Animals and treatments

All the experiments were approved by the Ethics Committee of Biomedical Science of Peking University (approval no. LA20210509). Twelve-week-old female C57BL/6 mice were used in this study. The mice were anesthetized and subjected to bilateral ovariectomy (OVX) as described previously.46 Two months later, the mice were further subjected to tibia fracture or sham operation as previous description with modification.47 Briefly, the mice were anesthetized with 2%-4% isoflurane gas and a syringe needle was inserted into the tibia canal for fixation. Fractures were created at the mid-diaphysis using scissors. X-ray confirmed the position of the intramedullary pin and fracture. OVX mice with fractures were further randomly assigned to four treatment groups: iPTH (rhPTH1-34; Shenzhen Salubris Pharmaceuticals Co, China), propranolol (MedChemExpress, New Jersey, USA), iPTH + propranolol, or vehicle. PTH was intermittently administrated by subcutaneous injection at 80 μg/kg once a day at approximately 10 am. Propranolol hydrochloride was dissolved in drinking water (0.5 g/L) and was delivered daily (changed once per 3 days). Mice were euthanized after 4 weeks of treatment. Tissues, including tibias, femurs, lumbar vertebrae and serum, were collected for further analyses.

μCT

The bone samples were fixed in 4% paraformaldehyde for 24 h prior to scanning using a micro-CT scanner (Siemens, Erlangen, Germany). The scanner was set at a voltage of 80 kV, a current of 500 μA, a resolution of 8.82 μm per pixel, and an exposure time of 1 500 ms. To determine the bone mass and microstructure, the trabecular bone in the L5 vertebra beginning from the cranial growth plate and extending to the caudal growth plate was analyzed. For the femur analysis, the region of interest started from 0.15 mm proximal to the distal epiphyseal growth plate and was extended by 0.5 mm. To analyze callus formation, 100 continuous slices (50 slices up and 50 slices down from the fracture line) covering the middle of the newly formed callus were selected as the region of interest (ROI). Micro-CT software was used to calculate the bone volume (BV) and bone volume fraction (BV/TV) of the callus, excluding the native cortical bone.

Biomechanics

A three-point bending test was performed to determine the mechanical properties of the fractured tibiae using a mechanical testing system (Landmark, MTS, Inc., Eden Prairie, MN, USA). The loading point is located in the middle of the tibiae, with two pivots spaced 8 mm apart. Each sample is loaded at 6 mm/min until the breaking point was reached. The ultimate strength was calculated from the load-deformation curve.

Calcein double labeling

To assess dynamic bone formation, mice in different groups received intraperitoneal injections of 0.1% calcein solution (10 mg/kg body weight; Sigma) in PBS at 10 and 3 days before euthanasia. After euthanasia, the femurs were collected and fixed with 4% paraformaldehyde for 24 h, dehydrated in graded ethanol and embedded in methyl methacrylate. Using a hard sectioning microtome (EXAKT Cutting & Grinding System; EXAKT Advanced Technologies, Norderstedt, Germany), 50-μm-thick bone slices were obtained. Calcein double labeling was examined under a fluorescence microscope (Nikon). ImageJ v1.52 software was used to quantify the mineral apposition rate (MAR) of the trabecular bone.

Histological and immunohistochemical analyses

The femora were decalcified using a 10% EDTA solution with constant shaking for a period of 21 days. The samples were subsequently dehydrated using graded ethanol, embedded in paraffin and cut into 5-μm-thick sections. Immunostaining was conducted using a standard protocol, in which the sections were incubated overnight at 4 °C with an anti-osteocalcin (OCN) antibody (1:100; Abcam; Britain). The primary antibody was detected using a Rabbit-specific HRP/DAB Detection IHC Kit (Abcam; UK), followed by counterstaining with hematoxylin. TRAP staining was performed using a commercial kit (Sigma-Aldrich; USA) in accordance with the manufacturer’s recommendations. The nuclei were stained using methyl green, and TRAP+ cells (red) were identified as osteoclasts. The OCN+ and TRAP+ areas on the trabecular bone surface were calculated in three sections per mouse using ImageJ v1.52 software.

Immunofluorescence

OCN and Bmal1 double immunofluorescence staining was performed to evaluate the expression of BMAL1 in osteoblasts. Briefly, decalcified femora were dehydrated in 30% sucrose, embedded in OCT compound and cut into 5-μm-thick sections. After blocking with 5% donkey serum at room temperature for 30 min, the samples were incubated with an anti-BMAL1 antibody (1:100; Abcam; Britain) and an anti-OCN antibody (1:100; Abcam; Britain) overnight at 4 °C. Double-immunofluorescence staining was performed with a Treble-Fluorescence Immunohistochemical Mouse/Rabbit kit (Immunoway, USA) according to the manufacturer’s protocol. The number of BMAL1+ osteoblasts on the trabecular bone surface was calculated in three sections per mouse using ImageJ v1.52 software. The proportion of BMAL+ osteoblasts to total osteoblasts was also calculated.

ELISA

We used commercial ELISA kits from Elabscience (Wuhan, China) to measure the serum OCN, CTX-I and NE levels following the manufacturer’s instructions. We read the optical density of each well at 450 nm using a microplate reader (Bio-Rad 680, Hercules, USA) and calculated the protein concentration for each sample based on the standard curve.

