Following the Animal Experimentation Ethics Guidelines, the Ethics Committee of the Affiliated Hangzhou First People's Hospital approved all the procedures in the study (no. ZJCLA-IACUC-20080010). A total of 24 Sprague-Dawley female rats (12 weeks old, weighing 200-240 g) were purchased from the Beijing Weitong Lihua Experimental Animal Technology Co. Ltd. (Beijing, China). Then, the rats were housed in a specific pathogen-free (SPF) facility (temperature, 22±2°C; humidity, 50±10%) with a 12/12-h light/dark cycle, and standard laboratory animal chow and tap water were available ad libitum. To assess the effects of peptide 11R-VIVIT on osteoporotic fracture healing, a rat model of osteoporosis [model of ovariectomy (OVX)-induced osteoporosis] was established at first by removing the ovaries bilaterally (16). At 12 weeks after the OVX surgery, a unilateral open femur fracture model was established in all rats by an experienced operator to reduce pain in the animals. Briefly, an osteotomy fracture was established using an oscillating sagittal saw the middle of the left femur and 25-G needles were inserted intramedullary for fixation. A total of four groups (n=6 for each group) were included in the study as follows: i) A group of whole-stage saline treatment (Saline/Saline group, saline treatment only during ovariectomy and sacrifice); ii) a group of late-stage VIVIT (intraperitoneal injection, 100 µg/kg, Tocris Bioscience) treatment (Saline/VIVIT group, saline treatment following OVX and 11R-VIVIT treatment following OVX); iii) a group of early-stage 11R-VIVIT following saline treatment (VIVIT/Saline group, 11R-VIVIT treatment following OVX and saline treatment following OVX); and iv) a group of whole-stage 11R-VIVIT treatment (VIVIT/VIVIT group, 11R-VIVIT treatment only during OVX and sacrifice). Food and water were provided without limits to all rats during the fracture healing process. All animals were anesthetized by an intraperitoneal injection (50 mg/kg) of 1% sodium pentobarbital during the surgeries. The rats were then euthanized by cervical dislocation under anesthesia. The femurs were collected for X-ray, micro-CT and histological analyses. All wounds healed well and no death or adverse events were observed at the experimental endpoint.

Femurs were fixed in 4% paraformaldehyde for 24 h and then scanned with an X-ray scanner or a micro-CT system. The region of interest was defined as 2 mm above and below the fracture. All the slices derived from the micro-CT scanning were collected for 3D reconstruction. The bone formation area, including callus and cortex bone around the fracture line was selected to measure fracture healing. Following X-ray and micro-CT evaluation, the femurs were decalcified using EDTA, sliced into 4-µm-thick sections and stained with hematoxylin and eosin (Sigma-Aldrich; Merck KGaA) at room temperature for 30 min. Histological images were observed under a microscope (XSP-C204, China Investment Corporation).

The BMSCs were isolated and obtained by referring to a previous study (17). In brief, after the rats were anesthetized, the rat tibia was separated and obtained. The ends of the rat tibia were cut with scissors, and the tibia bone marrow contents were flushed and washed with phosphate-buffered saline (PBS) supplemented with 5% fetal bovine serum (FBS). Thereafter, the bone marrow contents were centrifuged at 450 × g for 10 min at room temperature and the cell pellet was re-suspended, which was then layered using the same volume of Ficoll-Paque (inno-train Diagnostik GmbH) and centrifuged at 850 g for 25 min at 4°C. Subsequently, BMSCs was collected and re-suspended in α-MEM containing 10% FBS and 1% penicillin-streptomycin (all from HyClone; Cytiva). Cell cultures were maintained in a 37°C incubator with 5% CO₂, and the medium was replaced twice weekly in the present study. Cells at passage 3-6 were utilized throughout the present study.

Flow cytometric analysis for the characterization of BMSCs was conducted by referring to the method described in previous studies (18,19). Briefly, 1×10⁶ BMSCs were incubated with (FITC or PE)-conjugated antibody CD45 (cat. no. 559135, BD Biosciences), CD11b (cat. no. 554982, BD Biosciences), CD73 (cat. no. 11-0739-42, Invitrogen; Thermo Fisher Scientific, Inc.) and CD90 (cat. no. 554898, BD Biosciences) (1 µg/10⁶ cells) in washing buffer for 30 min at 4°C. All fluorescently-labeled antibodies used in flow cytometric analysis were purchased from BD Biosciences. Thereafter, both the stained and unstained cells were washed, and their fluorescence intensity was quantified using a FACSCalibur flow cytometer (BD Biosciences). FlowJo software (Ver. 6.2) was utilized for data analysis.

