Mice
Feminine C57BL/6 mice (JAX #000664), obtained from the Jackson Laboratory, have been used on this research. All procedures involving animal topics have been carried out in strict accordance with the accredited tips and underneath the protocols sanctioned by the Institutional Animal Care and Use Committee (IACUC) of the College of Pennsylvania (#805478).
Antibodies
Anti-mouse cleaved caspase-3 (#9661), Anti-mouse Runx2 (#8486), Anti-mouse phospho-p44/42 MAPK (Erk1/2) (#4370), Anti-mouse p44/42 MAPK (Erk1/2) (#9102), Anti-mouse PI3 Kinase p110 (#4249), Anti-mouse PI3 Kinase p85 (#4292), Anti-mouse NF-κB p65 (#8242), Anti-mouse phospho-NF-κB p65 (#3033) antibodies have been bought from Cell Signaling Know-how (Danvers, MA, USA) and diluted to 1:1000 earlier than utilizing. Anti-mouse Integrin alpha 5 antibody (#EPR7854), Anti-mouse Calreticulin (#EPR3924), Anti-mouse Calnexin (#ab22595), Anti-mouse ALP (#ab229126), Anti-mouse phospho-mTOR (#ab109268), Anti-mouse mTOR (#ab134903). Anti-mouse TGF-β Receptor I (#ab235578), Anti-mouse TGF-β Receptor II (#ab259360) antibodies have been bought from Abcam (Danvers, MA, USA) and diluted to 1:300 or 1:1000 (anti-mouse ALP antibody) earlier than utilizing. Anti-mouse ALP (#sc-28904) and anti-mouse PPAR-γ (#sc-7273) antibodies have been bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and diluted to 1:500 earlier than utilizing. Anti-mouse LPL (#SAB5702421), Anti-mouse β-actin (#A1978), Anti-mouse phospho-Smad2/3 (#SAB4504208), Anti-mouse Smad2/3 (#07-408), Anti-mouse DKK1 (#WH0022943M1) antibodies have been bought from Sigma-Aldrich (St. Louis, MO, USA) and diluted to 1:1000 earlier than utilizing. Anti-mouse active-β-catenin (#05665) and anti-mouse β-catenin (#06-734) antibodies have been bought from EMD Millipore (Billerica, MA, USA) and diluted to 1:1000 earlier than utilizing. Antibodies utilized in move cytometry have been all bought from Biolegend (San Diego, CA, USA) and diluted to totally different concentrations in keeping with the producer’s directions earlier than utilizing.
Institution of animal mannequin of postmenopausal osteoporosis
8-weeks-old C57BL/6 mice have been intraperitoneally injected with 10Â mg/kg physique weight of ketamine for anesthesia. After anesthesia, an ophthalmic scissors have been used to make a longitudinal incision of about 1.5Â cm on the middle of again, then bluntly separated the subcutaneous tissue, and sequentially lower the subcutaneous fascia and muscle layer of the facet stomach. Ovarian was pulled out and electrocautery was used to cease bleeding. Then the tissues have been reset, the muscle layer and epidermal layer have been sutured in sequence. After the mice recovered from train, they have been positioned again within the cage and saved underneath common care. After 8 weeks of operation, X-ray scan of femoral was carried out to validate the success of modeling.
Isolation of mouse BMMSCs
BMMSCs have been remoted from the limbs of C57BL/6 mice, and adherent cells have been cultured for 2 passages to complement bone marrow mesenchymal stem cells (BMMSCs) in keeping with the strategy beforehand described [27, 101]. Briefly, a suspension containing 1.5 × 107 nuclear cells from bone marrow was ready and seeded onto 10 cm tradition dishes. These dishes have been positioned in an incubator at 37 °C with a 5% CO2 environment. After 48 h, non-adherent cells have been eliminated, leaving the adherent cells, which have been subsequently cultured for a further 16 d. The tradition medium used was alpha Minimal Important Medium (α-MEM) provided by Invitrogen, USA, enhanced with 20% fetal bovine serum (FBS, Gemini Bio, USA), 2 mM L-glutamine, 55 µM 2-mercaptoethanol, and a mix of 100 U/mL penicillin and 100 µg/mL streptomycin, all sourced from Invitrogen (USA). To attenuate hematopoietic cell contamination, the adherent cell colonies have been passaged as soon as, with frequent medium adjustments. For experimental functions, MSCs from the third to fifth passages have been utilized.
