Synthesis and Characterization of the DSPE-PEG-ALN Conjugate
ALN was bought from Aladdin (Shanghai, China). DSPE-PEG-NHS was ordered from Ruixi Bio-Tech Co, Ltd (Xi’an, China). The conjugating process was in line with earlier publications with slight modification [67]. Briefly, DSPE-PEG-NHS and ALN (at a 1:5 M ratio) have been dissolved in DMSO, after which the pH of the answer was adjusted to eight.2 through the use of triethylamine. The ensuing combination was gently agitated at room temperature for twenty-four h, adopted by being transferred to a dialysis bag (molecular weight cutoff = 3.5 kDa) and dialyzed in opposition to deionized water for 72 h to take away the unconjugated ALN. The obtained product (DSPE-PEG-ALN) was freeze-dried and saved at − 20 ℃ till required. The Fourier rework infrared spectroscopy (FTIR, BrukerOptic Gmbh, Germany), and 1H-nuclear magnetic resonance (H-NMR, Bruker 400 M, USA) spectra have been used to research the modifications in chemical bonding alteration from the preliminary reagents to the ultimate merchandise.
Extraction and ALN modification of PL-exosomes
Human platelet lysates (PL) have been bought from StemEry Hematopoietic Tech Co, Ltd (Fuzhou, China). Protocol for exosome isolation was based mostly on a beforehand described methodology by Torreggiani et al. with delicate emendation [19]. In short, the PL was diluted 5 instances utilizing PBS firstly, adopted by serial low-speed centrifugation (300×g for 10 min, 2000×g for 10 min and 10,000×g for 60 min) in 4 °C to take away cell particles. Then, the supernatant was collected and filtered by a 0.22 µm sterilized filter (Merck-Millipore, Darmstadt, Germany). The filtrate was pelleted by ultracentrifugation (Beckman ultracentrifuge, Beckman Coulter, USA) at 100,000×g for 70 min. The sediment was washed and resuspended in a big quantity of PBS, and ultra-centrifuged once more on the identical excessive velocity for 70 min. The ultimate precipitated PL-exos was fastidiously resuspended in PBS, and the protein content material of exosomes and native PL have been quantified through the use of BCA assay in line with the manufactural directions. Lastly, they have been saved at − 80 °C for subsequent experiments.
Modification of exosomes’ membrane with phospholipid polymer referred to earlier research [68]. The purified DSPE-PEG-ALN was dissolved in HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) buffer for 15 min at 60 °C to kind micelles. Then, the obtained suspension was combined with PL-exo resolution at a 1:1 mass ratio for two h at 40 °C. After cooling to room temperature, exosomes have been instantly purified by size-exclusion chromatography (Exo-spin™, XP Biomed Ltd. Shanghai, China) to get ALN-modified exosomes (PL-exo-ALN). The morphology, measurement distribution and zeta potential of exosomes have been recognized through the use of TEM (Hitachi, Tokyo, Japan), dynamic gentle scattering (DLS) (Beckman delsa, Brea, USA) and Zetasizer (Malvern, UK).
In vitro hydroxyapatite binding assay
Quartz crystal microbalance with dissipation (QCM-D, Q-sense E1, Biolin, Sweden) integrated with hydroxyapatite (HAp) coated sensor (QSX 327, Biolin, Sweden) have been first used to find out the in vitro bone-targeting capacity of exosomes. Briefly, the sensors have been cleaned for 30 min in 99% ethanol, rinsed with deionized water, and uncovered below UV for 10 min. They have been then mounted within the move cells, and PBS was injected for five min (100 µL/min) to permit the sign to be stabilized. Subsequently, a ten µg/mL resolution of PL-exo or PL-exo-ALN in PBS was injected on the move velocity of 20 µL/min for 150 s to allow the movie floor to keep up a correspondence with the exosome resolution. Lastly, the surfaces have been rinsed with PBS for five min to exclude unstable binding of exosomes and to make sure sign stability. The third overtone frequency of the sensor was used to evaluate the exosomes’ disposition on the HAp floor. Modifications within the resonance frequency (Δf) and dissipation (ΔD) have been recorded.
