Human samples
Samples had been collected from sufferers on the Division of Oral and Maxillofacial Surgical procedure, Hospital of Stomatology, Solar Yat-sen College, Guangzhou, China. After acquiring knowledgeable consent from the sufferers, gingival tissues had been collected throughout the surgical procedure carried out on sufferers with continual periodontitis who required the extraction of free enamel or wholesome members who wanted to extract their third molars. The samples had been divided into an experimental group (sufferers with continual periodontitis) and a management group (systemically wholesome sufferers). The inclusion standards for the experimental group had been: (1) Sufferers aged 18–80 who had been newly recognized with periodontitis based mostly on the 2017 classification phases III and IV decided utilizing scientific attachment loss (CAL) and radiographic bone loss for the prognosis of periodontitis [59]; (2) No systemic therapy inside the final 6 months for continual periodontal illnesses equivalent to subgingival scaling, supragingival scaling, or these on anti-inflammatory medicine; (3) No systemic illness (e.g., most cancers, kidney or liver failure, or coronary heart illness), different acute or continual inflammatory illnesses, or psychological sickness. (4) Capable of signal the knowledgeable consent kind. Topics with one of many following situations weren’t included within the trial: (1) Age underneath 18 years outdated or over 80 years outdated; (2) Sufferers with different systemic illnesses, acute or continual inflammatory illnesses, or psychological diseases; (3) Pregnant or lactating girls; (4) Systemic therapy for continual periodontal illness inside the final 6 months. The inclusion standards for the management group had been as follows: (1) Wholesome topics over 18 years outdated (together with those that had been 18 years outdated); (2) Gender: male or non-pregnant, non-lactating feminine; (3) The load of male topics was not lower than 50 kg, the burden of feminine topics was not lower than 45 kg, and the physique mass index (BMI) of the themes was between 18.0 and 28.0 kg/m2, the place BMI = weight (kg)/top2 (m2), together with the essential worth; (4) No historical past of main illnesses and drug allergic reactions; (5) No smoking or long-term alcohol consumption; (6) Medical and imaging examination confirmed the wholesome periodontal situation and no inflammatory illness. Contributors had been recruited in keeping with a pre-determined minimal pattern measurement utilizing a priori evaluation utilizing a PPD index estimate based mostly on sort I error = 0.05, energy = 0.80, and impact measurement (ES) f = 0.25.
Quantitative real-time PCR
RNAzol reagent (Genecopoeia, USA) was used to extract whole RNA from the samples, and the PrimeScript RT Grasp Combine (Takara, Japan) was used for reverse transcription. Quantitative real-time PCR was carried out with an ABI QuantStudio5 (USA) and the AceQ qPCR SYBR Inexperienced Grasp Combine (Vazyme, China). cDNA of the microRNA was generated utilizing a miRNA cDNA Synthesis Package (CoWin Biosciences, China), and gene expression was measured by qPCR with a miRNA qPCR Assay Package (CoWin Biosciences, China). GAPDH was used as the interior management of the entire RNA, and U6 was the management for microRNA. The primer sequences are proven in Desk S1. The relative amount of genes was calculated utilizing the two−ΔΔCt technique.
Immunohistochemistry (IHC)
The animal samples had been decalcified in ethylene diamine tetra-acetic acid (EDTA, Servicebio, China) for 4 weeks after microcomputed tomography evaluation. The samples had been dehydrated by a graded sequence of alcohol, embedded in paraffin, and reduce into 6-µm sections for staining. The sections had been stained with hematoxylin & eosin (H&E) and tartrate-resistant acid phosphatase (TRAP, Servicebio, China) in keeping with the producer’s protocol. For immunohistochemistry, the sections had been dewaxed, rehydrated, and antigen retrieval was carried out earlier than being incubated with the anti-CD68 or the anti-CD206 (1:200, Servicebio, China) major antibodies in a single day at 4℃, then incubated with the secondary antibodies for 1 h at room temperature. Diaminobenzidine (DAB, Servicebio, China) was used for visualization, adopted by counterstaining with hematoxylin (Servicebio, China). Non-specific IgG was used as a unfavorable management. Then the pattern sections had been imaged with an Aperio AT2 slide scanner (Leica Biosystems, Germany).
MicroRNA scope
To localize miR-126 expression within the tissue sections, a miRNAScope™ HD (RED) Assay Package (Bio-techne, USA) was used in keeping with the producer’s directions and established protocols. Briefly, after dewaxing, hydrogen peroxide blocking, and heat-mediated antigen retrieval utilizing a steamer, Protease III was utilized. After the hybridization probe and Hybridize Amp1-6 had been added dropwise in sequence, diaminobenzidine (DAB, Servicebio, China) was used for visualization of miR-126, and the slides had been then counterstained and mounted. Scrambled miRNA was used as a unfavorable management.
Cell tradition
293T/17 cells [purchased from Procell Life Science&Technology Co., Ltd (China)] had been cultured in Dulbecco’s Modified Eagle Medium, excessive glucose, with Sodium Pyruvate (Biosharp, China) containing 10% fetal bovine serum (FBS, BioInd, Israel) at 37℃ in a 5% CO2 humidified cell tradition incubator.
