Animals and materials preparation
Fourteen-week-old C57BL/6 female mice (~ 18 g, Animal Center of Academy of Military Medical Sciences, China) were employed. The animals were housed in a temperature-controlled room (22–25 °C, 50–60% humidity) on a dark-light cycle of 12:12-h under pathogen-free conditions. They were handled by a professional person and had access to water and food. All experiments were carried out in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Ethics Committee of Tianjin Medical University. We acquired minimum essential medium alpha (MEM-α), fetal bovine serum (FBS), penicillin, streptomycin, and trypsin from Invitrogen (Carlsbad, CA, USA), while other agents were obtained from Sigma (St. Louis, MO, USA) unless otherwise stated.
After 1 week of acclimation, one hundred and ten mice were randomly sorted into five groups: the control group fed with a standard chow diet (SC; N = 22), high-fat diet group (HF; N = 22, D12492, Beijing Huafu Kang Biological Co. Beijing, China), high-fat diet and knee loading group (HFL; N = 22), high-fat diet in combination with ovariectomy (OVX) group (HO; N = 22), and high-fat diet in combination with OVX and knee loading group (HOL; N = 22). Two OVX groups (HO and HOL) underwent bilateral ovariectomy, while three pseudo-OVX groups (SC, HF, and HFL) underwent sham surgery. In addition to SC, the other four groups received a high-fat diet. After 4 weeks, two groups (HFL and HOL) received knee loading for the next 4 weeks (Fig. 1a).
Mice were anesthetized using 1.5% isoflurane (IsoFlo; Abbott Laboratories, North Chicago, IL, USA). We used a scalpel to make an incision on the dorsal skin of the midline, and removed the ovaries with scissors (Fig. 1b). For mice undergoing pseudo-OVX surgery, the same procedure was conducted without isolating the ovaries. To alleviate the surgery-linked pain, mice received 0.05 mg/kg buprenorphine hydrochloride every 8 h during the first 3 days. Also, 1% pramoxine hydrochloride ointment was applied daily at the incision site [19,29].
After 4 weeks of high-fat diet and OVX, mice in HFL and HOL groups were mask-anesthetized using 1.5% isoflurane and loaded onto both knees in the lateral-medial direction using a custom mechanical loader. Daily dynamic loads (1 N at 5 Hz) were applied every day to each of the right knees and then the left knee for 3 minutes (loading total time was 6 min/day), for 4 weeks (Fig. 1c). The lateral and medial sides of the femur and tibia were in contact with the loading rod and the stator, respectively (Fig. 1d). The sham-treated control mice (SC) and obese mice (HF and HO) were given sham loading, in which mice were placed on the loading table and anesthetized, but did not receive any loads.
Body weight and body fat composition analysis
We measured the body weight and food intake weekly and daily, respectively, by a blinded person. Body fat content and body mass index were determined by a systemic composition analyzer (ImpediVet, Pinkenba, Qld, Australia) .
Histology, MacNeal’s and immunohistochemistry assays
We harvested femurs fixed them using 10% neutral buffered formalin. The samples were decalcified in 10% ethylenediaminetetraacetic acid (EDTA, PH = 7.4) for 3 weeks, and embedded in paraffin after dehydration. We prepared 5-μm thick sections along the coronal plane using a Leica RM2255 microtome (Leica Microsystems Inc., Bannockburn, IL). Using H&E stained femoral sections, we determined A.ar/T.ar (in %, T.ar = total tissue area, and A.ar = adipocytes area), adipocyte perimeter, adipocyte number  and B.ar/T.ar (in %, T.ar = total tissue area, and B.ar = trabecular bone area), as well as bone trabecular circumference in the 1.9 mm2 sample area of the proximal femoral growth plate. Furthermore, MacNeal’s stained sections were used to determine the osteoblast number on the trabecular bone surface (osteoblasts number/bone surface, N.Ob/BS/mm) .