One other HAp binding methodology was additionally used to imitate the binding affinity of Aln functionalized exosomes to bone floor in vitro through the use of DiD (a type of lipophilic fluorochrome) labeled exosomes. Firstly, the PL-exo and PL-exo-ALN options have been incubated with 3 μM DiD for 1 h at room temperature to acquire fluorescent exosomes. Then, they have been incubated with HAp (10 mg/mL) suspensions with gently shaken for five h at 25 °C, adopted by centrifugation at 4000 rpm for 10 min to spin down HAp and exosomes sure to them, the gross photos have been captured by IVIS imaging (Lumina Sequence III, PerkinElmer, USA). And the fluorescence intensities of the supernatants have been measured utilizing fluorimetry (Edinburgh Devices FS920, Ex 474 nm, Em 533 nm). The lower within the depth relative to the preliminary depth suggests the diploma of exosome sure to Hap.
Cell tradition and therapy
Rat bone marrow mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs) have been harvested from the marrow of wholesome 2‐week‐previous Sprague‐Dawley rats in line with our earlier research [69, 70]. BMSCs have been cultured with α‐MEM (HyClone, Shanghai, China) supplemented with 1% penicillin/streptomycin (Gibco, Shanghai, China) and 10% fetal bovine serum (FBS, Gibco, Shanghai, China). EPCs have been cultured in endothelial cell progress media (EGM-2) supplemented with EGMTM-2 MV SingleQuots™ (Lonza, Basel, Switzerland). Each cells have been seeded in 5% CO2 at 37 ℃. All of the procedures have been authorized by the Institutional Ethics Evaluation Committee of Wenzhou Medical College. A excessive dose of dexamethasone (Dex) was utilized to imitate the GC-induced detrimental results on cells. The focus of PL (50 µg/mL), exosomes (50 µg/mL) and Dex (10 μM) have been in line with earlier publications [23].
To guage the impact of PL, PL-exo and PL-exo-ALN on osteoclasts, bone marrow monocytes (BMMs) have been harvested from the marrow of wholesome 6-week-old C57BL/6 mice in line with earlier examine [71]. BMMs have been cultured with α‐MEM (HyClone, Shanghai, China) supplemented with 1% penicillin/streptomycin (Gibco, Shanghai, China), 10% fetal bovine serum (FBS, Gibco, Shanghai, China) and 50 ng/mL M-CSF (Peprotech, NJ, USA). As for the oteoclast differentiation, the BMMs (6 × 103 cells/effectively) have been seeded on 96-well plates and incubated with 50 ng/mL RANKL (Peprotech, NJ, USA) after 24 h. PL (50 µg/mL) and exosomes (50 µg/mL) have been added to the associated medium within the experimental teams, whereas the management group was cultured in α‐MEM full medium containing M-CSF with none addition. The medium was changed each two days till osteoclasts shaped on the sixth day. Cells have been then fastened and visualizated by tartrate-resistant acid phosphatase (TRAcP) staining. TRAcP-positive multinucleated cells that had greater than three nuclei have been counted as osteoclasts. The variety of osteoclasts per effectively was used to guage the impact on the inhibition of osteoclastogenesis.
In vitro and in vivo distribution of exosomes
To guage the internalization of exosomes in vitro, the PL-exo and PL-exo-ALN have been labeled with the DiD firstly as talked about above. Then, BMSCs have been uncovered to the labeled exosomes for 12 h. After that, the cells have been fastened with 4% paraformaldehyde (PFA) for 30 min at room temperature, adopted by permeabilized in 0.1% Triton-X 100 in PBS for five min, and stained cytoskeleton and nucleus with FITC labeled phalloidin (Yeasan, Shanghai, China) and 4′,6-diamidino-2- phenylindole (DAPI, Beyotime Biotechnology, China) respectively. The pictures have been obtained utilizing an invert fluorescence microscope (Olympus, Japan).
The bone focusing on impact of PL-exo-ALN was evaluated in vivo by assessing the biodistribution of the intravenous supply of DiD labeled exosomes. Six feminine Sprague–Dawley rats (200–250 g, 8-week-old) have been obtained from the Animal Middle of the Chinese language Academy of Science (Shanghai, China). The rats have been divided into two teams: PL-exo and PL-exo-ALN (n = 3 in every group). After anesthesia with sodium pentobarbital, the rats have been acquired 100 μg exosomes (dissolved in 200 μL of PBS) by way of tail vein injection. Six hours later, the rats have been sacrificed and the femur together with main organs (femur and coronary heart, liver, spleen, lung, kidney) have been collected for IVIS imaging (Lumina Sequence III, PerkinElmer, USA).
