Cell Culture, Regents and Antibodies
The hBMSCs provided by Cyagen Biosciences (HUXMA-01001; Guangzhou, China) had the potential to differentiate into osteoblasts, chondrocytes, and adipocytes under appropriate conditions. Adherent hBMSCs were cultured in flasks with hBMSC growth medium (Cyagen Biosciences, Guangzhou, China) in an incubator at 37°C under 5% CO2 and were passaged after reaching 80% confluence. The medium was replaced every 3 days; cells from passages 3–5 was used in subsequent experiments.
Recombinant human MFG-E8 (rhMFG-E8) protein was purchased from R&D Systems (Minneapolis, MN). Primary antibodies against GAPDH (CST#5174), COL1A1 (CST#72026), GSK3β (CST#12456), and phospho-GSK-3β (Ser9) (CST#5558) were purchased from Cell Signaling Technology (Danvers, MA). Primary antibodies against RUNX2 (ab192256), active β-catenin (ab246504), and total β-catenin (ab223075) were obtained from Abcam (Cambridge, UK). AR-A014418, a competitive and selective ATP inhibitor of GSK3β, was purchased from Selleck Chemicals (Houston, TX).
Cell proliferation assay
To assess the effects of rhMFG-E8 on hBMSC proliferation and viability, the cells were seeded into 96-well plates (2000 cells/well) and allowed to adhere for 18 h. The medium was exchanged for hBMSC growth medium with rhMFG-E8 (0, 0.1, 1, 10, 100, or 1000 ng/mL) for 1, 3, or 5 days. The medium was next changed for 10% Cell Counting Kit-8 (CCK-8) solution (Dojindo, Kumamoto, Japan) in 100 µL of low-sugar Dulbecco’s modified Eagle’s medium (L-DMEM) without fetal bovine serum (FBS) (Gibco, Waltham, MA) for 4 h at 37°C. The absorbance at 450 nm (A450), which is proportional to the number of viable cells, was measured using a microplate reader (ELX808; BioTek, Winooski, VT).
Osteogenic differentiation protocol
For osteogenic differentiation, hBMSCs were cultured in osteogenic induction medium (OIM) consisting of L-DMEM, 10% FBS, 100 IU/mL penicillin/streptomycin, 100 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate. First, hBMSCs were cultured in hBMSC growth medium in 6- or 12-well cell-culture plates (Corning, Shanghai, China) at a density of 3 × 104/cm2 and incubated at 37°C under 5% CO2. After cells reached about 80–90% confluence, the culture medium was replaced with fresh OIM. Subsequently, the OIM was replaced every 2 days.
Small Interfering RNA (siRNA) transfection targeting MFG-E8
To knock down the expression of MFG-E8 in hBMSCs, small interfering RNA (siRNA) transfection was performed. siRNAs for the human MFG-E8 gene were purchased from GenePharma Inc. (Shanghai, China). The sequences were as follows: siRNA 1, GGUUUAUGCGAGGAGAUUUTT; siRNA 2, GCCUUAAUGGACACGAAUUTT; siRNA 3, CCCACAAGAAGAACUUGUUTT. hBMSCs were cultured in six-well plates for 18 h prior to siRNA transfection. The medium was replaced with Opti-MEMTM I Reduced Serum Medium (Thermo Fisher Technology Co., Ltd., China) with 20 nM targeting siRNA or negative control using Lipo6000™ transfection reagent (Beyotime Biotechnology, Shanghai, China). After culturing for 6 h at 37°C under 5% CO2, the medium was exchanged for fresh hBMSC growth medium. The MFG-E8 mRNA and protein levels were determined using real-time PCR (RT-PCR) and Western blotting.
