Culture of macrophages
The Institutional Review Board at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology approved this study. C57BL/6J mice (6-week-old) from the Center of Experimental Animals, Tongji Medical College, Huazhong University of Science and Technology were used for this study. BMDMs were generated using M-CSF as in past reports.[26] Briefly, bone marrow cells isolated from these mice were grown in RPMI-1640 (#11875093, Gibco, Grand Island, USA) containing 10% FBS (#10099141, Gibco, Grand Island, USA), 1% penicillin/streptomycin (#SV30010, Hyclone, USA), and 50 ng/ml M-CSF (#216-MC, R&D System, Minneapolis, MN, USA) for 7 days. Subsequently, BMDMs were harvested for flow cytometric and transcriptomic analysis. M1 and M2 macrophages differentiation was initiated by treating BMDMs for 24 hours using either 100ng/ml LPS (#L2630, Sigma–Aldrich, St Louis, MO, USA) + 20ng/ml IFNγ (#285-IF-100/CF, R&D Systems), or 20ng/ml IL-4 (#204-IL-010, R&D Systems), respectively. M1 and M2 macrophages were then harvested for flow cytometric and transcriptomic analysis.
Flow Cytometric Analysis
This procedure was performed as descripted previously.[27] Antibodies were purchased from eBioscience company. BMDMs, M1 macrophages, and M2 macrophages were stained using lineage-specific antibodies, with F4/80 (#123107, Biolegend, San Diego, CA, USA) and CD 209 (#12-2092-80, Sigma–Aldrich) used for BMDM identification, F4/80 and CD11c (#117307, Biolegend) used for M1 macrophage identification, and F4/80 and CD206 (#141705, Biolegend) used for M2 macrophage identification. FlowJo software (version: FlowJo™ v10.6.1) was used for data analysis.
BMSCs Culture and Transfection
BMSCs were kindly donated by the Huazhong University of Science and Technology, Wuhan, China. Cells were grown in a specific media designed for C57BL/6 mouse mesenchymal stem cells (#MUBMX-03011-440, Cyagen, Guangzhou, China) at 37℃ in a 5% CO2 incubator. Cells were maintained for a maximum of 3 passages. Lipofectamine 3000 (#L3000001, ThermoFisher Scientific, USA) was used to transfect cells with siRNAs or miRNAs based on provided directions. agomiR-5106, agomiR-NC, antagomiR-5106, and antagomiR-NC (GenePharma, Shanghai, China) were transfected into cells using a 20 uM final concentration, whereas SIK2 siRNA, and SIK3 siRNA constructes (RIBOBIO, Guangzhou, China) were transfected at 50 nM. Plasmid-NC, plasmid-SIK2, and plasmid-SIK3 were synthesized by GenePharma (GenePharma, Shanghai, China).
Exosome purification, characterization and uptake
Following a 72 hours culture step, we removed debris and dead cells from samples via a 10 minutes spin at 1000 xg, followed by filtration with a 0.2 μm filter (122-0020PK ThermoFisher Scientific). Media then underwent ultracentrifugation for 6 hours at 100,000 xg at 4°C. Samples were then washed with PBS and spun for an additional 20 minutes at 100,000 xg, after which PBS was used to resuspend the pellet, which contained exosomes. Transmission electron microscopy (TEM; EFI, TECNAI G2) was used to assess morphology, while a Nanosizer™ instrument (Malvern Instruments, Malvern, UK) was used for Dynamic light scattering (DLS) analyses and flow cytometry was used to assess exosome surface marker expression. BMSC uptake of these exosomes was confirmed by labeling isolated exosomes using PKH 26 (#MINI26-1KT, Sigma, MO, USA) and then collecting them via centrifugation at 110,000 x g for 20 minutes. Briefly, exosomes were suspended in 1 mL diluent C containing 5 μM PKH26 and incubated for 5 minutes. The labeling action was stopped after 1 minute incubation with an equal volume of 1% bovine serum albumin (Bovogen, Melbourne, Australia). The exosomes were washed three times with Amicon ultrafilter (10 KDa cut-off, Millipore, MA, USA) with cold PBS and then resuspended in 200 μL PBS. These labeled exosomes were used to treat BMSCs for 12 hours followed by analysis under a confocal microscope.
qRT-PCR analysis
Before extracting miRNAs, callus samples were preserved using RNA Later (#76104, QIAGEN, Germany). Trizol (#15596018, Invitrogen, USA) was used to isolate total cell RNA, after which a VersoTM cDNA Synthesis Kit (#AB-1054/A, ThermoFisher Scientific) was applied to reverse transcribe RNA. miRNA expression was assessed using the SeraMir Exosome RNA purification Kit (System Biosciences, USA) to extract exosomal miRNAs, followed by use of a TaqMan microRNA assay kit (Applied Biosystems, USA) for cDNA synthesis. All qRT-PCR reactions were performed using a Thermal Cycler C-1000 Touch system (#10021377, Bio-Rad CFX Manager, USA), and U6 or GAPDH were used to normalize target expression as appropriate. Data were expressed as fold changes over controls. Primers used herein are compiled in Table 1.
Luciferase reporter assays
BMSCs were added to 24-well plates (2.5×105 cells/well) followed by transfection with dual-luciferase vectors (SIK2 WT, SIK2 Mut, SIK3 WT, and SIK3 Mut) along with either miR-5106 agonist (AgomiR-5106) or negative control (AgomiR-NC). A Quik Change Site-Directed Mutagenesis Kit (Strata gene) was used to insert mutations in the binding-region and dual luciferase reporter assay (Promega) was performed according to the manufacturer’s instructions. Ultimately, a luminometer (Glomax, Promega) was used to quantify luminescence, normalizing each value from the firefly luciferase construct to the corresponding Renilla signal.