Cell viability assay
Cell counting kit-8 (CCK-8, Dojindo, Japan) was carried out to evaluate the cell viability. A complete of 5000 BMSCs per effectively have been seeded into 96-well plates. One group with out remedies served because the management. The remaining teams have been handled with Dex (10 μM). In the course of the Dex publicity, PL, PL-exo and PL-exo-ALN (50 µg/mL) instantly added to every group. After culturing for 3 days, in every effectively, 90-µL medium and 10-µL CCK-8 resolution have been topped up and incubated at 37 °C for one hour. A microplate reader (Thermo Fisher Scientific, MA, USA) was used to find out the viability of the cells at absorbance 450-nm.
Osteogenic differentiation
The tactic for osteogenic differentiation was relying on a earlier examine [69]. Briefly, the BMSCs (1 × 104 cells/cm2) have been seeded on 24-well plates and incubated with osteogenic induction medium (OM, containing 1 nM dexamethasone, 50 μM L-ascorbic acid-2-phosphate and 20 mM β-glycerophosphate), after reaching 80% frequency. Dex and exosomes have been added to the associated medium within the experimental teams, whereas the management group was cultured in OM with none addition. The medium was changed each two days. Alkaline phosphatase (ALP) exercise was measured after 5 days of tradition by stained with BCIP/NBT ALP Coloration Growth Equipment (Beyotime, Shanghai, China) and quantified with an alkaline phosphatase assay equipment (Beyotime, Shanghai, China) after lysed. The calcium deposits have been evaluated by Alizarin purple staining (Cyagen Biosciences, Guangzhou, China) after 14 days of tradition, and the stained mineralized nodules have been desorbed with 10% cetylpyridinium chloride (Sigma-Aldrich) and the OD worth was measured at 570 nm for quantification.
Western blotting
After therapy, whole cell protein was extracted through the use of RIPA lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF). The nuclear protein extraction was achieved through the use of the nuclear protein and cytoplasmic protein extraction equipment from Thermo Fisher Scientific. The extracts have been lysed on ice for 30 min, adopted by therapy with ultrasound, after which centrifuged at 12,000 rpm for 20 min at 4 °C. After quantified by BCA kits, a complete of 30 μg of the mobile protein was diluted with ddH2O and loading buffer (Beyotime, China) to ascertain a 20 ul pattern system. However for exosome, solely 10 μg exosomal protein is contained in 20 ul pattern system. The samples have been separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) after which blotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). Blocking was carried out for two h utilizing 5% BSA, and subsequent incubation of the membranes along with a main antibody in opposition to Col-I (1:1000, abcam, USA), Runx-2 (1:1000, Bioworld, USA), OCN (1:500, santa, USA), p-PDGFRβ1(1:1000, Cell Signaling Expertise, USA), PDGFRβ1(1:1000, Cell Signaling Expertise, USA), p-FAK(1:1000, abcam, USA), FAK(1:1000, abcam, USA), PDGF-BB(1:1000, LifeSpan BioSciences, USA), VEGF-A(1:1000, abcam, USA), BMP-2(1:1000, abcam, USA), OPG(1:1000, abcam, USA) and GAPDH (1:10,000, abcam, USA) and exosomal markers CD9 (1:1000; Abcam, Cambridge, UK), CD81 (1:1000; Abcam), TSG101 (1:1000; Santa Cruz, Dallas, USA), the platelet marker CD41 (1:1000; Santa Cruz) and cytoplasm marker Calnexin (1:1000; Santa Cruz) at 4℃ in a single day. The subsequent day, the membrane was incubated for two h with the respective secondary antibodies at room temperature. After being washed 3 times utilizing TBST, the blots have been then developed utilizing the Bio-rad ChemiDoc MP (Bio-Rad, USA). Lastly, band depth was evaluated utilizing picture lab 3.0 software program (Bio-Rad, USA). The information have been normalized and introduced because the fold change of the protein degree of the therapy group in comparison with the management group.
Immunofluorescence
The cells have been plated on crystal 6-well slice on the density of 5 × 105/ml within the tradition plates, and handled with Dex and completely different teams of exosomes for twenty-four h. The samples have been washed thrice in PBS, fastened in 4% paraformaldehyde, and permeabilized for 15 min with 0.1% TritonX-100 in PBS. The 5% bovine serum albumin was used to dam cells at 37 °C for 1 h, adopted by washing with PBS, and culturing with main antibodies in opposition to Col-I (1:200, abcam, USA), or in a single day at 4 °C. The TRITC Phalloidin, Alexa Fluor®488-labeled secondary antibodies (1:400, abcam, USA) and DAPI resolution (1:10) have been added in sequence for 20 min, 1 h and 5 min at room temperature respectively, adopted by 3 instances rinsing in PBS. The pictures have been captured by an inverted fluorescence microscope (Olympus, Japan). As for histofluorescence, the sections have been deparaffinized and hydrated and incubated with a combination of EMCN (1:200, Invitrogen, USA) and CD31(1:200, Invitrogen, USA). After washed with PBS, sections have been incubated with secondary antibodies: Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated (1:200, Abcam) at room temperature for 30 min. The nuclei have been stained with DAPI resolution. The pictures have been captured by an inverted fluorescence microscope (Olympus, Japan).
