All animal care and studies were approved by the Animal Research Committee of Sichuan University (Chengdu, China) and conducted in accordance with international standards. Young adult (1 month old) and aged (12 months old) C57BL/6 male mice were obtained from the Animal Centre of Sichuan University.

Young adult mice were used for primary extraction of mesenchymal cells. After the mice were sacrificed, the femurs and tibias of both sides were obtained and immediately immersed into phosphate buffer saline (PBS, Gibco) containing 10% penicillin–streptomycin (Liquid, Gibco). The epiphyses were cut off and the marrow cavities were syringed with a sterile medium using a #25-gauge needle. Cells were washed with PBS by centrifugation and then cultured in alpha minimum Eagle's medium (α-MEM, HyClone) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin. BMSCs were incubated at 37 °C with 5% CO₂, and the culture medium was exchanged every 2 days. When the confluence rate reached 80%–90%, BMSCs were detached by 0.25% trypsin and then passaged. BMSCs were employed for the subsequent research at passage 4 (18). For osteogenic induction, osteogenic medium was supplemented with the basic medium 10 mM β-glycerophosphate (Sigma), 50 μM ascorbic acid (Sigma), and 10 nM dexamethasone (Sigma).

Bmal1 siRNA and negative control RNA were synthesized from Shanghai Sangon Biotech Co. (China). The sequence of Bmal1 siRNA is GCAAACUACAAGCCAACAU, the bases of which were shuffled for control siRNA. According to the manufacturer's protocol, the siRNA and Lipofectamine® RNAiMAX (Invitrogen) were dissolved in Opti-MEM (Gibco). The two transfection solutions were mixed and then incubated at room temperature in dark for 30 min. When the confluence rate of BMSCs reached 40%–50%, the culture medium was exchanged into α-MEM with 10% FBS without penicillin or streptomycin 2 h in advance. Then, the transfection mixture with Bmal1 siRNA or control siRNA was added into the culture medium. The knockdown efficiency was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) after 12-h incubation. If BMAL1 was successfully knockdown, cells were applied for tests as described below.

A cell counting kit-8 (CCK-8) (Beyotime, China) was used to measure the proliferation ability of BMSCs. At the time point of 1 and 3 days after being transfected with siRNA, cells were cultured into a 96-well plate in five duplicated wells. Then, a 100 µl CCK-8 working solution was added into each well. After incubating for 2 h, absorbance of the culture medium was detected at 450 nm.

A senescence-associated β-galactosidase (SA-β-gal) staining kit (Beyotime, China) was used to detect cell senescence of BMSCs. Passage 4 BMSCs were incubated in a six-well plate. After being transfected with siRNA, the medium was removed and cells were rinsed with PBS twice. Then, BMSCs were fixed with 4% paraformaldehyde and stained with a working mixture of β-galactosidase and X-Gal at 37 °C overnight in dark. Senescent cells were inspected with an optical microscope and counted in random field of vision.

In the in vitro test, total RNA was isolated using Trizol Reagent (Invitrogen) 7 days after osteogenic induction. The total RNA was treated with DNase and then reverse transcribed via a PrimeScript RT reagent Kit (Takara). Real-time PCR was conducted in a 20-µl mixture using SYBR Premix Ex Taq (Takara) and LightCycler 96 (Roche). Bmal1 and osteogenesis-related genes such as Sp7, Runx2, and Bmp2 were examined quantitatively using the housekeeping gene GAPDH as baseline. The primers are shown in Table 1 (18).

After 7 days of osteogenic induction, BMSCs were fixed with 4% paraformaldehyde and then incubated with a working mixture of BCIP/NBT (BCIP/NBT Alkaline Phosphatase Color Development Kit, Beyotime, China) for 15 min. Images were taken with the Epson Perfection V370 Photo Scanner. For the alkaline phosphatase (ALP) protein assay, the total protein of BMSCs was collected with a protein extraction kit (PE001, Sab-biotech) and quantitatively assayed using the BCA Protein Assay Kit (Beyotime, China). An alkaline phosphatase assay kit (Beyotime, China) was used for the protein assay. After reaction with 0.5 mM p-nitrophenyl phosphate for 10 min, absorbance of p-nitrophenol formed by hydrolysis was detected at 405 nm. The corresponding of ALP activity was converted according to the standard curve.

After 14 days of osteogenic induction, BMSCs were fixed with 4% paraformaldehyde and stained with Alizarin Red S (ARS) solution (Solarbio, China). Images were taken with the Epson Perfection V370 Photo Scanner.