Lentiviral packaging and cell infection
MFG-E8-overexpression lentiviral vector particles (MFG-E8-overexpression group, OE), and lentiviral GFP vector particles (MFG-E8-overexpression control group, OE-NC) were provided by Cyagen Biosciences. Approximately 50–60% confluent hBMSCs were incubated with lentiviral particles and 5 µg/mL polybrene in hBMSC growth medium at a multiplicity of infection of 50 (the optimum according to GFP expression after lentiviral GFP particle infection). For infection, hBMSCs were incubated with lentiviral particles and polybrene (5 µg/mL) in hBMSC growth medium. After about 18 h, the infection medium was exchanged for fresh growth medium. After 3 days, the cells were screened using puromycin (4 µg/mL) and passaged for use in subsequent experiments. The expression of MFG-E8 was detected using RT-PCR, Western blot, and immunofluorescence.
ALP staining and ALP activity assay
ALP staining was used to investigate early mineralization. For ALP staining, cells were fixed with 4% paraformaldehyde (Sangon Biotech, Shanghai, China) for 30 min. The cells were then washed with double distilled water (ddH2O) three times and stained using an Alkaline Phosphatase Color Development kit (Beyotime, Shanghai, China). ALP activity was determined using an ALP Activity Assay kit (Beyotime) according to the manufacturer’s instructions. Briefly, cells were lysed for 1 h with radioimmunoprecipitation assay (RIPA) buffer. The appropriate amount of supernatant and 50 µL of chromogenic substrate (para-nitrophenyl phosphate) were added to wells of a 96-well plate, to which testing buffer was also added to a volume of 100 µL. We prepared standard samples (para-nitrophenol 0.5 mM) to generate an ALP standard curve. Next, the 96-well plate was incubated at 37°C for 5–10 min. Finally, to each well was added 100µL of reaction termination solution to stop the reaction, and the A405 was measured using a microplate reader.
Alizarin red staining and quantification assay
Alizarin red staining (ARS; Cyagen Biosciences) was performed to assess late mineralization. For ARS, cells were fixed in 4% paraformaldehyde for 20 min at room temperature and subsequently washed three times with ddH2O. Finally, the cells were treated with Alizarin red stain (0.5%, pH 4.1–4.2) for 20 min and rinsed with distilled water. To quantify the staining intensity, stained mineralized nodules were incubated with 10% cetylpyridinium chloride (Sigma, Shanghai, China), the solution was collected, and the A570 was measured using a microplate reader.
Western blot analysis
Cells were lysed in RIPA buffer supplemented with proteasome and phosphatase inhibitors (Boster Biological Technology, Wuhan, China). Equal amounts of proteins were separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 1 h, the membranes were incubated overnight at 4°C with antibodies specific against GAPDH (1:10,000), COL1A1 (1:1,000), RUNX2 (1:1,000), active β-catenin (1:1,000;), total β-catenin (1:1,000), phospho-GSK-3β (1:1,000), and total GSK3β (1:1,000). A stripping method was used to measure two antibodies of identical molecular weight. After washing four times (5 min each) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with a horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibody (Boster Biological Technology) for 1 h at room temperature. After washing three times (5 min each) with TBST, proteins were detected using enhanced chemiluminescence blotting reagents (Millipore). Signal intensities were measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA).
Cells were cultured in 12-well plates with OIM. After the induction of osteogenesis, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized in 0.1% Triton X-100 for 30 min and blocked in 5% bovine serum albumin for 60 min. Fixed cells were washed and incubated overnight with anti-RUNX2 (1:500), anti-COL1A1 (1:500), or anti-active β-catenin (1:500) antibody. The cells were incubated with a fluorescence-conjugated secondary antibody (DyLight 647 Conjugate; Boster Biological Technology) for 2 h, and nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Beyotime) for 5 min. Samples were observed under a fluorescence microscope (EU5888; Leica, Wetzlar, Germany).
