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 based on earlier publications with slight modification [67]. Briefly, DSPE-PEG-NHS and ALN (at a 1:5 M ratio) had 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 towards 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 had been used to research the adjustments in chemical bonding alteration from the preliminary reagents to the ultimate merchandise.

Extraction and ALN modification of PL-exosomes

Human platelet lysates (PL) had been bought from StemEry Hematopoietic Tech Co, Ltd (Fuzhou, China). Protocol for exosome isolation was primarily based on a beforehand described technique 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 way of 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 similar 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 had been quantified through the use of BCA assay based on the manufactural directions. Lastly, they had 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 blended with PL-exo answer at a 1:1 mass ratio for two h at 40 °C. After cooling to room temperature, exosomes had 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 had been recognized through the use of TEM (Hitachi, Tokyo, Japan), dynamic mild 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) included with hydroxyapatite (HAp) coated sensor (QSX 327, Biolin, Sweden) had been first used to find out the in vitro bone-targeting potential of exosomes. Briefly, the sensors had been cleaned for 30 min in 99% ethanol, rinsed with deionized water, and uncovered beneath UV for 10 min. They had been then mounted within the circulation cells, and PBS was injected for five min (100 µL/min) to permit the sign to be stabilized. Subsequently, a ten µg/mL answer of PL-exo or PL-exo-ALN in PBS was injected on the circulation velocity of 20 µL/min for 150 s to allow the movie floor to keep up a correspondence with the exosome answer. Lastly, the surfaces had 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. Adjustments within the resonance frequency (Δf) and dissipation (ΔD) had been recorded.

One other HAp binding technique was additionally used to imitate the binding affinity of Aln functionalized exosomes to bone floor in vitro through the use of DiD (a sort of lipophilic fluorochrome) labeled exosomes. Firstly, the PL-exo and PL-exo-ALN options had been incubated with 3 μM DiD for 1 h at room temperature to acquire fluorescent exosomes. Then, they had 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 pictures had been captured by IVIS imaging (Lumina Sequence III, PerkinElmer, USA). And the fluorescence intensities of the supernatants had 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) had been harvested from the marrow of wholesome 2‐week‐previous Sprague‐Dawley rats based on our earlier research [69, 70]. BMSCs had 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 had been cultured in endothelial cell development media (EGM-2) supplemented with EGMTM-2 MV SingleQuots^™ (Lonza, Basel, Switzerland). Each cells had been seeded in 5% CO₂ at 37 ℃. All of the procedures had been authorised by the Institutional Ethics Assessment 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) had been based on earlier publications [23].

To guage the impact of PL, PL-exo and PL-exo-ALN on osteoclasts, bone marrow monocytes (BMMs) had been harvested from the marrow of wholesome 6-week-old C57BL/6 mice based on earlier examine [71]. BMMs had 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/nicely) had 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) had 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 had been then mounted and visualizated by tartrate-resistant acid phosphatase (TRAcP) staining. TRAcP-positive multinucleated cells that had greater than three nuclei had been counted as osteoclasts. The variety of osteoclasts per nicely 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 had been labeled with the DiD firstly as talked about above. Then, BMSCs had been uncovered to the labeled exosomes for 12 h. After that, the cells had been mounted 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 had been obtained utilizing an invert fluorescence microscope (Olympus, Japan).

The bone concentrating 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) had been obtained from the Animal Middle of the Chinese language Academy of Science (Shanghai, China). The rats had been divided into two teams: PL-exo and PL-exo-ALN (n = 3 in every group). After anesthesia with sodium pentobarbital, the rats had been obtained 100 μg exosomes (dissolved in 200 μL of PBS) by way of tail vein injection. Six hours later, the rats had been sacrificed and the femur together with main organs (femur and coronary heart, liver, spleen, lung, kidney) had 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 nicely had been seeded into 96-well plates. One group with out remedies served because the management. The remaining teams had 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 nicely, 90-µL medium and 10-µL CCK-8 answer had 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 strategy for osteogenic differentiation was relying on a earlier examine [69]. Briefly, the BMSCs (1 × 10⁴ cells/cm²) had 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 had 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 Colour Improvement Equipment (Beyotime, Shanghai, China) and quantified with an alkaline phosphatase assay package (Beyotime, Shanghai, China) after lysed. The calcium deposits had been evaluated by Alizarin crimson staining (Cyagen Biosciences, Guangzhou, China) after 14 days of tradition, and the stained mineralized nodules had 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 package from Thermo Fisher Scientific. The extracts had 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 had 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 towards 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 following 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 had 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 info had been normalized and offered because the fold change of the protein degree of the therapy group in comparison with the management group.

Immunofluorescence

The cells had 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 had been washed thrice in PBS, mounted 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 towards 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 answer (1:10) had 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 had been captured by an inverted fluorescence microscope (Olympus, Japan). As for histofluorescence, the sections had been deparaffinized and hydrated and incubated with a mix of EMCN (1:200, Invitrogen, USA) and CD31(1:200, Invitrogen, USA). After washed with PBS, sections had 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 had been stained with DAPI answer. The pictures had been captured by an inverted fluorescence microscope (Olympus, Japan).