Induction of MSCs apoptosis and isolation of apoVs
MSCs have been induced to bear apoptosis utilizing a modified protocol primarily based on our earlier report [102]. Upon reaching full confluency, the MSCs have been handled with a fundamental medium devoid of FBS, however containing 500 nM staurosporine (STS, Enzo Life Sciences, USA) for 16Â h. Put up-treatment, the cells have been examined underneath a microscope. For the evaluation of morphological adjustments and the expression of apoptosis-related markers, the cells underwent immunofluorescent co-staining using cleaved caspase-3 (Cell Signaling Know-how, USA) and Hoechst 33,342 (Sigma-Aldrich, USA).
Subsequently, apoVs have been remoted utilizing an optimized gradient centrifugation methodology, detailed in Supplementary Fig. 2. The process commenced with sequential centrifugation: initially at 800 g for 10 min, adopted by 2000 g for an additional 10 min, each carried out at 4 °C. These steps have been designed to take away apoptotic cell particles. Subsequently, the supernatant, which contained EVs, was collected. To isolate apoVs, this supernatant was additional centrifuged at 16,000 g for 30 min at 4 °C. The ensuing apoVs have been then washed as soon as with PBS and resuspended in both PBS or recent tradition medium for subsequent experimental functions.
Characterization of MSCs derived apoVs
Transmission electron microscopy (TEM) was employed to watch the morphology of apoVs. ApoV pellets, obtained post-centrifugation, have been mounted in 1% glutaraldehyde (Sigma-Aldrich, USA) for 30 min at 4 °C. Then, 10 µL of the apoVs pattern was positioned on carbon-coated formvar copper grids (ProSciTech, Australia) at room temperature. After a minute, the samples have been stained with 2.5% uranyl acetate (Electron Microscopy Sciences, USA) for 3 min. Photos of the apoVs have been captured utilizing a JEM-1200EX electron microscope (JEOL, Japan).
For nanoparticle monitoring evaluation (NTA), the apoVs have been initially diluted in PBS and additional diluted with ultrapure water at a ratio of 1:1000. The focus, particle dimension, and zeta potential of the apoVs have been measured using a ZetaView PMX120 (Particle Metrix, Germany). The obtained knowledge have been analyzed with the ZetaView software program (model 8.02.31).
For immunofluorescent staining of apoVs, the vesicles have been initially mounted in 2% paraformaldehyde (PFA) for 30 min and blocked using 5% BSA for 1 h. The apoVs have been then incubated in a single day at 4 °C with main antibodies focusing on integrin alpha-5, calreticulin, calnexin, and Annexin V (all sourced from Cell Signaling Know-how, USA). Put up-incubation, the apoVs have been washed with PBS and centrifuged at 16,000 g for 30 min. The apoVs pellets have been subsequently incubated for 1 h with an Alexa Fluor 488-conjugated secondary antibody (Invitrogen, USA). After a remaining wash and centrifugation, the apoVs have been counterstained for cytomembranes with CellMask. Imaging was carried out utilizing a Zeiss Elyra 7 with Lattice SIM.
For nano move cytometry (nFCM) detection, freshly remoted apoVs suspended in PBS have been incubated with antibodies towards integrin alpha-5, calreticulin, and calnexin. Following a beforehand established protocol, this step was performed to investigate the floor molecules [103]. The usual curve and acquisition parameters, together with detector achieve, laser energy, and detection threshold, have been arrange. The apoVs have been then launched into the nFCM instrument (NanoFCM, Xiamen, China). Evaluation of the full quantity and the proportion of positively stained particles was carried out utilizing the NanoFCM software program (NanoFCM Skilled Suite V1.15).
