mouse anti-human antibodies containing CD34-PE, and CD45-PE, CD73-PE, CD29-APC, CD90-FITC, CD105-PerCP-Cy5.5TM (BD Biosciences, USA). Fluorescence analysis was evaluated in flow cytometer (Thermo Fisher Scientific).
2.2.3 Colony Forming Assay
After 10 days of culture, cells were stained with 0.1% toluidine blue (Sigma Aldrich, MO, USA).
2.2.4 Chondrogenic Differentiation
SCAPs were cultured in chondrogenic medium for 1 month. After that, cells were immersed in paraformaldehyde for 48 hours, then imbedded in paraffin, and sliced into 5 μm sections. Then, cells were stained with Alcian blue staining for 30 min.
2.2.5 Osteogenic differentiation
Cells were seeded in 12-well plates. Afterward, mineral induction medium consisting ofβ-glycerophosphate10 mM, ascorbate-2-phosphate50 mM, and dexamethasone 100 nM (Sigma), 10% FBS and 1% Pen-Strep was added to the wells. The culture plates were incubated in humidified condition for 21 days.
2.2.6 Adipogenic Differentiation
SCAPs were cultured in adipogenic medium (Cyagen, China) for 28 days. After stained with Oil Red O reagent (Cyagen) for 5 min, lipid droplets were observed under microscope.
The miR‐24‐3p oligos (inhibitor, mimic, and inhibitor NC and mimic NC) and control siRNA (NC), siRNA for MACF1(siMACF1) were designed by Ribio (Ribio CO., China). Cells were transfected with oligos and siRNAs via riboFECTTM CP following the manufacturer’s protocols. Western blot and qRT-PCR were used to verify the interference efficiency. Lentiviral vectors overexpressing MACF1 were constructed and produced by Shanghai Genechem Company (Shanghai, China). SCAPs were inoculated overnight and infected with lentiviruses with polybrene.
2.4 Cell Counting Kit-8 Assay (CCK-8)
SCAPs were respectively transfected with miR-124-3p.1 inhibitor, mimic, inhibitor NC and mimic NC for 48 h. Next, cells were seeded on 96-well plates and cultured for 0, 1, 3, 5 and 7 d. Afterward, 10 μL CCK-8 (Dojindo, Japan) and 90 μL α-MEM were added to each well for 2 h. The optical densities (OD) were measured at 450 nm.
2.5 5-Ethynyl-2ʹ-Deoxyuridine (EdU) assay
The EdU DNA Proliferation Detection kit (Ribo Biotechnology, China) was used to detect cell proliferation ratio. After culture, cells were fixed and administrated with EdU labeling solution. Cells were photographed under inverted fluorescence microscope (Leica, Germany).
2.6 Alizarin red staining
After washed and fixed, SCAPs were stained with ARS solution (Sigma, USA). The red sediments of calcium deposition were observed under microscope. For quantitative analysis, mineralized nodules were dissolved by 10% cetylpyridinium chloride (CPC; Sigma, USA). Then OD value was measured at 562 nm.
2.7 Alkaline phosphatase activity
Cells were harvested and permeated in Triton X-100. After centrifugation, the supernatant transferred into fresh 1.5 ml tubes with relative working solution and then were incubated. The OD value was measured at 520 nm.
2.8 ALP staining
After washed and fixed, cells were stained using the BCIP/NBT ALP staining kit (Beyotime, China). Cells were observed with microscope.
2.9 RNA extraction and quantitative real- time polymerase chain reaction (qRT-PCR)
RNA isolation was performed using the TRIzol™ (Invitrogen) method. The cDNAs were synthesized and used for PCR. qRT-PCR reactions were performed by SYBR Green method. GAPDH and U6 were used as internal controls. Bulge-Loop miRNA qPCR Primer kit (RiboBio) was used for measuring miRNA-124.3p.1 expression. Primers in this study are listed in Table 1.
2.10 Western blotting
RIPA buffer was used to extract proteins with protease inhibitor cocktail. After PAGE, the proteins were blotted onto PVDF membranes. Membranes were blocked, incubated with specific primary antibody and next day with the secondary HRP-conjugated antibody
2.11 Immunofluorescence staining
Cells were fixed, permeabilized. Then, cells were blocked and incubated with primary antibodies, fluorescent dye-labeled designated secondary antibody and DAPI above
2.12 Luciferase Assay
The potential binding site of MACF1-wt and mutant sequence MACF1-mut was synthesized into pmiR-GLO (Promega, Madison, WI, USA). SCAPs were co-transfected with the MACF1-wt or MACF11-mut reporter gene plasmid and miR-124-3p.1 mimic. The activities were measured by a Promega luciferase assay (Promega, USA) were normalized against the activity of the Renilla luciferase gene.
2.13 Co-immunoprecipitation (Co-IP) assay
After cells lysed and centrifuged, supernatants were incubated with the anti-MACF1 antibody in rotation. Next day, the supernatants were mixed with protein G plus A agarose (Beyotime, China) at in rotation. Then, the immunocomplexes were washed. Beads complexes were resuspended in 2 × loading buffer.
2.14 Animal procedures
SCAPs with miR-124-3p.1 mimic and miR-124-3p.1 mimic NC induced in MM for 7 days were harvested for the in vivo study. About 6.0 × 106 SCAPs were mixed with Bio-Oss Collagen scaffolds (Geistlich, Germany). And then the mixtures were implanted into the dorsal surface of BALB/c homozygous 5-week-old nude mice. Two months later, the implants were harvested and were detected under micro-CT analysis. After that, implants were decalcified and embedded with paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E) staining and Masson’s trichrome staining.
2.15 Statistical analysis
All data are expressed as the mean ± SD and were analyzed by one-way analysis of variance (ANOVA). p<0.05 was considered statistically significant.