RNA isolation and real time quantitative PCR
Total RNA was isolated from cells cultured with OIM using RNAiso reagent (TaKaRa Bio Inc., Dalian, China) and quantified by measuring the A260 (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA). First-strand cDNA was synthesized using PrimeScript RT Master Mix (TaKaRa Bio Inc.) according to the manufacturer’s instructions. Total RNA (≤ 1,000 ng) was reverse-transcribed into cDNA in a reaction volume of 20 µL using a Double-Strand cDNA Synthesis kit (TaKaRa Bio Inc.). The levels of mRNAs encoding COL1A1, RUNX2, OCN, Osterix, OPN, ALP, and GAPDH were determined using the StepOnePlus Real-Time PCR System (Applied Biosystems Inc., Warrington, UK) and SYBR Premix Ex Taq (TaKaRa Bio Inc.) with the following program: 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. GAPDH was used as an internal control and for normalization. DNA concentrations were calculated using the 2 − ΔΔCt method24. The primers were synthesized by Sangon Biotech and are listed in Table 1.
MFG-E8 levels in the hBMSC culture supernatants were assessed via enzyme-linked immunosorbent assay (ELISA) using the Human MFGE ELISA Kit (Boster Biological Technology) following the manufacturer’s protocol.
In vivo evaluation in animals
All animal experiments and procedures were conducted in accordance with the principles of the Institutional Animal Care Use Committee of the Second Affiliated Hospital of Zhejiang University and approved by the same committee. A rat tibial-defect model was used to assess the bone-forming ability of MFG-E8.[25, 26]. Fifteen rats were divided randomly into three groups: the blank group, normal saline (NS) (negative control group treated with NS) group, and rhMFG-E8 group. First, rats were anesthetized via inhalation of 2–5% isoflurane, with the anesthesia maintained via 2% isoflurane inhalation during surgery. After anesthesia, an incision was made lateral to the tibia, away from the bone. An intramedullary fixation pin (1.2-mm-diameter stainless-steel syringe needle) was inserted inside the medullary canal of the tibia for fixation. Osteotomy to create a transverse 1.5-mm-wide defect approximately 7 mm from the proximal tibial growth plate was performed using an electronic saw. The same leg was used in each group. Next, rhMFG-E8 (20 µg) was injected locally at the fracture site on days 0, 3, 5, 8, and 11 (i.e., immediately and at 72-h intervals thereafter); NS was used as a vehicle control. Rats were sacrificed at 1 month after surgery, and samples were collected and fixed in 4% paraformaldehyde for 72 h at room temperature.
A range of 3 mm above and below the bone-defect area of tibia samples was scanned using the µCT-100 Imaging System (Scanco Medical, Brüttisellen, Switzerland) with the following parameters: 70 kVp; reconstruction matrix, 1,024; slice thickness, 14.8 µm; and exposure time, 300 ms. The trabecular bone volume fraction (BV/TV), mean trabecular thickness (Tb.Th), mean trabecular number (Tb.N), and mean trabecular separation (Tb.Sp) were evaluated via standard three-dimensional microstructural analysis[25, 27].
After micro-computed tomography (CT), samples were decalcified using 10% ethylene diamine tetra acetic acid (Sigma) in 0.1 M phosphate-buffered saline, with the solution changed once a week for 6 weeks, before embedding in paraffin. Serial sections of 3 µm thickness were cut and mounted on polylysine-coated slides and deparaffinized. The consecutive tissue sections were stained with hematoxylin and eosin (HE) or Masson’s trichrome stain.[25, 28] Images were obtained using a microscope (Leica DM4000B; Leica, Wetzlar, Germany).
Data and statistical analysis
Statistical analysis was performed using Prism (version 8.0; GraphPad Software, San Diego, CA). All experiments were conducted at least three times and the data are presented as the means ± SDs. Differences between two groups were analyzed using the two-tailed Student’s t-test. For comparisons of more than two groups, one-way analysis of variance followed by Bonferroni post hoc tests was used. In all analyses, P < 0.05 was taken to indicate statistical significance.
Role of the funding source
The funding agencies had no further role in study design, in the collection, analysis and interpretation of data, in the writing of the report and in the decision to submit the paper for publication.