In vitro apoVs stimulation and systemic infusion of MSCs
Cells have been plated at a density of 6 × 105 cells per properly in a 6-well plate. As soon as the cells have been adherent, the medium was changed with full α-MEM supplemented with apoptotic vesicles (apoVs), sustaining a cells-to-apoVs ratio of 1:50 as talked about in our earlier research [27], and this ratio holds true for all in vitro experiments. The cells have been then incubated at 37 °C in an environment containing 5% CO2 for 72 h. Following this incubation, MSCs, each with and with out in vitro stimulation by apoVs, have been harvested, resuspended in PBS at a focus of 4 × 106 cells per 200 µL, getting ready for the following in vivo administration. For in vivo experiments, MSCs (4 × 106 apoVs in 200 µL PBS) have been administered intravenously through the tail vein on a weekly foundation. After a four-week remedy interval, the mice have been euthanized for additional evaluation as talked about earlier than [27]. In accordance with our earlier research [104], 5 mice have been ready for every group and any three of them have been randomly chosen for statistical evaluation.
X-ray scanning
The femurs have been excised from mice after which mounted in 4% PFA. The mounted bones have been immersed in PBS for a day to take away residual PFA. Following this, the femurs underwent a dehydration course of utilizing a graded collection of ethanol. Any extra tendon and cartilage tissues have been rigorously trimmed utilizing ophthalmic scissors. This trimming was important to make sure secure positioning of the femurs within the X-ray scanner (Carestream Well being, Canada) for subsequent radiographic scanning and imaging.
Micro CT evaluation
The preparation of femur specimens adopted the beforehand talked about pretreatment procedures. The femurs have been imaged and analyzed utilizing a high-resolution Scanco µCT35 scanner (Scanco Medical AG, Switzerland), as detailed in our prior research [27]. Briefly, the specimens specializing in the metaphysis of femurs have been scanned using a voxel dimension of 20 μm at 200 µA and 70 kVp. A area of curiosity, particularly focusing on the trabecular bone, was manually outlined close to the endocortical floor. The scanned knowledge have been then reconstructed utilizing Scanco software program. For picture reconstruction, datasets have been imported into Amira 5.3.1 software program (Visage Imaging, Germany). Parameters such because the bone quantity to whole quantity ratio (BV/TV) and bone mineral density (BMD) have been additionally computed.
Hematoxylin and eosin (H&E) staining
Femurs from every experimental group have been harvested and initially mounted in 4% PFA. Following fixation, the bones have been decalcified utilizing 5% EDTA. The decalcified femurs have been then embedded in paraffin and sectioned into slices with a thickness of 6 μm. These sections have been stained utilizing hematoxylin and eosin (H&E) for histological examination. The stained sections have been analyzed with a concentrate on trabecular bone utilizing ImageJ software program (Nationwide Institutes of Well being, USA).
Enzyme-linked immunosorbent (ELISA) assay
Peripheral blood serum was collected from mice and analyzed for the expression ranges of TNF-α, IFN-γ, and IL-17 proteins utilizing the Mouse ELISA Prepared-SET-GO kits (eBioscience, USA). Equally, the supernatant from the MSC tradition medium was additionally collected and assessed for TGF-β1 expression utilizing the identical Mouse ELISA Prepared-SET-GO equipment (eBioscience, USA). Particular protocol was referred to literature [105].
