2.1 Animals
This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals of Nanfang Hospital Southern Medical University. The protocol was approved by the Animal Care and Use Committee of Nanfang Hospital. Pathogen-free mice were maintained under standard conditions in a 12h light and 12h dark cycle, at 25 ± 3°C, with a relative humidity of 40-60%, and with food and tap water available ad libitum. PDE during GD 12-14 was applied according to the procedure previously described [6]. Briefly, virgin C57BL/6 female mice at 10-12 weeks old were mate with male mice overnight. The day on which the presence of a vaginal plug was set as GD 0, the pregnant mice were randomly assigned to the PDE group or vehicle treatment (control) group. To construct PDE mice model, dexamethasone sodium phosphate (Cat. 2392-39-4, Tianxin, China) was injected subcutaneously (1.2 mg/kg/day) during GD 12-14. To construct the vehicle control of PDE model, pregnant mice were treated with the same amount of vehicle (normal saline) daily during GD 12-14. The pregnant mice were housed individually in cages with freely available food and water. Two female offspring were selected randomly from each litter for postnatal bone development investigation. Femurs and tibias from mice offspring at 2-, 4-, 6-, and 12-week-old were dissected for further analysis. To evaluate the effect of dasatinib (S1021, Selleck Chemicals,Houston,TX, USA) and quercetin (S2391, Selleck Chemicals,Houston,TX, USA), PDE pregnant mice during GD 12-14 were treated with vehicle (200 μl 1% methyl cellulose) or dasatinib (5 mg/kg/day) plus quercetin (50 mg/kg/day) by oral gavage, respectively.
2.2 Microcomputed Tomography (μCT) analysis
Tibias from 12-week-old mice offspring were dissected free of soft tissue, fixed and stored in 70% ethanol, and imaged using a μCT specimen scanner (Scanco Medical, AG, Switzerland). The scan was performed using an X-ray energy of 55 kV and current of 145 mA, with a voxel size of 12μm and an integration time of 400 msec. Trabecular bone measurements consisting of 250 slices (3mm) were performed from 0.215 mm (18 image slices) below the growth plate. Bone volume (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th) and trabecular separation (Tb. Sp) were determined. Quantitative analyses were carried out using IPL software (Image Processing Language V5.15, Scanco Medical AG, Switzerland).
2.3 Histochemistry
To study the morphology of postnatal long bone growth in offspring after PDE, femurs and tibias of mice offspring at 2-, 4-, and 6-week-old were fixed in 4% paraformaldehyde, decalcified in 0.5M ethylenediamine-tetraacetic acid (EDTA, pH 7.4), followed by paraffine embedding or frozen embedding. Hematoxylin-eosin (H&E) staining, Goldner’s trychrome staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed on 4 μm paraffin sections according to standard procedures. The number of osteoblasts per square millimeter of metaphyseal area (N. per mm2) were quantified in the area from 0-0.5mm below growth plate.
For detecting the osteoclastic activity in bone, TRAP staining was performed on the deparaffinized and rehydrated sections using a Leukocyte Acid Phosphatase kit (Cat. 387A-1KT, Sigma-Aldrich, USA). The TRAP+ multinucleated cells containing at least three nuclei were identified as osteoclasts under light microscope (Olympus, BX53). The number of TRAP+ cells per square millimeter of metaphyseal area (N. per mm2) in the area from 0-0.5mm below growth plate was quantified.
For detecting senescence associated β-galactosidase (SA-β-Gal) activity, frozen sections were stained using SA-β-Gal staining kit (Cat. 9860, Cell Signaling Technology, USA) according to manufacturer’s instructions. Senescent cells were identified as blue-stained cells under light microscope (Olympus, BX53). The number of SA-β-Gal+ cells per square millimeter of metaphyseal area (N. per mm2) in the area from 0-0.5mm below growth plate was quantified.
2.4 Immunofluorescence
For immunofluorescence staining, frozen sections were incubated in blocking buffer (3% BSA in PBS with Tween (PBST)) for 1 hour at room temperature, incubated with primary antibodies overnight at 4°C. The primary antibodies for immunostaining include: Nestin (ab134017, Abcam, Cambridge, MA, USA), CD31 (FAB3629G-100, R&D Systems, Minneapolis, MN, USA), Endomucin (Emcn, SC-65495,Santa Cruz, Dallas, TX USA), Ki67 (ab15580, Abcam, Cambridge, MA, USA). Sections were washed 3 times in PBS and then incubated with secondary antibodies at room temperature for 1 hour. The secondary antibodies for immunostaining include: 488-conjugated secondary antibody (703-546-155, Jackson ImmunoResearch, West Grove, PA, USA), 594-conjugated secondary antibody (712-586-153, Jackson ImmunoResearch, West Grove, PA, USA), 488-conjugated secondary antibody (A21206, ThermoFish Scientific, USA). Nuclei were counterstained with DAPI (S2110,Solarbio, China). Images were captured using a fluorescence microscrope (Olympus, BX53, Japan). Positive-stained area or the number of positive-stained cells per square millimeter of the metaphyseal area was measured from 0-0.5mm below growth plate.
2.5 Flow cytometric analysis
Bone marrow cells were collected from femurs and tibias of mice offspring at 4-week-old. Cell numbers were determined after removal of red blood cells with ACK Lysis Buffer (CS0001, Leagene, China). After washed with PBS twice, pellets were resuspended and blocked in 1% BSA on ice for 15min. Cells were then washed twice with PBS and incubated with primary antibody (for cell surface marker) solution diluted by 0.5% BSA for 30min on ice in the dark. After being fixed by 4% paraformaldehyde and permeabilized by PBST and washed with PBS, cells were incubated with primary antibody (for intracellular antigen) solution diluted by 0.5% BSA for 30min on ice in the dark. The primary antibody used were PE-conjugated Nestin Antibody (MA5-23574, ThermoFish Scientific, Rockford, IL, USA), and BV421−conjugated anti-mouse CD45 (563890, BD Biosciences, San Jose, CA, USA). Cells were then washed once and re-suspended in 300 μl PBS and transferred to flow tubes. For BMSCs, CD45-CD29+CD105+Sca-1+ bone marrow cells were detected using a Mouse mesenchymal stem cell Multi-color Flow kit (FMC003, R&D systems, Canada) following manufactures protocol. Flow cytometric analysis was performed on a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (BD Life Sciences San Jose, CA, USA).
2.6 Statistics
The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology. All quantitative data were presented as mean ± S.E.M. For comparisons between two groups, independent Student’s t-test was performed. For multiple comparisons, one-way analysis of variance (ANOVA) with Bonferroni post hoc test was used. Statistical analysis was performed using SPSS, version 20 software (International Business Machines Corporation, IBM Corp.). Significant level was defined as P < 0.05.