BMSC culture

We isolated primary BMSCs from the femurs and tibias of C57BL/6 mice as described previously.48 Briefly, the femurs and tibias were extracted under sterile conditions and immersed in α-MEM (HyClone, Logan, USA) supplemented with 1% penicillin-streptomycin (PS; Beyotime, Shanghai, China). The bone marrow was flushed with α-MEM + 1% PS. The cells were washed and cultured in α-MEM supplemented with 10% fetal bovine serum (FBS) and 1% PS. Non-adherent cells were removed after 24 h of culturing with fresh complete α-MEM medium while the adherent cells were further cultured until they reached 90% confluence and passaged as needed.

Osteogenic differentiation

BMSCs and BMSCs transfected with Bmal1-siRNA or NC-siRNA were plated in 24-well plates until 80% confluence. The BMSCs were then incubated with osteogenic differentiation medium, and treated under the following conditions: intermittent PTH (iPTH;100 ng/mL), iPTH + NE (10 μmol/L), iPTH (100 ng/mL) + NE (10 μmol/L) + propranolol (1 μmol/L) or vehicle (PBS). The iPTH treatment was conducted as previously described.49 Briefly, during the 48-hour incubation cycle, BMSCs were treated with PTH for the initial 6 hours, followed by 42 h of culture without PTH. The osteogenic differentiation medium was changed every 48 h. The conditioned media (CM) from the different groups were harvested and centrifuged at 2 000 × g for 10 min to collect the supernatant, which was used for subsequent experiments. After 7–14 days of differentiation culture, the cells were stained or harvested for total RNA and protein extraction for further analysis.

ALP and ARS staining

After 7 days of osteogenic induction, the differentiated BMSCs were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and stained for alkaline phosphatase (ALP) with a commercially available ALP staining kit (Solarbio, Beijing, China). Alizarin red S (ARS) staining was carried out after 14 days of differentiation, after which the cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and then incubated with 2% ARS solution (Solarbio, Beijing, China) according to the manufacturer’s instructions. After washing with PBS, the stained cells were examined using an inverted microscope (Leica DMI6000B, Solms, Germany). The fractions of ALP+ and ARS+ areas were calculated using ImageJ v1.52 software.

qRT‒PCR

Total cellular RNA was extracted with TRIzol Reagent (Invitrogen, USA) and cDNA was synthesized via reverse transcription of 1 μg of the total RNA using a commercial kit (Qiagen, Dusseldorf, Germany). qRT‒PCR (20 μL) was conducted on an ABI PRISM® 7900HT System (Applied Biosystems, USA). Relative mRNA levels were calculated by the comparative Ct (2–ΔΔCT) method using Gapdh for normalization. The sequences of primers used are listed in Table S1.

Western blot

Total protein samples were separated via sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% nonfat milk for 1 h at room temperature, the membranes were incubated overnight at 4 °C with the appropriate primary antibodies, which included BMAL1 (1:1 000, Abcam, Cambridge, Britain), RUNX2 (1:500; ProteinTech, Chicago, USA), and tubulin (1:1 000; Affinity, Liyang, China). Then, the membranes were incubated for 1 h at room temperature with the appropriate secondary antibodies (1:5 000; Cell Signaling Technology, Danvers, USA). The blots were then detected with an enhanced chemiluminescence kit (Thermo Fisher Scientific).

Transfection of siRNA

Four Bmal1 siRNAs (Bmal1-siRNA #1, 2, 3 and 4) were synthesized by GenePharma (Shanghai, China). Cells transfected with negative control siRNAs (NC-siRNAs) were used as controls. The siRNAs were transfected into BMSCs using Lipofectamine® 3000 (Invitrogen, USA) according to the manufacturer’s instructions. The transfection efficiency was determined by qRT‒PCR and Western blot analysis. All siRNA sequences are listed in Table S2.

BMM culture

BMMs were isolated from the long bones of 6-week-old C57BL/6 mice as described previously.46 Briefly, the bone marrow cells were flushed out using an injector and then cultured in α-MEM supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Solarbio, China), 30 ng/mL recombinant murine macrophage colony stimulating factor (M-CSF; PeproTech, USA). After 18 hours, we discarded the adherent cells and collected the floating cells that were subsequently cultured in a new flask to obtain the macrophages.

Osteoclastogenic induction

The BMMs were cultured in 48-well plates in α-MEM (HyClone, Logan, USA) supplemented with 50 ng/mL of receptor activator for nuclear factor κB ligand (RANKL; Novoprotein, Shanghai, China) in the presence of vehicle (PBS), PTH (100 ng/mL), PTH + NE (10 μmol/L), PTH (100 ng/mL) + NE (10 μmol/L) + propranolol (1 μmol/L) or conditioned media from BMSCs treated with vehicle iPTH, iPTH + NE or iPTH + NE + propranolol as described above. Osteoclast formation was identified using a commercial TRAP kit (Sigma‒Aldrich, USA) according to the manufacturer’s instructions. The areas of TRAP+ (red) osteoclasts were measured by ImageJ 1.8.0 software.

Statistical analysis

The data are presented as the mean ± SD. The unpaired, two-tailed Student’s t test was used for comparing mean differences between two groups. Two-way ANOVA combined with Turkey’s or Sidak post hoc test was used for multiple-group comparisons, as detailed in each figure legend. A difference with P < 0.05 was considered to indicate statistical significant. GraphPad Prism 9 software was used for statistical analyses.