Tradition of mouse splenocytes and T lymphocytes
Splenocytes have been remoted from mouse spleens by finely grinding the tissue till no discernible splenic materials remained after which straining the resultant suspension via a 70 nm cell strainer. Subsequently, ACK lysis buffer (Lonza Bioscience, Switzerland) was added for five min at room temperature to lyse pink blood cells. This was adopted by centrifugation to separate the splenocytes. Naive CD25−CD4+ T cells have been then remoted from the splenocytes utilizing magnetic cell sorting (MACS) know-how (Miltenyi Biotec, Germany). For cell tradition, the remoted T cells have been maintained in RPMI 1640 medium (Lonza Bioscience, Switzerland) supplemented with 10% FBS (Sigma-Aldrich, USA), 10 mM HEPES (Lonza Bioscience, Switzerland), 1 mM sodium pyruvate (Gibco, USA), 55 µM 2-mercaptoethanol, Non-Important Amino Acids (Gibco, USA), and 100 U/mL Penicillin and Streptomycin (Gibco, USA). T cell activation was achieved utilizing plate-bound anti-mouse CD3 antibody (BioLegend, USA) and soluble anti-mouse CD28 antibody (BioLegend, USA), each at a focus of two µg/mL.
Th1/Th17 subsets and Treg cells differentiation
To induce differentiation into Th1/Th17 subsets and Treg cells, naive CD25−CD4+ T cells have been stimulated with anti-CD3/CD28 antibodies in a polarizing atmosphere, as described beforehand [106]. The particular circumstances for every subset have been as follows, with a period of three days for the differentiation course of:
Th1 Polarization: The medium was supplemented with IL-12 (20 ng/mL, R&D Programs, USA) and anti-IL-4 neutralizing antibodies (10 µg/mL, BioLegend, USA).
Th17 Polarization: The cells have been cultured within the presence of IL-6 (50 ng/mL, R&D Programs, USA), TGF-β1 (2 ng/mL, R&D Programs), together with each anti-IFN-γ (10 µg/mL, BioLegend, USA) and anti-IL-4 neutralizing antibodies (10 µg/mL, BioLegend, USA).
Treg Polarization: This required TGF-β1 (2 mg/mL; R&D Programs, USA), IL-2 (2 mg/mL, R&D Programs, USA), and the identical concentrations of anti-IFN-γ and anti-IL-4 neutralizing antibodies as used for Th17 polarization.
Move cytometry
For the characterization of MSCs, the remoted cells have been incubated with a panel of antibodies: CD34, CD45, CD73, CD90, CD105, CD126, CD166, and Sca-1 (all from BioLegend, USA). This incubation was carried out at the hours of darkness at 4℃ for 30 min. Following incubation, the cells have been washed with a washing answer to take away any residual antibodies. They have been then resuspended and stuck in PBS containing 2% PFA. The samples have been subsequently analyzed utilizing a FACS Calibur Move Cytometer (BD Biosciences, USA) to find out the chances of constructive expression for the aforementioned markers. Knowledge evaluation was carried out utilizing FlowJo v10 software program (FlowJo LLC, USA).
To guage apoptosis in MSCs, together with these stimulated with STS, the cells have been first suspended in a binding buffer. They have been then incubated with Annexin V (BD Pharmingen, USA) and 7-AAD (BioLegend, USA) for 15Â min at the hours of darkness at room temperature. To halt the response, an equal quantity of binding buffer was added. Apoptosis charges have been then assessed utilizing a FACS Calibur Move Cytometer (BD Biosciences, USA). The gathered knowledge have been analyzed with FlowJo v10 software program (FlowJo LLC, USA).
For the evaluation of Th1/Th17 subsets and Treg cells, cells have been harvested and first incubated with CD4 and CD25 antibodies (BioLegend, USA) for 45 min on ice in darkish circumstances. Subsequently, the cells have been stained with particular antibodies: IFN-γ for Th1, IL-17 for Th17, or Foxp3 for Treg cells (all from BioLegend, USA). For cell fixation and permeabilization, a Foxp3 Staining Buffer Equipment (BioLegend, USA) was utilized. The stained cells have been then analyzed utilizing a FACS Calibur Move Cytometer (BD Biosciences, USA). The acquired knowledge have been processed and analyzed with FlowJo v10 software program (FlowJo LLC, USA).
