Cells and reagents
human bone marrow stromal cells (hBMSCs) were obtained from four donors who gave their written informed consent. Briefly, marrow was extracted from the femoral midshaft and then suspended in minimum essential medium containing 10% fetal bovine serum (Hyclone; GE Healthcare, Little Chalfont, UK), 100 U/mL penicillin and 100 mg/L streptomycin. Subsequently, the non-adherent cells were discarded; the adherent cells converged to 80–90% confluence and were then replated as passage one (P1) cells. P3 cells were used for experiments. A density of 1×105 cells/mL was used in the cellular tests. Recombinant DKK1 was purchased from PeproTech (Rocky Hill, NJ, USA). In accordance with a previous study, the applied concentration of DKK1 was 0.5 μ g/mL(20, 25).
Lentiviral packaging and cell infection
Lentivirus knockdown MEG3 particles and lentiviral RFP particles were described as previously(9). The lentiviral RFP particles were used as control group in this study. For infections, hBMSCs were incubated with lentiviral particles and polybrene (5 μ g/mL) in growth medium. After 6 h, the infection medium was discarded. After 3 days, the cells were screened using puromycin (4 μ g/mL; Sigma, Shanghai, China) and then passaged for use in subsequent experiments. The expression of MEG3 was quantified by quantitative real-time polymerase chain reaction (qPCR) and immunofluorescence.
Cell Counting Kit-8 (CCK-8)
To assess the effect of MEG3 downregulation on the proliferation of hBMSCs, the cells were seeded into a 96-well plate (5000/well) and allowed to adhere for 24 h. After 24 h, the medium was removed, and the cells were treated with 10% CCK-8 (Dojindo, Kumamoto, Japan) in 100 μ L low-sugar Dulbecco’s modified Eagle’s medium (L-DMEM) without fetal bovine serum (FBS) for 2 h at 37 °C. Absorbance at 450 nm, which is directly proportional to cell proliferation, was measured using a microplate reader (ELX808; BioTek, Winooski, VT, USA).
Osteogenic differentiation protocol
BMSCs were cultured in growth medium [L-DMEM; 10% FBS (1495527; Gibco, Waltham, MA, USA) and 100 IU/mL penicillin/streptomycin] in 6- or 12-well cell culture plates (Corning, Shanghai, China), at a density of 3 × 104/cm2, and incubated for 48h at 37℃ under 5% CO2. The cells were subsequently cultured in osteogenic induction medium (L-DMEM with 10% FBS, 100 IU/mL penicillin/streptomycin, 100 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β -glycerophosphate). The cells were maintained by the addition of fresh osteogenic induction medium every 3 days.
Measurement of alkaline phosphatase (ALP) activity
For the measurement of ALP activity, cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China), and the lysate (10 μ L) was incubated with 90 μ L fresh solution containing p-nitrophenyl phosphate substrate at 37 °C for 30 min. The reaction was stopped by the addition of 0.5N NaOH (100 μ L), and the absorbance was measured at 405 nm using a microplate reader (ELX808; BioTek). The total protein concentration was measured using a BCA protein assay kit (KeyGen BioTECH, Nanjing, China). The relative ALP activity is expressed as the percentage change in optical density (OD) per unit time per milligram protein: (OD/15 min/mg protein) × 100.
Alizarin red staining (ARS)
After the induction of osteogenic differentiation, mineral deposition was assessed by ARS (Cyagen Biosciences). Cells were fixed in 4% paraformaldehyde (Sangon Biotech, Shanghai, China) for 15 min at room temperature and then washed with distilled water. A 1% solution of alizarin red was added and incubated for 30min at room temperature, followed by rinsing with distilled water. The solution was collected, and 200 μ L were plated on 96-well plates, which were read at 560 nm using a microplate reader (ELX808; BioTek). The readings were normalized to the total protein concentration.
