Isolation and culture of ASCs and DOP-ASCs
All procedures that involved animals were reviewed and approved by the Southwest Medical University Ethical Committee. Anesthesia and animal care were implemented by following the guidelines for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006). Adipose tissue in the inguinal region was collected from C57BL/6 DOP and control mice under sterile conditions. The adipose tissue was cut finely and fragments were seeded in 25-cm2 culture flasks (Corning Inc., NY) and cultured in alpha-modified Eagle’s medium (α-MEM, Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin (Hyclone) at 37°C with 5% CO2. The medium was changed every 3 days. Adherent cells were cultured and non-adherent cells were removed.
DOP-ASCs were passaged three times to obtain relatively pure ASCs. Osteogenic, adipogenic, and cartilage media (Cyagen, USA) were used to define the multipotential differentiation capacity of DOP-ASCs. DOP-ASCs (5×104 cells) were seeded in a 6-well plate for osteogenic induction. DOP-ASCs (1×105 cells) were also seeded for adipogenic induction. All cells were cultured for 21 days. Then, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 1 h. Alizarin Red-S (osteogenic dye) and 0.3% Oil Red-O (adipogenic dye) were used to stain mineralized nodules and lipid droplets, respectively, for 30 min. The stained cells were imaged under an inverted phase contrast microscope (Nikon, Japan). For cartilage induction, DOP-ASCs (2.5×105 cells) were centrifuged and cell aggregates were cultured in cartilage medium. After 21 days, the cell aggregates were washed three times with PBS and fixed with 4% paraformaldehyde. The cartilage pellets were imaged under a stereo fluorescence microscope (Carl Zeiss Microscopy, Germany). Then, they were embedded in paraffin and sections were stained with Alcian blue. Cartilage matrix was imaged under an optical microscope (Nikon).
A Cell Counting Kit-8 (CCK-8) assay (Sigma-Aldrich, St Louis, Missouri, USA) and xCelligence system for real-time cellular analysis (RTCA) (Roche Diagnostics GmbH, Basel, Switzerland) were used to assess cell proliferation. For the CCK-8 assay, cells were seeded in 96-well plates (Corning Inc.) at a density of 3×103 cells per well and cultured in α-MEM with 10% FBS for 5 days. A BioTek ELX800 (Bio-Tek, USA) was used to measure absorbance at 450 nm. For RTCA, cells were seeded in 96-well E-plates (Roche Diagnostics GmbH) at 3×103 cells per well. Cell proliferation in the RTCA SP xCelligence system was monitored in real-time as the impedance value over 5 days. Data were analyzed by the provided RTCA software.
Alizarin Red-S staining
Mineralized nodule formation in ASCs was stained by Alizarin Red-S (Cyagen). DOP-ASCs and control ASCs (5×104 cells) in 6-well plates were treated with osteogenic medium for 21 days. Cells were then washed with PBS three times, fixed in 4% paraformaldehyde for 1 h, and stained with Alizarin Red-S for 30 min.
Real-Time Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted using a Total mRNA Extraction Kit (Takara Bio, Japan). cDNA was synthesized by reverse transcription using a Prime Script Reverse Transcription Reagent Kit (Takara Bio). Then, RT-PCR was conducted to measure the gene expression of Runt-related transcription factor 2 (Runx2), osteopontin (Opn), DNA methyltransferase 1/3a/3b (Dnmt1/3a/3b), β-catenin, and lymphoid enhancer-binding factor-1 (Lef1). Primer sequences are shown in Table 1. Samples were analyzed using a SYBR Premix ExTaq kit (Takara Bio), following the standard procedure, in an ABI 7900 system (Applied Biosystems, USA), which included melting curve analysis and obtaining CT values. The results were normalized to Gapdh CT values and the 2−∆∆Ct method was used to calculate gene expression.
Western Blot Assay
A Total Protein Extraction Kit (Keygen Biotech, China) was used to extract total cellular proteins. A bicinchoninic acid protein assay kit (Thermo Fisher Scientific, MA, USA) was used to measure the protein concentration. Proteins were separated by 10% (v/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane at 200 mA for 1 h. Tris-buffered saline with 0.05% (v/v) Tween-20 (TBST) was used to dissolve dry skimmed milk (Keygen Biotech). PVDF membranes were blocked with 5% dry skimmed milk for 1 h and then incubated with antibodies against GAPDH (ab181602), DNMT3a (ab188470), DNMT3b (ab79822), and OPN (ab91655) (Abcam, UK), RUNX2 (12556s), DNMT1 (5032S), β-catenin (D10A8), or LEF1 (2230p) (Cell Signaling Technology, USA) for 1 day at 4°C. Then, PVDF membranes were washed three times with TBST and incubated with a goat anti-rabbit secondary antibody (Beyotime, Shanghai, China) for 1 h. They were then washed again with TBST and developed with an enhanced chemiluminescence detection system (Bio-Rad, USA).