THP-1 cells [purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd (China)] had been cultured in Roswell Park Memorial Institute Medium (RPMI)-1640 (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA), 1% v/v penicillin/streptomycin (Gibco, USA) and 1% v/v GlutaMAX (Gibco, USA). To acquire M0 macrophages, THP-1 cells had been handled with 10 mg/mL of Phorbol 12-myristate 13-acetate (PMA, Solarbio, China) for twenty-four h. To induce M0 macrophage polarization into M1 macrophages, M0 macrophages had been additional handled with 1 µg/mL LPS (Sigma, USA) and 50 ng/mL IFN-γ (Peprotech, USA) for twenty-four h (Determine S8).
MicroRNA transfection
MiR-126 mimics from two species (miR-126 [Homo sapiens] (Gene ID: 406913) and miR-126 [Rattus norvegicus] (Gene ID: 100314237)) and their unfavorable controls had been bought from Genepharma, China. MiR-126 mimics (Genepharma, China) and unfavorable management (Genepharma, China) had been transfected into THP-1 cells and 29T3/17 cells by the RFect siRNA/miRNA Transfection Reagent (Bio-generating Biotechnology, China) in keeping with the producer’s directions. After transfection for twenty-four h, cells had been cultured for an additional 48 h. In line with preliminary experiments carried out, the ultimate concentrations of transfected miRNAs had been 30 nM (miR-126 mimics) and 30 nM (unfavorable management) (Determine S9), and the effectivity of transfection was verified by qRT-PCR.
Western blot
RIPA Lysis Buffer (Robust, CoWin Biosciences, China) with 1% Protease Inhibitor Cocktail (CoWin Biosciences, China) was used to extract proteins from M1 macrophages. The focus of whole protein was measured utilizing a Bicinchoninic Acid Protein Assay Package (BCA, CoWin Biosciences, China). SDS-PAGE Loading Buffer and the protein samples had been combined and heated in a boiling water bathtub for 3–5 min. Then, the proteins had been resolved by SDS-PAGE (Genscript, China) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) with a moist switch blotting system (Bio-Rad, China). The membranes had been blocked with TBS-Tween (CoWin Biosciences, China) plus 5% bovine serum albumin for 1 h, then had been incubated at 4℃ in a single day with major antibodies: Polyclonal anti-IL 1α (1:500, Affinity Biosciences, China), Polyclonal anti-IL 4 (1:500, Affinity Biosciences, China), Polyclonal anti-IL 6 (1:500, ZENBio, China), Polyclonal anti-IL 10 (1:500, Affinity Biosciences, China), Polyclonal anti-TNFα (1:500, Affinity Biosciences, China), polyclonal rabbit anti-β-Actin (1:1000, Servicebio, China), monoclonal rabbit anti-Phospho-p38 MAPK (1:1000, CST, USA), and monoclonal rabbit anti-p38 MAPK (1:1000, CST, USA), then incubated with secondary antibodies (1:10000) for 1 h at room temperature. Indicators had been detected with the ECL western blotting substrate package (Millipore, USA), and the relative depth of each immunoreactive protein band was quantified with the ImageJ software program (NIH, USA, 1.8.0).
ELISA
The concentrations of IL-1α, IL-4 and IL-10 in cell-culture supernatant had been detected utilizing a commercially out there ELISA package (MMbio, China) in keeping with the producer’s directions and established protocols. Briefly, the ELISA reagents had been added to the samples and incubated at 37℃ for 1 h. After washing the wells 5 occasions with the wash resolution, the chromogenic reagent was added and incubated at 37℃ in the dead of night for 15 min. The response was stopped utilizing the cease resolution. The absorbance (OD worth) was measured at a wavelength of 450 nm utilizing a microplate reader.
Preparation of the CXCR4-overexpressing 29T3/17 cells
293T/17 cells had been cultured as described above. A PCR-amplified gene fragment encoding human CXCR4 (Gene ID: 7852) or rat Cxcr4 (Gene ID: 60628) was cloned right into a lentiviral vector (Genepharma, China). 293T/17 cells (1.5 × 106) had been seeded in 6-cm tradition dishes for 12 h after which transfected with the CXCR4 recombinant lentivirus or the unfavorable management at a multiplicity of an infection (MOI) of fifty for twenty-four h. Within the subsequent 5 days, lentivirus-free medium with 2 µg/mL Puromycin dihydrochloride (Puro, APExBIO, USA) was used for the collection of transfected cells, and the transfected cells had been expanded for additional experiments. The transfection effectivity of CXCR4 within the 293T/17 cells was detected by the GFP fluorescence (Determine S10).