BMSCs were pre-treated with 0.1, 1 and 10 µM 11R-VIVIT, 10 µM LY294002 (Sigma-Aldrich; Merck KGaA) and 200 nM rapamycin (Beijing Solarbio Science & Technology Co., Ltd.) for 1 h.

To detect the osteogenic ability, BMSCs were inoculated at approximately 1×10⁴ cells/cm² in 24-well plates and induced in osteogenic induction medium (OIM; cat. no. RASMX-90021, Cyagen Biosciences). Likewise, commercial standard adipogenic induction medium (AIM; cat. no. RASMX-90031, Cyagen Biosciences,) was employed for the induction of adipogenesis, which revealed the adipogenic ability of the BMSCs.

ALP staining and ALP activity assays were performed using BMSCs that had been cultured in OIM for 7 days. For ALP staining, BMSCs were washed with PBS, fixed in 4% paraformaldehyde for 20 min at room temperature, and then stained with an ALP staining kit (Nanjing Jiancheng Bioengineering Institute) for 30 min at room temperature in the dark. For ALP activity quantification, osteogenically differentiated BMSCs were examined using an EscAPeTM SEAP Chemiluminescence kit (Clontech Laboratories, Inc.), the value of which was normalized to the total cellular protein concentrations. Alizarin Red S staining and Oil Red O staining were performed at day 14 following differentiation. For Alizarin Red S staining, BMSCs were fixed with 4% paraformaldehyde for 20 min, washed PBS, and then stained with Alizarin Red (Cyagen Biosciences) for 5 min at room temperature. Images obtained following staining were analyzed using ImageJ software (ver. 1.6, Wayne Rasband, National Institutes of Health) and the Alizarin Red S percentage of the positively stained area was determined. Finally, Oil Red O staining was applied to determine the adipogenic ability of the BMSCs. In brief, BMSCs were fixed in 3.7% formaldehyde for 30 min, followed by staining with Oil Red O (Sigma-Aldrich; Merck KGaA) for 30 min. All images were acquired using a light microscope (Dmi8, Leica Microsystems GmbH).

Total RNA was extracted from the BMSCs with TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Reverse transcription was carried out using the Script™ RT-PCR kit (Takara Bio, Inc.), followed by PCR using SYBR PreMix Ex TaqTM (Takara Bio, Inc.) on the CFX96TM Real-time PCR Detection System (Applied Biosystems). GAPDH mRNA was used as an internal control. The reaction conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 94°C for 5 sec and 60°C for 35 sec. The 2^−ΔΔCq method (20) was used to determine the relative expression levels. The primers for rat mRNAs are listed in Table I. The amplified products were separated on 1% agarose gel stained with ethidium bromide and visualized by UV light

Western blot analysis was carried out by referring to the protocol described in a previous study (21). Total proteins were isolated from the BMSCs using RIPA-lysis buffer (Sigma-Aldrich; Merck KGaA) on ice, and protein concentrations were measured using a BCA protein assay kit (Sigma-Aldrich; Merck KGaA). A total of 20 µg of total protein were electrophoresed on 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked using non-fat milk for 1 h at room temperature and incubated overnight at 4°C with primary polyclonal antibodies including anti-LC3 (ab192890, 1:2,000), anti-p62 (ab109012, 1:10,000), anti-p-AKT (ab38449, 1:1,000), anti-AKT (ab8805, 1:500), anti-p-glycogen synthase kinase-3β (GSK-3β, ab131097, 1:1,000), anti-GSK-3β (ab93926, 1:1,000), anti-NFATc1 (ab25916, 1:2,000), anti-Lamin B (ab32532, 1:500), anti-Runt-related transcription factor 2 (Runx2; ab76956, 1:2,000) (all from Abcam), anti-ALP (PA1004, 1:1,000, Boster Biological Technology Co., Ltd.) and anti-GAPDH (ab8245, 1:1,000, Abcam). The proteins were then visualized following incubation with goat anti-rat IgG (H&L) secondary antibody (ab182018, 1:2,000, Abcam) for 60 min at 25°C, with extensively washing with TBST and detecting using a chemiluminescence system (Pierce; Thermo Fisher Scientific, Inc.). The intensity of protein bands was measured by ImageJ 1.6 software and the signal intensity of each band was normalized to its corresponding GAPDH or Lamin B control.