The expression of Wnt3a was analyzed by immunohistochemistry. After deparaffinization and rehydration, the distal femur sections were incubated with primary antibodies and an immunohistochemical color reaction was performed using a 3, 3′-diaminobenzidine substrate kit and hematoxylin as a counterstain. One of every 10 slices (5 slices in total) was chosen for quantification in a blinded fashion. We determined the ratio of positively stained cells in 10 fields at 200× magnification per section .
Isolation of bone marrow mesenchymal stem cells and their culture
After sacrifice, the femur and tibia were washed with MEM-α (Thermo Fisher Scientific) for collecting BMSCs. The cells were isolated by centrifugation using a Ficoll low-density gradient and cultured in MEM-α containing 10% FBS .
Adipogenic differentiation assay and oil-red O staining
For adipogenic differentiation, bone marrow-derived cells (2×106 cells/ml in 6-well plates) were cultured for 72 h in adipogenic medium (MEM-α containing 10% FBS), with 0.5 μM dexamethasone, 0.25 mM methylisobutylxanthine, 5 μg/ml insulin, and 50 μM indomethacin, and then treated for an additional 48 h with 5 μg/mL insulin.
Differentiated adipocytes were fixed in 4% paraformaldehyde and stained in a 60% saturated oil red O solution. For quantification, isopropanol was employed to extract oil red O and optical absorbance at 520 nm was measured .
Measurements of bone mineral density and bone mineral content in vivo
Mice were anesthetized by 1.5% isoflurane to maintain in the prone position, and measurements were performed in about 5 min per mouse. In the femur, we determined bone mineral density (BMD, g/cm2) and bone mineral content (BMC, g) using peripheral dual-energy X-ray absorptiometry (pDEXA; PIXImus II; Lunar, Madison, WI, USA) .
Osteoblast differentiation assay and ALP staining
For osteoblast differentiation, bone marrow-derived cells (2×106 cells/ml) were cultured in osteogenic differentiation medium (MEM-α containing 10% FBS, 10 nM dexamethasone, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate). The medium was changed every 2 days, and cells were cultured for 14 days .
We performed alkaline phosphatase (ALP) staining (Sigma). We fixed cells in citrate-buffered acetone, incubated in the alkaline-dye mix, and counterstained with Mayer’s Hematoxylin. Five fields (400×) were randomly selected in each well, and the ratio of the number of ALP-positive cells to that of total cells was determined.
Western blotting analysis
After sacrifice, the femurs were removed. Protein samples were isolated from the femurs using a mortar and pestle and lysed in a radioimmunoprecipitation assay lysis buffer with protease inhibitors and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). The isolated proteins were electro-separated and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Primary antibodies specific to Wnt3a (Abcam, Cambridge, MA, USA), β-catenin, LRP5, Runt-related transcription factor 2 (Runx2), PPARγ, C/EBPα (Cell Signaling, Danvers, MA, USA), ALP (Proteintech, Wuhan, China) and β-actin (Sigma, St Louis, USA) were employed. After incubation with horseradish peroxidase-conjugated secondary antibodies, chemiluminescence signals were detected and processed with reference to β-actin .
We expressed data as mean ± standard error of mean (SEM). For comparisons among more than 2 groups, we employed one-way analysis of variance (one-way ANOVA) and a post-hoc test of least significant difference (LSD) using SPSS software (version 20.0). We also conducted correlation analysis using Pearson correlation coefficients. The relative parameters (% change) such as body weight, body fat content, BMD, and BMC were calculated as ((S-B)/B × 100 in %, where S= “sacrifice” and B = “baseline”). All comparisons were two-tailed and we assumed statistical significance at P < 0.05. The asterisks (*, ** and ***) represent P < 0.05, P < 0.01, and P < 0.001, respectively. The pound signs (#, ## and ###) represent P < 0.05, P < 0.01, and P < 0.001, respectively.