In vitro tube formation
The tubule formation capacity of EPCs after remedies was examined by transferring the cells seeding on Matrigel™ (BD Bioscience). In short, a 96-well plate was coated with 50 μL of chilly Matrigel per effectively and gelatinized at 37 °C for 30 min. Then, EPCs suspension of two.0 × 104 cells/mL have been digested and seeded on the matrix. After 6 h of incubation, the variety of full capillaries and nodes of every gap have been calculated.
Migration assay
To measure the migration potential of EPCs after completely different remedies, a transwell assay was used. The cells have been seeded within the higher chambers of a 24-well transwell plate (Corning, USA) at a density of 5.0 × 104 cells/mL. Serum-free mediums have been added to the decrease chambers. And Dex and completely different teams of PL or PL-exosomes have been added to serum-free medium. After 24 h of incubation, the cells on the higher floor of the transwell membrane have been gently wiped with a cotton swab, and cells on the decrease floor have been fastened with 4% paraformaldehyde and stained for 10 min with 0.5% crystal violet. Lastly, 3 random decrease surfaces of every filter have been chosen and counted twice.
Preparation of conditioned media from BMSCs and EPCs
BMSCs or EPCs have been handled with PL, PL-exo, or PL-exo-ALN for twenty-four h or not with out therapy, then the serum-containing conditioned media (CM) have been collected. After centrifugation (2500 rpm for 10 min at 4 °C), we obtained the supernatants and saved them at -80 °C for downstream experiments. After seeding the BMSCs or EPCs, we modified to the corresponding CMs and added Dex (10 μM) for tradition. As for the migration experiment, we additionally harvested several types of serum-free CM from BMSCs after 24 h of the above remedies. After inoculating EPCs within the higher chambers of a 24-well transwell plate, Dex (10 μM) and corresponding serum-free CMs have been added to the decrease chambers.
Animal mannequin
All procedures adopted in our examine for animal care and use complied with the Guides for the Care and Use of Laboratory Animals of the Nationwide Institutes of Well being and was authorized by the Animal Care and Use Committee of Wenzhou Medical College. A complete forty male Sprague–Dawley rats (220–250 g, 6-week-old) have been supplied by the Animal Middle of the Chinese language Academy of Science (Shanghai, China). The rats have been randomly divided into 5 teams: Management, MPS, MPS + PL, MPS + PL-exo, MPS + PL-exo-ALN (n = 8 in every group) and housed in commonplace temperature situations with a 12-h gentle/darkish cycle and usually fed with meals and water. The institution of GIOP mannequin was in line with earlier research [72]. Rats within the MPS-related teams have been acquired an intramuscular injection of methylprednisolone (MPS) (30 mg/kg/day in 0.9% NaCl resolution) for 60 days, whereas a management group was injected every day with equal quantity car (saline). After 3 weeks injection of MPS, the therapy teams have been intravenously administrated with PL, PL-exo, PL-exo-ALN (100 μg in PBS) as soon as per week. In distinction, the MPS teams acquired equal quantity PBS therapy solely. The injection dose and frequency of PL and PL-exo are carried out by referring to related literature [23, 73, 74].
To watch dynamic bone formation and mineralization, 5 rats of every group have been intraperitoneally injected with 30 mg/kg alizarin purple S (Macklin, Shanghai, China) and 10 mg/kg calcein (Macklin, Shanghai, China) at week 3 and 6 in the course of the experiment. On the finish of the therapy interval, these 5 rats have been humanely sacrificed and femurs have been collected for micro-CT scanning. Then, the best femurs have been used for undecalcified histological examination, whereas the left femurs have been decalcified in 10% EDTA for hematoxylin and eosin (H&E), tartrate‐resistant acid phosphatase (TRAcP) and immunohistological staining. Then again, the remainder three rats in every group have been anaesthetized and perfused with Microfil to evaluate intraosseous vessels. Then, the femurs have been collected and decalcified with 10% EDTA for 2 months. After micro-CT scanning, femurs have been paraffin-embedded for immunofluorescence staining. The investigators weren’t blinded to allocation throughout experiments and end result evaluation. Pattern sizes have been chosen on the idea of earlier experiments. No animals have been excluded from evaluation.