Exosome isolation and characterization
For exosome assortment, 293T/17 cells had been allowed to achieve 80% confluence in DMEM basal medium with out FBS for 48 h, after which, the supernatant was collected and centrifuged at 300×g for 10 min at 4℃ to take away any cells or giant mobile fragments. Differential centrifugation was used to isolate the exosomes: the supernatant was first centrifuged at 2000×g for 20 min at 4℃ then 10,000×g for 30 min at 4℃ to take away microvesicles, and filtered by way of a 0.22 μm filter. The samples had been rigorously transferred to ultracentrifuge tubes (Beckman Coulter, USA) and centrifuged at 110,000×g for 70 min at 4℃. The supernatant was eliminated, leaving a residual quantity of 1 mL containing the exosome pellet within the tubes. After replenishment with filtered PBS, the ultracentrifuge tubes had been centrifuged once more at 110,000×g for 70 min at 4℃. The exosomes had been reconstituted in filtered PBS and saved at -80℃.
A micro BCA Protein Assay Package (CoWin Biosciences, China) was used to measure the focus of exosomes. Western blotting was carried out to substantiate the presence of exosome markers equivalent to CD9, CD63, CD81, TSG101 and HSP70. The exosomes had been added drop-wise on a copper grid and stained with 2% (w/v) phosphotungstic acid for five min. Then the morphology of the exosomes was noticed with a transmission electron microscope (TEM, Tecnai Spirit, FEI, USA). The exosomes had been stained with 10µL Allophycocyanin- (APC) labeled anti-human CXCR4 Antibody (Biolegend, USA) in the dead of night for 20 min, then the particle measurement distribution and CXCR4 expression on the exosome membrane had been examined by Movement NanoAnalyzer (Nanofcm, China).
Exosome labelling and internalization
A PKH26 purple fluorescent dye package (Umibio, China) was used to label the exosomes. Purified exosomes had been incubated in 50µL PKH26 for 10 min at 37℃, washed, and centrifuged at 110,000×g for 70 min at 4℃ to take away unbound dye. Labeled exosomes (10 µg for each 105 cells) had been added to the M1 macrophages, human bone marrow mesenchymal stem cells (HBMSC), human gingival epithelial cells (HGEC) and human umbilical vein endothelial cells (HUVEC) seeded on glass slides and incubated at 37℃ for 12 h. Subsequent, the cells had been fastened in 4% paraformaldehyde (PFA) for 15 min and permeabilized in 0.1% Triton-X in PBS for 15 min. After washing twice with PBS, the samples had been stained with Hoechst 33,342 (Beyotime Biotechnology, China) for 15 min. The uptake of exosomes by completely different cells was noticed and imaged through the use of a laser scanning confocal microscope (Olympus, Japan).
Animal samples
Eight-week-old male Sprague-Dawley (SD) rats weighing 300–400 g had been bought from Solar Yat-sen College. The SD rats had been randomly divided into 4 teams: (1) CXCR4-miR126-Exo; (2) miR126-Exo; (3) Ctrl-Exo; and (4) Clean Ctrl. An experimental periodontitis mannequin in rats that employed the ligation of molars was incessantly utilized in dental analysis to analyze the pathology of periodontitis. Ligatures across the cervical space of the molars in rats allowed for plaque accumulation, periodontal irritation and alveolar bone loss within the periodontal tissues, just like the results noticed in human periodontitis [60,61,62,63]. The periodontitis fashions had been established in SD rats as beforehand described [64], with modifications to make sure profitable mannequin development (Determine S11). Briefly, the rats had been anesthetized with 1% pentobarbital (0.4 mL/100 g) by way of intraperitoneal injection. A 1 mm metallic ligature was wrapped across the neck of the bilateral maxillary second molars for 4 weeks to ascertain continual periodontitis. Plaque Disclosing Resolution (Miradent, Germany) was used to detect biofilm formation across the ligature. The answer was utilized across the rat molars and gently rinsed with physiological saline after 5 minutes. The purple coloring of the enamel indicated plaque biofilm. For exosome therapy, the ligature was eliminated and exosomes (300 µg) from every group or PBS had been injected into the periodontium of the second maxillary molar of every rat. After 2 weeks, the rats had been euthanized to reap their maxillae and gingiva tissues. The samples had been fastened in 4% paraformaldehyde resolution for twenty-four h washed 3 times in PBS after which saved in 75% ethanol for subsequent evaluation.
Micro-CT
The maxillae of SD rats had been positioned in pattern containers and scanned by a micro-CT system (Scanco Medical µCT 50, Switzerland), with the voltage set at 70 kV and electrical present at 114µA. The three-dimensional microstructure was reconstructed and analyzed utilizing the RadiAnt dicom Viewer (Poland). The gap between the cementoenamel junction and the alveolar bone crest (CEJ-ABC distance) was measured at six websites across the maxillary second molars.
Statistical evaluation
All knowledge are introduced as imply ± commonplace deviation (SD). A minimum of three unbiased experiments had been carried out. The GraphPad Prism software program (USA) was used for the statistical analyses. Comparisons between two teams had been carried out utilizing unbiased unpaired two-tailed Scholar’s t-test, and comparisons between greater than two teams had been carried out utilizing one-way evaluation of variance (ANOVA). P values < 0.05 had been thought-about to point statistical significance.