The mouse Bmal1 sequence (NM_007489) was retrieved from GeneBank, and the lentiviral vector was obtained from GENECHEM Co. (China). Mice were divided into three groups: (1) young group: young adult mice without lentiviral vector injection; (2) aged group: aged adult mice without lentiviral vector injection; and (3) transfection group: aged adult mice with lentiviral vector injection. Rod-shaped titanium implants (commercial pure titanium, grade 4, SLA surface modification, 2 mm in length and 1 mm in diameter) were obtained from WEGO CO. (China). Mice were anesthetized through intraperitoneal injections of ketamine (70 mg/kg) and xylazine (10 mg/kg), and implants were inserted into the distal aspect of femurs as described by Xiang et al. (20) and Xue et al. (21). Briefly, after dissecting the skin and muscles, the implant sites were prepared by sequential drilling with 0.8/1.0 mm-diameter stainless-steel twist drills under continuous sterile saline irrigation. Then, one implant was press-fitted into the prepared hole (Figure 3A). In the transfection group, BMAL1 lentiviral vector was injected into the holes before implant insertion. Finally, the muscles and skin were sutured tightly with 5-0 nylon suture (Huawei, China) to protect the implant sites from the surrounding environment. Figures 3B,C show the representative actual and x-ray inspection of femurs with implants.

To confirm the functionality of BMAL1 lentiviral vector transfection system in the peri-implant sites, the IVIS Spectrum imaging system (PerkinElmer, Inc.) (absorbance of 465 nm) and immunofluorescence labeling of GFP (Abcam) were used to observe the expression of GFP in the transfection group at day 14 after the implant surgery.

Two weeks after implant surgery, femurs containing implants were harvested and scanned with a µCT 80 micro-CT system (SCANCO 50, Switzerland) at 8 µm resolution (90 kV, 200 µA, 500 ms integration time). A radius of µ20 m around implants was defined as the peri-implant site for volume of interest (VOI). After three-dimensional reconstruction, a few basic parameters were analyzed as follows: (1) BIC: bone–implant contact; (2) BV/TV: bone volume fraction; (3) Tb. N: trabecular number; (4) Tb. Sp: trabecular separation.

The specimens were fixed with 4% paraformaldehyde, dehydrated with a graded series of ethanol solutions (60%, 80%, 90%, and 100%), and embedded in light-curing epoxy resin (Technovit 7200VLC, Hereaus Kulzer, Germany) for 1 week. Then, the specimens were sliced perpendicular to the long axis of the implants and sanding to about 50 µm thickness using a grinding system (Exakt Apparatebau, Germany). Sections were stained with Stevenel’s blue and Van Gieson’s Picrofuchsin stain (20), and images were taken under a light microscope (OLYMPUS BX43F, Japan). For histomorphometric analysis, BIC and BV/TV were measured using the NIH IMAGE J software (21). BIC was defined as the line percentage of the implant interface that directly contacts the bone. As for the BV/TV, it indicated the proportion of bone to total tissue volume around the implants.

All data were presented as mean ± standard error (SD). Statistical differences were calculated via Student's t test for independent samples or one-way ANOVA for multiple comparisons using SPSS 17.0 software (SPSS, Inc., Chicago, IL, United States). A P value of less than 0.05 was considered as a statistically significant difference.

BMAL1 was knockdown in BMSCs to verify its effect on cell senescence. qRT-PCR tests showed the efficiency of BMAL1 knockdown (Figure 1A). CCK-8 assay demonstrated that deficiency of BMAL1 led to reduced cell proliferation capacity (Figure 1B). In the knockdown group, more blue-dyed products catalyzed by β-galactosidase were observed than another group under optical microscope via SA-β-gal staining (Figure 1C). It indicated that the knockdown of BMAL1 in BMSCs accelerated cell senescence. The counting result of senescent cells is shown in Figure 1D.

The osteogenesis of BMSCs was examined with ALP staining after 7 days osteoinduction, which indicates reduced capacity of osteogenic differentiation in the knockdown group (Figure 2A). The quantitative analysis of ALP activity also showed the downregulated osteogenesis under BMAL1 deficiency (Figure 2B), and the ARS staining demonstrated the consistent result (Figure 2A). Moreover, qRT-PCR tests at 7 days after osteoinduction revealed that a few osteogenesis-related genes (Sp7, Runx2, Alp, Dlx-5, Col1a1, and Bglap) were downregulated significantly (Figure 2C). The results above indicated reduced osteogenic differentiation of BMSCs under BMAL1 depletion.

To investigate the overexpression of BMAL1 around titanium implants in aged mice, micro-CT and histomorphometric analyses were conducted. First of all, immunofluorescence proved that GFP is expressed around the implant sites in the transfection group (Figure 3D). The three-dimensional reconstruction of the implant and newly formed bone tissue indicated that 2 weeks after implant surgery, bone–implant contact was obviously detected in the young group, while in the aged group, less bone–implant contact could be observed. After overexpressing BMAL1, samples in the transfection group exhibited increased BIC (Figure 4A). Histomorphometric analyses demonstrated likewise that the transfection of BMAL1 reversed bone–implant contact, which decreased while aging (Figure 5A). Figures 4B and 5B show the calculation results of BIC. As shown in Figure 4C, the BV/TV and Tb. N of mice femurs around implants in the aged group were significantly lower than that in the young group, but they increased in the transfection group. Otherwise, the Tb. Sp of mice femurs around implants in the aged group were significantly higher than that in the young group, but they decreased in the transfection group. Histomorphometric analyses demonstrated consistent results of BV/TV (Figure 5C). Results above indicated that overexpression of BMAL1 facilitates osseointegration, which decreases while aging.