2.1 Experimental animal
The current study employed 40 healthy female SD rats (12 weeks of age, weighing 230±25g). All animals were housed in groups of five in cages in a temperature- controlled environment (25±1° C, 55-65% relative humidity; 12 h of artificial lighting) in the central laboratory of yijishan hospital. All experimental rats were kept on pellet feed with standard laboratory diet and tap water, ad libitum. All surgical procedures and drug treatment during the course of the experiment, as well as the sacrifice of the rats at the end of the experiment were approved by the Animal Research Committee of Southern Wannan Medical College.
2.2 Preparation of VPA Hydrogel scaffolds
The VPA hydrogel was synthesized as described[18]. Briefly, 200 μl poloxamer 407 hydrogel (BASF, Ludwigshafen, Germany)(1/4 volume ratio) was mixed with 50μg Valproic acid and 0.01 M phosphate-buffered saline (PBS) (pH 7.4, at 4° C). The VPA hydrogel solutions were introduced in Teflon molds (1.5 mm diameter x 4.0 mm height), and samples were immersed in 0.9 wt.% of NaCl solution at 37° C for setting.
2.3 Animal experiments
In order to establish an osteoporosis model, the rats were subjected to bilateral ovariectomy(OVX, n=25) or sham operation(Sham, n=15) according to a previously-described protocol[19] and were kept for 12 weeks. Subsequently, each five rats from OVX group and Sham group were randomly selected and executed. Bilateral femora were collected and measured by Micro-CT and HE staining to verify the establishment of standard postmenopausal osteoporotic animal models. Then all animals were randomly divided into three groups of 10 rats each: Sham group, OVX group and VPA treatment group(VPA group). Once osteoporosis was confirmed, a drilling bone defect with 1.5 mm external diameter of anteroposterior channel was created by an electric motor with a speed of 1500 rpm in the femoral condyle of the remaining rats according to previous reports[20, 21]. The rats were classified to VPA group and were implanted and treated with VPA hydrogel. Two intraperitoneal injections of calcein(20 mg/kg) were injected on the 3rd and 10th day before the rats were sacrificed. After 12 weeks of treatment, the rats undergoing bone defect surgery were sacrificed using an overdose of chloral hydrate. Femur samples were harvested. Femurs were fixed at 4°C with 4% paraformaldehyde.
2.4 Cell culture and Alkaline phosphatase (ALP) staining and Alizarin Red(RES) staining
As an osteoblast precursor cell line, MC3TE‐E1 was obtained from the Institute of Biochemistry and Cell Biology, CAS (Shanghai, China). MC3TE‐E1 were cultured in 24-well plates at 1X104 cells per well with growth culture medium. After culturing for 24 h, MC3TE‐E1 cells were plated at a density of 1X104 cells/ml in 24 well plates and cultured in growth medium supplemented with 10-8 M dexamethasone (Sigma), 50 μg/ml ascorbic acid (Sigma) and 5 mM β-glycerol phosphate (Sigma). Then the medium was added with phosphate-buffered saline (PBS) or valproic acid (10−6 M). The medium was changed every four days during osteogenic differentiation. After induction for 14 and 21 days, osteogenesis was evaluated by staining MC3T3-E1 osteoblasts with ALP substrate mixture (ALP staining kit, Sigma) and Alizarin Red reagent (RES, Cyagen Biosciences, Guangzhou, China) as protocol described, respectively.
2.5 Micro-CT evaluation
Formation of new bone in defect areas was evaluated by Micro-CT (Bruker Skyscan 1272 system, Kontich, Belgium). The parameter is set to 55 kV and 114 m A with a thickness of 0.048 mm per slice in medium-resolution mode, 1024 reconstruction matrix, and 200 ms integration time. These images and parameters of trabecular bone parameters with a distance of 1 mm proximal from the end of the growth plate in femoral metaphysis were compared between the Sham group and OVX group to confirm the osteoporosis rat model. For evaluation of bone formation in the defect area, a 1.5 mm diameter area in the center of each bone defect was selected as the volume of interest (VOI). After 3D reconstruction, bone mineral density(BMD), bone mineral content(BMC), bone volume fraction(BV/TV), trabecular number(Tb.N), trabecular thickness(Tb.Th), trabecular separation(Tb.Sp) were automatically determined for identification of osteoporosis model while BMD, BV/TV, Tb. N, Tb. Th, Tb. Sp, the mean connective density (Conn.D) in VOI regions were used to evaluate new bone formation, using a protocol provided by the manufacturer of the Micro-CT scanner as previously described[22, 23].