Micro-CT scanning
Micro-CT scanning and corresponding evaluation for femurs have been carried out utilizing SkyScan1178 system and bundled software program (Bruker MicroCT, Kontich, Belgium). The 14-micron voxel measurement of photos have been acquired with a set at 35 kV of power and 220 mA of depth. 2D and 3D reconstructions have been carried out utilizing DataViewer and CTVox software program respectively. We selected the trabecular bone on the metaphysis of the distal femur under the expansion plate as a area of curiosity (ROI). The bone mineral density (BMD), trabecular bone quantity fraction (BV/TV), trabecular quantity (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) have been calculated through the use of CTAn software program.
Histology
For undecalcified sections, after embedding in resin, specimens have been sawn coronally into 100-μm thickness, fastened on plastic slide, and punctiliously grinded and polished into about 50-μm thickness. The immunofluorescent alerts about dynamic bone formation have been captured utilizing a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany). The later Von Kossa staining was carried out by immersing slices into 5% silver nitrate for 3 h in 60 ℃ oven and cleaned with sodium thiosulfate resolution. The pictures have been recorded utilizing microscope (Olympus, Japan).
For decalcified specimens, after being dehydrated utilizing an alcohol gradient, cleared, and embedded in paraffin, the tissues have been minimize into 5-μm-thick sections. H&E and TRAcP have been carried out as described beforehand [75]. The osteoclast floor per bone floor (Oc.S/BS) have been calculated utilizing BIOQUANT OSTEO 2011 software program in line with a way proposed by Sawyer et al. [76]. For immunohistochemical (IHC) staining, sections have been deparaffinized, antigen retrieved, blocked and incubated with main antibodies of COL I (1:200, abcam, USA), OCN (1:100, santa, USA) and VEGF (1:100, abcam, USA) and related biotinylated secondary antibodies. Lastly, sections have been stained with DAB and counterstained with hematoxylin. The sunshine photos have been captured utilizing microscope (Olympus, Japan). Semi-quantitative evaluation was carried out utilizing Picture-Professional Plus software program model 6.0 (Media Cybernetics, Rockville, MD, USA).
Micro-fil perfusion
Three rats of every group have been utilized for Micro-fil perfusion to evaluate intraosseous vessels, in line with earlier publications [23, 77]. After anesthesia, opened the thoracic and belly cavity to reveal and dissect the belly aorta and vein and the proximal aorta. After ligating the proximal aorta and chopping off the belly vein, a needle was inserted into the belly aorta. And the vasculature was flushed with heparinized saline (0.9% regular saline containing 100 U/ml heparin sodium), 4% paraformaldehyde (PFA) was then injected for fixation. After the decrease limbs have been fastened and stiff, Micro-fil (MV-122, Carver, MA, USA) have been injected into the belly aorta till a continuing outflow was proven within the belly vein. About 25 ml Micro-fil was consumed per rat to make sure ample filling of intraosseous vessels. The rats have been saved at 4 °C in a single day to make sure polymerization. Then, the bilateral femurs have been eliminated, fastened and decalcified with 10% EDTA resolution for two months. Lastly, inside femoral vessels have been imaged and analyzed by Micro-CT. The scanner was set at a decision of 9 μm per pixel. The 3D photos of the vasculature have been reconstructed utilizing CTVox software program. The overall vessel quantity was calculated utilizing CTAn software program.
ELISA assay
ELISA kits detecting the extent of bFGF, PDGF-AB, VEGF, TGF-β1 and PDGF-BB have been obtained from R&D methods. Assays have been carried out in line with the producer’s directions. Absorbance at 450 nm within the ELISA assays was detected on a microplate reader (Thermo Fisher Scientific, MA, USA).
Statistical evaluation
No less than three impartial replicates have been carried out. The information are introduced because the imply ± commonplace deviation (SD). We used unpaired, two-tailed Pupil’s t-tests for comparisons between two teams and one-way evaluation of variance (ANOVA) with Tukey’s a number of comparisons take a look at for a number of comparisons. All statistical analyses have been carried out by Prism software program model 8.0 (GraphPad, San Diego, CA, USA). A P worth < 0.05 was thought-about statistically important. Energy evaluation was carried out by G*energy software program (Model 3.1).