RNA isolation and qPCR
Total cellular RNA was isolated using RNAiso reagent (Takara, Dalian, China) and quantified by measuring the absorbance at 260 nm (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA). Total RNA (≤1000 ng) was reverse-transcribed into cDNA in a reaction volume of 20 μ L using the Double-Strand cDNA Synthesis Kit (Takara, Dalian, China). One microliter of cDNA was used as the template for the qPCR reaction. All gene transcripts were quantified by qPCR using the Power SYBR Green PCR Master Mix (Takara) on the ABI StepOnePlus System (Applied Biosystems, Warrington, UK). The mRNAs of the target genes and GAPDH were quantified in separate tubes. All primers were synthesized by Sangon Biotech (Shanghai, China). The primer sequences used are shown in Table 1. The cycle conditions were as follows: 95 °C for 30 s and then 40 cycles of 95 °C for 5 s and 60 °C for 30s. The relative target gene expression levels were calculated using the 2−△△Ct method.
Western blot analysis
Cells were lysed in RIPA lysis buffer supplemented with a proteasome inhibitor (Beyotime). Total proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 2 h, the membranes were incubated overnight at 4 °C with antibodies specific to β-actin (1:1000, Abcam Inc., USA), or β-catenin (1:1000, Cell Signaling Technology, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1500; Cell Signaling Technology, USA) was applied as a secondary antibody for 2 h at room temperature. The immunoreactive bands were detected using an enhanced chemiluminescent detection reagent (Millipore, Shanghai, China). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).
Cell seeding
Prior to seeding cells, the prefabricated PHMG scaffolds were sterilized using gamma irradiation. Cell suspension (100 μ L) was added to four groups at a density of 1×104 cells/scaffold. After 4 h, 100 μ L of culture medium was carefully added to the base of the culture plate until the scaffold was covered with sufficient culture medium.
In vivo evaluation in animals
Animal experiments were approved by the Research Ethics Committee of the Shanghai General Hospital, and performed in accordance with the Care and Use of Laboratory Animals protocols. Briefly, mature Sprague Dawley (SD) male rats (mean body weight 250–300 g) were provided with sterilized food and water and housed in a barrier facility with a 12-h light/dark cycle. These rats were randomly divided into three groups, each containing six rats: PHMG, PHMG + sh-Ctrl, and PHMG + sh-MEG3 group. For the surgical procedure, as previously described, the animals were anesthetized by intraperitoneal injection of chloral hydrate (4%; 9 mL/kg body weight) and all operations were performed under sterile conditions. A 1.5-cm sagittal incision was made in the scalp and the calvarium was exposed by blunt dissection. Two critical-sized calvarial defects with a bilateral diameter of 5 mm were created using a dental trephine, and the scaffolds were then implanted into the defects. Following the operation, the animals received intramuscular antibiotic injections, were allowed free access to food and water and were monitored daily for potential complications. Eight weeks after the operation, the rats were killed by an overdose of anesthetics and their craniums were harvested and fixed in a 4% paraformaldehyde solution buffered with 0.1 M phosphate solution (pH 7.2) overnight before further analysis.
Micro-computed tomography (CT) evaluation
All the harvested specimens were examined using the mCT-80 system to evaluate new bone formation within the defect region. Briefly, the undecalcified samples were scanned at a resolution of 18 μ m and decalcified samples perfused with Microfil® were scanned at a resolution of 9 μ m. After 3D reconstruction, the bone mineral density (BMD) and bone volume fraction (bone volume/total volume [BV/TV]) in the defect regions were used to calculate new bone formation using the auxiliary software of the mCT-80 system.
Histological and Immunohistochemical (IHC) analysis
The one part of calvarias were decalcified in 10% EDTA for 14 days, dehydrated with graded ethanol solutions, embedded in paraffin and sectioned at 5 μ m at the central area of the defect. Sections were stained with hematoxylin and eosin (HE) to observe new bone formation.
The other part of each cranium was decalcified for approximately 2 weeks, dehydrated using a graded alcohol series, embedded in paraffin and sectioned into 5 μ m sections. Osteocalcin (OCN) IHC was performed to evaluate osteogenesis in specimens.
Statistical analysis
Statistical analysis was performed using SPSS 17.0 software (IBM, Armonk, NY, USA). All experiments were performed at least in triplicate, and the data are presented as means ± standard deviation. Statistical significance was determined using a two-tailed Student’s t-test when comparing two groups, and one-way ANOVA followed by Bonferroni’s post hoc test when comparing more than two groups. P < 0.05 was considered to indicate statistical significance.