2.6 Biomechanical examination
Compression testing of bone samples was performed immediately after the micro-CT scan. The distal femoral metaphysis of each femur was placed in a 5 mm wide and 2 mm-deep notch of an aluminum alloy base, which was fixed to the mechanical testing system(Instron 5566; Instron, Norwood, MA, USA). The compression load was applied to the ventral aspect of the condyles at 2 mm/min until failure. Ultimate load (N) was calculated from the load deformation curve.
2.7 Histomorphometric analysis and immunofluorescence staining
Part of the femora was decalcified in 10% EDTA (pH 7.4) for 4 weeks and then embedded in paraffin. Four-micrometer-thick longitudinally oriented along the defect sections were used for staining. The specimens were stained with hematoxylin and eosin (H&E) according to a standard protocol, viewed under a light microscope and the stained areas were quantified using a BI-2000 medical image analysis system (Chengdu TME Technology Co, Ltd., Chengdu, China). Vascular endothelial growth factor (VEGFA) and recombinant Human Bone Morphogenetic protein-2(BMP-2) staining were used to quantify the expression of osteogenesis and vascularization factors in the defect area. In brief, fresh bone sections were stained with individual primary antibodies to rats VEGFA(Abcam, ab206887, 1:100) and BMP-2 (Abcam, ab214821, 1:100), overnight at 4°C. Subsequently, the secondary antibodies conjugated with fluorescence (Jackson Immuno Research, 415-605-166, 1:500; 315-605-003, 1:250) were used at room temperature for 1h while avoiding light and observed under a confocal microscope (FLUOVIEW FV300, Olympus). Calcein double labelling in undecalcified bone slices were observed under a fluorescence microscope(FLUOVIEW FV300, Olympus) to quantify bone mineralization in the defect area.
2.8 Western blot analysis
MC3TE-E1 cells 3 days after drug intervention were prepared for Western blotting as previously described. Bone tissue in the defect area was processed with liquid nitrogen. After that, spin-OUT columns (GT1200, G-Biosciences, St Louis, USA) were used for the rapid purification of protein. The membrane was incubated with Anti- alkaline phosphatase (ALP, Abcam, ab198554, 1:1000), Anti-RUNX family transcription factor 2(RUNX 2, Abcam, ab236639, 1:1000), Anti-osteopontin(OPN, Abcam, ab214050, 1:1000), Anti-Osteocalcin(OC, Abcam, ab133612, 1:1000), Anti-VEGFA(Abcam, ab214424, 1:1000), Anti-BMP-2 (Abcam, ab214821, 1:1000), Anti-Notch1 (Abcam,ab52627,1:1000), Anti-HEY1 (Abcam,ab154077,1:1000), Anti-Jag1 (Abcam,ab109536,1:1000) and Anti-HES1(Abcam, ab119776,1:1000) overnight at 4℃. Protein expression levels were normalised to Glyceraldehyde 3 phosphate dehydrogenase (GAPDH; Boster, Wuhan, China, 1:2000) protein levels. The next day, the membranes were washed and incubated with the corresponding secondary antibody, diluted at 1:1000 for 2 h at room temperature. The membrane was incubated with ECL-enhanced claim inesence solution and then exposed to X-ray films (Pierce Biotechnology Inc., Rockford, IL).
2.9 Reverse transcription and real time polymerase chain reaction (RT-PCR) analysis
According to the manufacturer’s instructions, Total messenger RNA (mRNA) was extracted using the total RNA extraction kit (Takara, Kusatsu, Japan). Complementary DNA (cDNA) was obtained from total RNA using first Strand cDNA Synthesis Kit (Toyobo, Osaka, Japan). Then synthetic cDNAs and specific primers were used for qRT PCR with the TB GreenTM Premix Ex Taq II (Tli RNaseH Plus) kit (Takara, Kusatsu, Japan) on the CFX ConnectTM Real-Time System (Bio-Rad, Singapore). GAPDH was used as an internal control. Sequences of primers for the reference gene (GAPDH) and interested genes are listed in table 1.
2.10 Statistical analysis
All data is shown as mean ± standard deviation, analyzed using SPSS 19.0 software(IBM SPSS Statistics for Windows, Armonk, NY, USA). One-way analysis of variance(ANOVA) was used for multiple between-group comparisons followed by Tukey’s post hoc test. Paired-samples t test was used for comparisons of normal groups and OVX groups. A value of P≤0.05 was considered to reflect significance.