The viability of BMSCs was measured using a CCK-8 kit (Dojindo Molecular Technologies, Inc.). In detail, normal BMSCs, osteoporotic BMSCs and osteoporotic BMSCs treated with 11R-VIVIT (10 µM) in 96-well plates were collected at the time points of 0, 24, 48 and 72 h. CCK-8 kit solution (15 µl) were added to each well, and the cells were incubated at 37°C for an additional 3 h. The solution was finally removed, and the absorbance at 450 nm was recorded using a microplate spectrophotometer (Bio-Tek Instruments, Inc.).

The tumor necrosis factor (TNF)-α and interleukin (IL)-1β concentrations of the normal BMSCs, osteoporotic BMSCs and osteoporotic BMSCs treated with 11R-VIVIT (10 µM) were measured by ELISA. The cells were cultured in 96-well plates for 3 days and the culture media were harvested and centrifuged at 1,200 × g for 10 min at room temperature. The TNF-α and IL-1β concentrations in the supernatants were detected using rat TNF-α (EK0526) and IL-1β (EK0393) ELISA kits (Boster Biological Technology Co., Ltd.), according to the standard instructions. The value at 450 nm was read using a microplate reader (SpectraMax 190, Molecular Devices, LLC). The concentrations were calculated using the standard curve.

All data were presented as the mean ± SD. Statistical analyses were calculated using GraphPad Prism (version 8.0) software. The Student's t-test was adopted for two-sample comparisons. One-way ANOVA followed by Tukey's post hoc analysis were adopted for multiple comparisons. All data were determined by at least three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

To determine whether the model of OVX was successfully established, bone mineral density in the rats was detected. The results revealed that rats subjected to OVX had a lower bone mineral density than the rats at baseline (Fig. 1A). Thereafter, the effects of 11R-VIVIT on the osteoporotic fracture healing process were examined (Fig. 1B). Micro-CT scans results revealed that the rats treated with 11R-VIVIT had a higher bone volume compared with those treated with saline (Fig. 1C and D) in at 6 and 16 weeks after the fracture. The reconstruction results of micro-CT analysis revealed that at 6- and 16-weeks post-fracture, the 11R-VIVIT-treated rats (particularly the rats from the VIVIT/VIVIT group) exhibited smaller fracture calluses and fracture gaps. At 16 weeks post-fracture, all fracture gaps in the whole-stage 11R-VIVIT-treated rats had almost disappeared, while the other groups exhibited obvious fracture gaps. H&E staining analysis also revealed that whole-stage 11R-VIVIT treatment increased the formation of bony connective junctions between the fracture gaps, which also suggested that 11R-VIVIT accelerated bone remodeling process in osteoporotic fracture healing (Fig. 1E).

As the osteogenic potential of BMSCs is a key factor during osteoporotic fracture healing, the normal and osteoporotic BMSCs were used to induce osteogenesis in vitro to determine whether 11R-VIVIT would affect the bone formation ability of osteoporotic BMSCs. The results of flow cytometric analysis revealed that the isolated cells were positive for CD73 and CD90, whereas they were negative for CD11b and CD45, indicating that the isolated cells were BMSCs (Fig. 2A). ALP staining, ALP activity assays and Alizarin Red staining were then performed. The osteoporotic BMSCs treated with 11R-VIVIT exhibited more mineralized nodules in the extracellular matrix than the cells from the OVX group (Fig. 2B and D). The results of Alizarin Red S staining revealed that the BMSCs from the OVX group had less calcium deposition than those from the normal group; treatment with11R-VIVIT increased calcium deposition (Fig. 2C and E). The results of semi-quantitative PCR revealed that the normal BMSCs expressed higher levels of ALP, osteocalcin (OCN) and osteopontin (OPN) than the osteoporotic BMSCs (Fig. 2F). The osteoporotic BMSCs were then treated with 11R-VIVIT, at the concentration of 0.1-10 µM. ALP staining and Alizarin Red S staining revealed that the ALP activity and the mineralized nodules of the osteogenic BMSCs gradually increased in response to the increasing 11R-VIVIT concentration in a concentration-dependent manner. Semi-quantitative PCR revealed higher expression levels of ALP, OCN and OPN mRNA in response to 11R-VIVIT treatment compared with the OVX control group. Hence, 11R-VIVIT at a concentration of 10 µM was employed for further analyses in the present study.