Cell proliferation assay
MSCs have been plated on chamber slides at a density of 1 × 104 cells per properly and cultured for 3 d. Subsequently, the cells have been incubated with a BrdU answer (1:100, Invitrogen, USA) for 20 h. After incubation, BrdU staining was carried out utilizing the BrdU Staining Equipment (Invitrogen, USA). The proportion of BrdU-positive cells was calculated as a proportion of the full cell rely primarily based on eight totally different picture fields per pattern. This assay was replicated in 5 unbiased samples for every experimental group.
Inhabitants doubling (PD) assay
Single colony cluster P0 cells, at their first passage, have been seeded at a density of 5 × 105 cells on a 60 mm dish (Corning, USA). Upon reaching confluence, the cells have been harvested and reseeded on the identical density. The PD quantity was calculated utilizing the formulation PD = log2 (variety of harvested cells/variety of seeded cells). This calculation was primarily based on the cumulative whole of cells obtained from every passage, persevering with till the cells ceased dividing. To make sure reliability, this assay was carried out with 5 independently remoted cell samples for every experimental group.
Osteogenic differentiation of MSCs
MSCs have been cultured in an osteogenic inductive medium, which comprised 100 µM L-ascorbic acid 2-phosphate, 2 mM β-glycerophosphate, and 10 nM dexamethasone (all sourced from Sigma-Aldrich, USA). Ten days after induction, whole proteins have been extracted from the cells, and the expression of osteogenic-associated proteins was assessed through western blot evaluation. Moreover, at 21 d post-induction, the cells underwent staining with 1% Alizarin Pink-S (Sigma-Aldrich, USA) to detect mineralized nodules. The extent of mineralization was quantified utilizing Picture J software program (NIH, USA), with the outcomes offered as the proportion of the full space coated by the constructive staining.
Adipogenic differentiation of MSCs
MSCs have been cultured in an adipogenic inductive medium composed of 500 nM hydrocortisone, 60 µM indomethacin, 500 nM isobutylmethylxanthine, 10 µg/mL insulin, and 100 nM L-ascorbic acid phosphate, all obtained from Sigma-Aldrich, USA. Seven days following the induction, whole proteins have been extracted from the cells for the aim of detecting adipogenic-associated proteins through western blot evaluation. Moreover, 21 d post-induction, the cells have been stained with Oil Pink O (Sigma-Aldrich, USA) to visualise lipid droplets, indicative of adipogenic differentiation. The positively stained cells have been noticed underneath a microscope, and the outcomes have been quantified because the proportion of oil-red O-positive cells relative to the full cell rely.
Western blot evaluation
Whole protein was extracted from the cells utilizing the M-PER Mammalian Protein Extraction Reagent (ThermoFisher, USA). The protein concentrations have been then decided utilizing a BCA Protein Assay Equipment (ThermoFisher, USA). For the western blot evaluation, 20 µg of the extracted proteins have been separated on NuPAGE BT gels (Invitrogen, USA) and subsequently transferred onto ImmobilonTM-P PVDF membranes (Millipore, USA). The membranes have been blocked with an answer containing 0.1% Tween 20 and 5% non-fat milk for 1 h. They have been then incubated in a single day at 4 °C with main antibodies at dilutions starting from 1:200 to 1:1000. These antibodies focused varied proteins, together with ALP, PPAR-γ, LPL, phospho-Smad2/3 (Santa Cruz, USA), Dickkopf 1 (Sigma-Aldrich, USA), lively β-catenin, β-catenin (Millipore, USA), phospho-ERK, ERK (ThermoFisher, USA), Smad 2/3, phospho-mTOR, mTOR, p85, p110, phospho-p65, p65 (Cell Signaling Know-how, USA), Runx2, β-actin, TGF-β receptor 1, and TGF-β receptor 2 (Abcam, USA). After main antibody incubation, horseradish peroxidase-conjugated IgG (1:10000 dilution, Santa Cruz, USA) was utilized for 1 h. Detection was enhanced utilizing a SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher, USA). The bands have been visualized on movies (Bioland, USA) and analyzed with Picture J software program (NIH, USA). β-actin was used as a loading management to quantify the quantity of protein loaded.