Western blotting
Lysis buffer (#AS1004, Aspen, South Africa) containing 1% protease inhibitor (#AS1008, Aspen) was used to lyse cells or callus samples, after which protein was separated via SDS-PAGE and transferred to NC membranes (#IPVH00010, Millipore, USA) that were blocked with 5% nonfat milk and stained overnight at 4℃ overnight with antibodies specific for collagen I (1:500, Sigma, USA, #ab34710), ALP (1:1000, Sigma, USA,#ab95462), Osteocalcin (1:500, Sigma, USA, #ab93876), RunX2 (1:500, Sigma, USA, #ab23981), SIK2 (1:1,000, Sigma, USA, #SAB1302059 ), SIK3 (1:1,000, Sigma, USA, #SAB3500695), and GAPDH (1:10,000, Sigma, USA, #ab37168). Blots were then stained with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) (#AS1058, Aspen), and proteins were detected with a chemiluminescence detection system. Each experiment was repeated three times.
Alizarin red staining
BMSCs were grown in 6-well plates in media containing 100 nM dexamethasone, 50 mM ascorbic acid, and 10 mM b-glycerophosphate to promote osteogenesis (#HUXMA-90021, Cyagen, USA). Briefly, cells were washed twice by using PBS, after wich 10% formalin was added to fix the cells for 15 minutes. Subsequently, 1 mL 0.5% alizarin red staining solution was used to stain the cells at room temperature for 15 minutes. After rinsing with distilled water for 5 minutes, red mineralized nodules were analyzed via charge-coupled device microscope. Absorbance was then measured at 570 nm. Experiments were repeated in triplicate.
ALP staining
A BCIP/NBT alkaline phosphatase color development kit (#C3206, Beyotime, China) was utilized based upon provided directions. Briefly, cells were washed twice by using PBS, after wich 10% formalin was added to fix the cells for 15 minutes. BCIP/NBT substrate was then used to treat cells for 24 h, and colorimetric changes were analyzed using a charge-coupled microscope, with a scanner used to image stained cells. Absorbance was then measured at 405 nm. Experiments were repeated in triplicate.
Mice fracture model and treatment
Male C57BL/6J mice (8-week-old) were obtained as above, with all studies being approved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology. Intraperitoneal pentobarbital sodium (50 mg/kg; Sigma-Aldrich) was used to anesthetize animals, and a model of femoral fracture was then generated via longitudinal incision and blunt separation of the underlying muscles without periosteum removal, as in previous reports28. The femur was then cut with a diamond disk, yielding a mid-diaphysis transverse osteotomy. Next, a 23-gauge intramedullary needle was used to stabilize the fracture site. On day 14 post-fracture, 50% of study animals were euthanized to harvest and analyze calluses, with the remaining mice being analyzed on day 21.
Radiographic images
On days 7, 14, and 21 post injury, all animals were subjected to X rays with an In-Vivo FX PRO imaging system (BRUKER, Karlsruhe, Germany) with a 10 second exposure time.
Micro-computer tomography (Micro-CT) analysis
A BRUKER SkyScan 1276 scanner micro-CT system (BRUKER, Karlsruhe, Germany) was used to image fracture regions in mice (2400 views, 5 frames/view, 37 kV, and 121 mA), with the platform software being used to assess segmentation, three dimensional morphometric analyses, density, and distance parameters (BRUKER, Karlsruhe, Germany). After scanning, preserved calluses were stored at -80℃ prior to further experimental use. Measure parameters, includeding bone volume (BV), total volume (TV), BV/TV, and bone mineral density (BMD), were analyzed by CT Analyser evaluation software (Version: 1.15.4.0) on day 14 and day 21 post-operation. Micro-CT analysis was done in a completely blinded manner with all mice assigned to coded sample numbers.
Therapeutic administration in fracture mice
Control mice received a local fracture site injection of 0.1 mL PBS on days 1, 3, and 7 post-fracture, while other mice were instead administered equivalent volumes of M1D-Exos, M2D-Exos, M2D-ExosantagomiR-5106. At the end of the experiment, bone and callus samples were analyzed as above.
Histological analysis
Following decalcification, 5-7 µm thick paraffin-embedded tissue samples were prepared and subjected to H&E and Alcian blue staining. Sections were then imaged and measured with an Olympus BX51 microscope and a DP73 CCD Olympus Imaging System (Olympus Corporation, Tokyo).
Microarray analysis
RNA sequencing from M1 and M2 macrophages’ supernatant was conducted using a BGISEQ500 platform (BGI). Downstream library was constructed by using RNA samples with RNA integrity number values > 8.0. mRNAs were isolated and reverse transcription with PCR amplification was performed. Then, cDNA was obtained as the final sequencing library, and verified on a bioanalyzer (Agilent 2100). DNA nanoballs were produced by phi29 and the library was amplified, which were loaded into the patterned nanoarray followed by SE50 sequencing. Differentially expressed miRNAs (DEMs)with statistical significance between the two groups were identified by volcano plot filtering and fold change filtering was used to identify DEMs between the two samples. The Gene Cluster 3.0 software (Stanford University) was applied to make hierarchical cluster analysis. Database of annotation, visualization, and integrated discovery (DAVID 6.8; https://david.ncifcrf.gov/) was used to conduct functional analysis. The p value was set to 0.05 to denote the significance of GO enrichment of DEMs.
Statistical analysis
Data are means ± SD, and GraphPad Prism 8.0 (GraphPad Software, CA, USA) was used for all statistical testing. Data were compared via Student’s t-tests and one-way ANOVAs with Tukey’s post-hoc test as appropriate. P < 0.05 was the significance threshold.