Cells were seeded on round coverslips (Corning Inc.) and cultured for 4 days. After various treatments, the cells were carefully washed three times with PBS, fixed with 4% paraformaldehyde for 1 h, and permeabilized with 0.5% Triton X-100 for 10 min. Then, they were blocked with 5% goat serum (Beyotime) for 1 h and incubated for 1 day at 4°C with antibodies against RUNX2, OPN, DNMT1, DNMT3a, DNMT3b, 5-MC (28692S), β-catenin, or LEF1. The next day, the samples were incubated with a fluorescent dye-conjugated secondary antibody (Beyotime) for 1 h. Nuclei were counterstained with 4ʹ6-diamidino-2-phenylindole (Beyotime) for 10 min and phalloidine (Beyotime) was used to stain microfilaments for 10 min. Cells were imaged under a laser scanning confocal microscope (Olympus, Japan).
Transfection of small interfering RNA (siRNA)
Small interfering RNA (siRNA) that targeted Dnmt1, Dnmt3a, and Dnmt3b was designed and provided by GenePharma Co., Ltd (Shanghai, China). siRNA sequences are shown in Table 2. DOP-ASCs (5×104 cells) were seeded in a 12-well plate before siRNA transfection. The transfection reagent (Lipofectamine 2000; Thermo Fisher Scientific) was diluted with Opti-MEM I Reduced Serum Medium (Hyclone) and incubated at room temperature for 5 min. The siRNA was added to the diluted Lipofectamine 2000 and gently mixed to form the siRNA-lipofectamine-Opti-MEM complex. Then, the mixture was added to cells at 1 ml per well and incubated at 37°C with 5% CO2.
Transduction of shRNA-Dnmt3a and LVRNA-Dnmt3a
The Dnmt3a overexpression lentiviral vector (pLenti-EF1a-EGFP-P2A-Puro-CMV-Dnmt3a-3Flag) and Dnmt3a-silencing lentiviral vector (pLDK-CMV-EGFP-2A-Puro-U6-shRNA Dnmt3a) were designed and manufactured by OBiO Technology Corp., Ltd. (Shanghai, China). The oligonucleotide sequences of shRNA with Dnmt3a RNA interference targets are shown in Table 3. Various virus concentrations were used to determine the multiplicity of infection (MOI). The transduction efficiency was evaluated by analyzing the percentage of green fluorescent protein (GFP)-positive cells under a fluorescence microscope. ASCs at a density of 5×104/ml were seeded in a 6-well plate at 2 ml per well. After 12 h of culture, the medium was replaced with a lentivirus suspension medium (MOI: 80; 0.6 μg/ml puromycin; 5 μg/ml polybrene). The gene and protein expression were analyzed by RT-PCR and western blotting, respectively, after 4 days of osteogenic induction and their osteogenic ability was assessed by Alizarin Red-S staining after induction for 21 days.
Analysis of DOP‑ASCs seeded on BCP by scanning electron microscopy (SEM)
Before seeding DOP-ASCs, scaffolds sterilized by ultraviolet light were placed in 12-well plates. Then, 1 ml of passage 2 DOP-ASCs at a density of 5×104/ml was seeded on the surface of BCP in each well. After culture at 37°C with 5% CO2 for 3 days, samples were fixed with paraformaldehyde. After alcohol gradient dehydration, critical point drying, and spraying the cells with gold, scaffolds were observed by SEM.
Implantation of BCP seeded with DOP‑ASCs transduced with shRNA into a DOP mouse model with critically sized calvarial defects
DOP-ASCs were divided into DOP-blank, negative control, and Dnmt3a shRNA groups. DOP-ASCs infected with the silence-Dnmt3a lentivirus were cultured in osteogenic induction medium. A 1-ml cell suspension (5 × 104 cells/ml) was added to the surface of BCP in a 12-well plate and cultured for 48 h. Nine DOP mice received calvarial surgery to establish critically sized calvarial defect models. After anaesthetization, the DOP mice were subjected to prone fixation, skin preparation, and disinfection at the top of the skull. An incision was made along the median of the calvarium and the periosteum was bluntly separated to expose the calvarial bone surface. Then, a 4-mm diameter trephine bur was applied to drill a standardized round defect on the side of the sagittal suture. A 0.9% saline solution was used to irrigate the skull surface during drilling. Subsequently, the BCP seeded with DOP-ASCs was implanted into the skull defect area and the periosteum and dermis were sutured in position. After 8 weeks, mice were euthanized and skull specimens were obtained.
Micro-computed tomography (Micro‑CT), hematoxylin and eosin staining (HE) staining, and Masson staining
At 8 weeks, the calvarium was removed intact and fixed in freshly prepared 4% formaldehyde for 24 h at 4°C. Micro-CT scans of skull defects were performed to observe new bone formation. Then, three-dimensional reconstructed images were analyzed. The ratio of the bone volume to total volume available in the scaffold (BV/TV) was calculated. A high ratio indicated that more bone had grown into the scaffolds. Then, tissue samples of the mouse skull defect were decalcified for HE and Masson staining. Next, the samples were dehydrated in an alcohol gradient, clarified, and embedded in paraffin for sectioning. Lastly, the sections were stained with hematoxylin and eosin and Masson trichrome.
All experiments were repeated at least three times independently. Two group comparisons were made by the independent-samples t-test and multiple comparisons were made by one-way ANOVA with SPSS 18.0 software (SPSS Inc., Chicago, USA). P < 0.05 was regarded to be statistically significant.