Bone marrow-derived macrophages (BMDMs) and BMSCs were isolated from 4-week-old C57BL/6 male mice and Sprague Dawley (SD) male rats, respectively, by flushing the bone marrow from femurs and tibias with phosphate buffer saline (PBS; HyClone, USA). BMDMs were cultured in Dulbecco’s Modified Eagle’s Medium (HyClone) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 1% penicillin-streptomycin (P/S; Gibco), and 20% conditioned media collected from L929 cells. BMSCs were cultured in Modified Eagle’s Medium Alpha (HyClone) supplemented with 10% FBS and 1% P/S. All cells were cultured at 37 ℃ in a humidified atmosphere with 5% CO2.
BMDMs were stimulated with AGEs (200 μg/ml; BioVision, USA) for 48 h in the presence or absence of ADM2 (1 μM; Phoenix Pharmaceuticals, USA). An equal volume of PBS was added to the control group. In addition, GW9662 (2 μM, pretreatment for 2 h; Beyotime) was administrated along with AGEs and ADM2 treatment to verify the molecular mechanism by which ADM2 regulates AGE-induced macrophages.
Flow cytometry analysis
After treatment, BMDMs were fixed with 4% paraformaldehyde (PFA), blocked with 5% bovine serum albumin (BSA), and then incubated with FITC-conjugated F4/80 antibody (eBioscience, USA), APC-conjugated CD206 antibody (eBioscience), and PE-conjugated CD86 antibody (eBioscience) for 30 min. The candidate cells were detected using a BD FACS Caliber flow cytometer and analyzed using FlowJo v10.0 software. F4/80+ cells were identified as macrophages, and the expression levels of CD86 and CD206 were detected to evaluate the M1 and M2 polarization states of BMDMs.
Enzyme-linked immunosorbent assay (ELISA)
The media supernatant was collected from the culture of BMDMs and stored at -80 ℃. The concentrations of tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) were measured using ELISA kits (Proteintech, China), according to the manufacturer’s protocols.
BMDMs were fixed with 4% PFA, washed with PBS thrice, blocked with 5% BSA for 1 h, and then incubated with the primary antibody against p65 (Beyotime) at 4 ℃ overnight. The cells were then incubated with the Cy3-conjugated secondary antibody (Beyotime) at 25 ℃ for 1 h and then stained with 4',6-diamidino-2-phenylindole (DAPI) for 5 min. The activation and nuclear translocation of p65 were observed using a fluorescence microscope.
Osteogenic differentiation and detection
To determine the effects of ADM2 on the osteogenic differentiation of AGE-induced BMSCs, both alkaline phosphatase (ALP) and mineral deposition were detected. Briefly, BMSCs were inoculated in 24-well plates (5×104/well). Upon 80% confluence, the medium was replaced with osteogenic induction medium (OIM: 20 mM β-glycerophosphate, 1 nM dexamethasone, and 50 μM L-ascorbic acid-2-phosphate in the complete medium; Sigma-Aldrich, USA) containing AGEs (200 μg/ml) in the presence or absence of ADM2 (1 μM), and the medium was replenished every 2 d. ALP staining and activity assays were performed 7 d after osteogenic induction according to the manufacturer’s instructions (Beyotime). On the 14th day of differentiation, alizarin red S (ARS; Cyagen Biosciences, China) staining was performed to evaluate mineral deposition. For quantitative analysis of the mineralization, calcium deposition was eluted with 10% (v/v) cetylpyridinium chloride (Sigma-Aldrich), and the OD value was measured at 570 nm.
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
Total cell RNA was extracted using an RNA Purification Kit (EZBioscience, USA) and cDNA was obtained from 500 ng of total RNA using the Reverse Transcription Kit (EZBioscience). Thereafter, qRT-PCR was performed using SYBR Green qPCR Master Mix (EZBioscience). Relative gene expression levels were calculated by the 2-△△CT method and GAPDH was used as the reference gene for normalization. The primer sequences are shown in Table. 1.
Western blot analysis
Total cell protein was extracted using RIPA lysis buffer with protease inhibitor and protein phosphatase inhibitor (Solarbio, China) at 4 ℃. Protein concentration was determined using a BCA Protein Assay Kit (EpiZyme, China). Equal amounts of protein (30 μg) were subjected to 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, USA). After blocking with 5% BSA, the membrane was incubated with primary antibodies at 4 ℃ overnight. Afterward, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, USA) at 25 ℃ for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagent (Millipore) and the grayscale of protein bands were semi-quantified using ImageJ software.
The primary antibodies used in this study included anti-PPARγ (Cell Signaling Technology, USA), anti-IκBα (Cell Signaling Technology), anti-p65 (Cell Signaling Technology), anti-phosphorylated p65 (Cell Signaling Technology), anti-BMP2 (Abcam, UK), anti-OSX (Abcam), anti-OCN (ABclonal, China), and anti-GAPDH (Cell Signaling Technology).