As the adipogenic potential of BMSCs also plays vital roles in osteoporotic fracture healing, the normal and osteoporotic BMSCs were used to induce adipogenesis in vitro to determine whether 11R-VIVIT can affect the adipogenic ability of osteoporotic BMSCs. The results of RT-qPCR and Oil Red O staining revealed that the osteoporotic BMSCs had a greater adipogenic capacity than the normal BMSCs (Fig. 3). After the osteoporotic BMSCs were treated with 11R-VIVIT, Oil Red O staining revealed that the adipogenic activity of the osteoporotic BMSCs was inhibited (Fig. 3A). Likewise, the results of RT-qPCR revealed lower mRNA expression levels of peroxisome proliferator-activated receptor (PPAR)γ and lipoprotein lipase (LPL) in response to 11R-VIVIT treatment compared with the OVX control group (Fig. 3B).

ELISA and CCK-8 assay were used to determine whether 11R-VIVIT can affect the viability and inflammatory levels of osteoporotic BMSCs. CCK-8 assay revealed that the viability of osteoporotic BMSCs was markedly lower than that of normal BMSCs. In addition, 11R-VIVIT treatment enhanced the viability of osteoporotic BMSCs compared with the BMSCs from the OVX group (Fig. 4A). Finally, the results of ELISA revealed a same pattern as CCK-8 assay. In detail, 11R-VIVIT treatment eliminated the levels of inflammatory factors in osteoporotic BMSCs (Fig. 4B and C).

Subsequently, western blot analysis was performed to determine whether autophagy is involved in the reversion of osteogenic capacity under 11R-VIVIT treatment. The results revealed a significant downregulation in the LC3-II/LC3-I ratio and the upregulation of p62 protein expression in osteoporotic BMSCs during osteogenesis compared with the normal ones, which indicated that osteoporosis impaired the autophagy of BMSCs. By contrast, a significant upregulation in the LC3-II/LC3-I ratio and the downregulation of p62 activity were observed in response to treatment with 11R-VIVIT (Fig. 5A), suggesting that 11R-VIVIT acted as an activator of autophagy in osteoporotic BMSCs.

The present study then attempted to elucidate the molecular mechanisms through which 11R-VIVIT led to the enhancement of autophagy and the osteogenesis of osteoporotic BMSCs. As the AKT/GSK-3β signaling pathway plays a vital role in the autophagy and osteogenic differentiation of BMSCs, the present study determined whether there was a crosstalk between 11R-VIVIT and AKT/GSK-3β signaling. Western blot analysis revealed a significant upregulation of p-AKT and p-GSK-3β in osteoporotic BMSCs during osteogenesis compared with the normal BMSCs (Fig. 5B). Thereafter, the osteoporotic BMSCs were treated with 11R-VIVIT, which resulted in the downregulation of p-AKT in osteoporotic BMSCs, while the level of p-GSK-3β exhibited no significant changes in the osteoporotic BMSCs. In order to confirm these results, LY294002 was used to inhibit AKT signaling and rapamycin (RAPA) was used to activate autophagy. Western blot analysis revealed that the activation of autophagy and the inhibition of AKT signaling exerted similar effects as observed with 11R-VIVIT treatment. Finally, western blot analysis revealed that the nuclear expression of NFATc1 was significantly increased in osteoporotic BMSCs compared with the normal ones. The nuclear expression of NFATc1 was decreased in the 11R-VIVIT treatment group (Fig. 5C), indicating that 11R-VIVIT inhibited the activation of NFATc1. Combined with AKT expression inhibition and autophagy activation, these results demonstrated that 11R-VIVIT activated autophagy and inhibited NFATc1 activation by inactivating AKT expression.

Subsequently, Alizarin Red S and ALP staining were performed to confirm the osteogenic potential of the BMSCs after the various treatments. The osteoporotic BMSCs treated with 11R-VIVIT, LY294002 and RAPA exhibited an increased osteogenic potential when compared with the osteoporotic BMSCs without treatment (Fig. 6A-D). Western blot analysis of Runx2 and ALP also revealed that the activation of autophagy and the inhibition of AKT signaling exerted similar effects as those observed with 11R-VIVIT treatment (Fig. 6E).