Induction of T cell apoptosis by MSCs
T cells have been activated as described in Sect. 4.10. Subsequently, activated T cells (1 × 106 cells per properly) have been cocultured with MSCs (0.2 × 106 cells per properly) in a 24-well plate utilizing a T cell tradition medium. After a 3-day coculture interval, the cells within the supernatant have been collected. The apoptosis of T cells was then evaluated by figuring out cells constructive for each Annexin V and 7-AAD utilizing a FACS Calibur Move Cytometer (BD Biosciences, USA).
Actual-time polymerase chain response (PCR)
Whole RNA, inclusive of small RNA, was extracted from the cells utilizing the miRNeasy Mini Equipment (Qiagen, Germany). The quantification and purity evaluation of the extracted whole RNA have been carried out utilizing a NanoDrop ND2000 (ThermoFisher, USA). For cDNA synthesis, the miScript II RT Equipment (Qiagen, Germany) was employed. The expression of miR-93-5p, miR-145a-5p, miR-294-3p, and pri-miR-145 was then quantified via real-time PCR, using the miScript SYBR Inexperienced PCR Equipment (Qiagen, Germany). This process included the usage of the miScript Primer Assay and miScript Precursor Assay (Qiagen, Germany), which integrated particular primers designed primarily based on the newest sequences in miRBase. GAPDH and snRNA U6 have been used as endogenous controls for pri-miRNA and mature miRNA expression in MSCs for normalization. The actual-time PCR detection was carried out on a CFX96™ Actual-Time PCR System (Bio-Rad), and gene expression was calculated utilizing the efficiency-corrected ΔΔCt methodology.
MicroRNA mimics and inhibitors transfection
miR-145a-5p mimics, inhibitors, and corresponding unfavorable controls have been bought from Genecopoeia, United States. miR-145a-5p mimics or miR-145a-5p mimics unfavorable management at remaining focus of 5 nM for over expression of miR-145a-5p have been transfected into OVX MSCs. And miR-145a-5p inhibitor or miR-145a-5p inhibitor unfavorable management at remaining focus of 5 nM for depletion of the miR-145a-5p expression have been transfected into WT MSCs. The transfection course of was carried out utilizing Lipofectamine RNAiMAX (Invitrogen, United States) in keeping with the producer’s directions. Particularly, sooner or later earlier than transfection, plate cells in 500 µL of progress medium in order that the cells will likely be 50–80% confluent on the time of transfection. For every transfection pattern, put together complexes as follows: Dilute the optimized quantity of plasmid RNA in 100 µL Opti-MEM® I Decreased Serum Medium. Combine completely. Provided that utilizing PLUS™ Reagent: Combine PLUS™ Reagent gently earlier than use, add the optimized quantity of PLUS™ Reagent on to the diluted RNA. Combine gently and incubate for five min at room temperature. Combine Lipofectamine® LTX gently earlier than use and add the optimized quantity on to the diluted RNA. Combine completely. Incubate for 30 min at room temperature. RNA-lipid complexes are secure for six h at room temperature. Add 100 µL RNA-lipid advanced dropwise to the properly containing cells. Combine gently by rocking the plate backwards and forwards. Incubate the cells at 37 °C in a CO2 incubator for 18–48 h previous to testing for transgene expression. Medium could also be modified after 4–6 h.
Statistics
Knowledge are offered as means ± commonplace deviation (SD), primarily based on a minimal of three unbiased measurements, until specified in any other case. Statistical analyses have been carried out utilizing GraphPad Prism 9 software program (GraphPad, USA). Normality assessments have been carried out earlier than deciding on the statistics methodology. For evaluating knowledge between two teams, unbiased unpaired two-tailed Pupil’s t-test was employed. For a number of group comparisons, one-way evaluation of variance (ANOVA) with Tukey’s submit hoc evaluation was utilized when knowledge have been regular distribution, and Kruskal-Wallis check was utilized when knowledge have been non-normal distribution. A p-value of lower than 0.05 was thought-about statistically important.