Induction of Type 1 Diabetes mellitus (T1DM) rat model
All experimental procedures were approved by the Animal Research Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. After fasting for 12 h, a single high dose (65 mg/kg) of streptozotocin (STZ, 10 mg/mL in 0.01 M citrate buffer; Sigma-Aldrich) was intraperitoneally injected to SD rats, weighing 350-400 g, to induce T1DM models. After 7 d of STZ injection, random plasma glucose levels (PGLs) were determined with a glucometer using blood from the tail vein. Rats with PGLs above 16.7 mmol/L were considered as diabetic individuals, and those that failed to reach the target glycemic index were excluded from the study.
Animal surgery and treatment
A total of 36 T1DM SD rats were used in this study and randomly assigned to the DM (n = 12), DM+ADM2 (n = 12) and DM+ADM2+GW9662 (n = 12) groups. Rats injected with an equal volume of citrate buffer were assigned to the non-diabetic control group (n = 12). To establish the DO model, a transverse osteotomy was performed at the midshaft of the right tibia after anesthesia and exposure. Then, a specially designed monoliteral external fixator (Xinzhong Company, China) was mounted to fix the proximal and distal segments of the tibia. Thereafter, surgical incisions were closed layer-wise. The periosteum was preserved as much as possible during the procedure. The DO procedures comprised three phases: latency phase for 5 d, distraction phase for 10 d (0.25 mm every 12 h), and consolidation phase for 4 weeks. ADM2 (200 μg/kg/d) was subcutaneously injected during the consolidation phase to the DM+ADM2 group and DM+ADM2+GW9662 group, and the latter was intraperitoneally administrated GW9662 (1 mg/kg/d). Equal-volume PBS was subcutaneously injected at the same time as the DM and control groups.
Digital radiography and micro-computed tomography (CT)
From the beginning of the consolidation phase, X-ray films which were focused on the distraction gaps were acquired weekly. The lengthened tibia specimens were harvested 2 (n = 6) and 4 (n = 6) weeks after distraction. Micro-CT scanning was performed to quantitatively evaluate bone regeneration in the distraction zone. Thereafter, three-dimensional (3D) reconstructions of the regenerated callus were produced using the CTVox software. Parameters including bone mineral density (BMD) and bone volume/tissue volume (BV/TV) of the regenerated bone were analyzed using the CTAn software.
The mechanical characteristics of the fresh tibia specimens (n = 3) were determined using a four-point bending device after 4-week consolidation. During the test, the tibia specimens were loaded in the anterior-posterior direction with the posterior side in tension. The modulus of elasticity (E-modulus), ultimate load, and energy to failure were recorded and analyzed using Vernier Graphical Analysis software.
Histological and immunohistochemical staining
For histological analyses, after 2 (n = 3) and 4 (n = 3) weeks of consolidation, tibia specimens were fixed in 4% PFA for 24 h, decalcified in 10% ethylene diamine tetraacetic acid (EDTA, pH = 7.4) for 21 d, dehydrated through graded ethanol of increasing concentration, and then embedded in paraffin. Samples were cut into 5 μm-thick longitudinally oriented sections and then subjected to hematoxylin-eosin (H&E), Masson’s trichrome, and Safranine O-Fast Green (SO-FG) staining.
For immunohistochemical staining, sections were incubated in 0.3% hydrogen peroxide for 20 min to quench endogenous peroxidase activity. After antigen retrieval in 0.01 mol/L citrate buffer (pH 6.0) at 65 ℃ for 20 min and blocking with 5% goat serum for 1 h, sections were incubated with anti-OCN antibody (ABclonal) at 4 ℃ overnight. After incubation with secondary antibodies conjugated with HRP at 25 ℃ for 1 h, an HRP-streptavidin system was used to detect positive areas followed by counterstaining with hematoxylin.
CD68 and CD86, or CD68 and CD206 double immunofluorescent staining were performed to detect M1 or M2 macrophages, respectively. After 2 weeks of consolidation, tibia specimens (n = 3) were decalcified in 18% EDTA for 3 d after fixation. Subsequently, the samples were dehydrated in 30% sucrose, embedded in optimal cutting temperature compound, and cut into 10-μm thick longitudinally oriented sections. After blocking with 5% BSA for 1 h, bone sections were incubated with primary antibodies overnight at 4 ℃, followed by incubation with fluorophore-conjugated secondary antibodies at 25 ℃ for 1 h. Nuclei were stained with DAPI. A fluorescent microscope was used for observation and image capture. For semiquantitative analysis, the ratios of CD86+CD68+ / CD68+ cells and CD206+CD68+ / CD68+ cells in the distraction area were calculated using Image-Pro Plus software.
The primary antibodies used in this study included anti-CD68 (Abcam), anti-CD86 (Novus, USA), and anti-CD-206 (Abcam).
All data are presented as mean ± standard deviation. The statistical differences were analyzed with Student’s t-test between two groups or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test among groups using GraphPad Prism 8 software. Results were considered statistically significant at a two-tailed P-value less than 0.05.