In vitro tube formation

The tubule formation potential 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 nicely and gelatinized at 37 °C for 30 min. Then, EPCs suspension of two.0 × 10⁴ cells/mL had been digested and seeded on the matrix. After 6 h of incubation, the variety of full capillaries and nodes of every gap had been calculated.

Migration assay

To measure the migration potential of EPCs after completely different remedies, a transwell assay was used. The cells had 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 had been added to the decrease chambers. And Dex and completely different teams of PL or PL-exosomes had been added to serum-free medium. After 24 h of incubation, the cells on the higher floor of the transwell membrane had been gently wiped with a cotton swab, and cells on the decrease floor had been mounted with 4% paraformaldehyde and stained for 10 min with 0.5% crystal violet. Lastly, 3 random decrease surfaces of every filter had been chosen and counted twice.

Preparation of conditioned media from BMSCs and EPCs

BMSCs or EPCs had 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) had 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 various kinds 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 had 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 authorised by the Animal Care and Use Committee of Wenzhou Medical College. A complete forty male Sprague–Dawley rats (220–250 g, 6-week-old) had been offered by the Animal Middle of the Chinese language Academy of Science (Shanghai, China). The rats had 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 customary temperature circumstances with a 12-h mild/darkish cycle and repeatedly fed with meals and water. The institution of GIOP mannequin was based on earlier research [72]. Rats within the MPS-related teams had been obtained an intramuscular injection of methylprednisolone (MPS) (30 mg/kg/day in 0.9% NaCl answer) for 60 days, whereas a management group was injected every day with equal quantity automobile (saline). After 3 weeks injection of MPS, the therapy teams had been intravenously administrated with PL, PL-exo, PL-exo-ALN (100 μg in PBS) as soon as every week. In distinction, the MPS teams obtained 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 observe dynamic bone formation and mineralization, 5 rats of every group had been intraperitoneally injected with 30 mg/kg alizarin crimson S (Macklin, Shanghai, China) and 10 mg/kg calcein (Macklin, Shanghai, China) at week 3 and 6 through the experiment. On the finish of the therapy interval, these 5 rats had been humanely sacrificed and femurs had been collected for micro-CT scanning. Then, the precise femurs had been used for undecalcified histological examination, whereas the left femurs had been decalcified in 10% EDTA for hematoxylin and eosin (H&E), tartrate‐resistant acid phosphatase (TRAcP) and immunohistological staining. Alternatively, the remaining three rats in every group had been anaesthetized and perfused with Microfil to evaluate intraosseous vessels. Then, the femurs had been collected and decalcified with 10% EDTA for 2 months. After micro-CT scanning, femurs had been paraffin-embedded for immunofluorescence staining. The investigators weren’t blinded to allocation throughout experiments and final result evaluation. Pattern sizes had been chosen on the premise of earlier experiments. No animals had been excluded from evaluation.

Micro-CT scanning

Micro-CT scanning and corresponding evaluation for femurs had been carried out utilizing SkyScan1178 system and bundled software program (Bruker MicroCT, Kontich, Belgium). The 14-micron voxel measurement of pictures had been acquired with a set at 35 kV of vitality and 220 mA of depth. 2D and 3D reconstructions had 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) had been calculated through the use of CTAn software program.

Histology

For undecalcified sections, after embedding in resin, specimens had been sawn coronally into 100-μm thickness, mounted on plastic slide, and punctiliously grinded and polished into about 50-μm thickness. The immunofluorescent indicators about dynamic bone formation had 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 answer. The pictures had been recorded utilizing microscope (Olympus, Japan).

For decalcified specimens, after being dehydrated utilizing an alcohol gradient, cleared, and embedded in paraffin, the tissues had been reduce into 5-μm-thick sections. H&E and TRAcP had been carried out as described beforehand [75]. The osteoclast floor per bone floor (Oc.S/BS) had been calculated utilizing BIOQUANT OSTEO 2011 software program based on a technique proposed by Sawyer et al. [76]. For immunohistochemical (IHC) staining, sections had 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 had been stained with DAB and counterstained with hematoxylin. The sunshine pictures had 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 had been utilized for Micro-fil perfusion to evaluate intraosseous vessels, based on 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 reducing 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 had been mounted and stiff, Micro-fil (MV-122, Carver, MA, USA) had been injected into the belly aorta till a relentless outflow was proven within the belly vein. About 25 ml Micro-fil was consumed per rat to make sure sufficient filling of intraosseous vessels. The rats had been saved at 4 °C in a single day to make sure polymerization. Then, the bilateral femurs had been eliminated, mounted and decalcified with 10% EDTA answer for two months. Lastly, inner femoral vessels had been imaged and analyzed by Micro-CT. The scanner was set at a decision of 9 μm per pixel. The 3D pictures of the vasculature had been reconstructed utilizing CTVox software program. The full 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 had been obtained from R&D methods. Assays had been carried out based on 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

At the very least three impartial replicates had been carried out. The info are offered because the imply ± customary 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 check for a number of comparisons. All statistical analyses had been carried out by Prism software program model 8.0 (GraphPad, San Diego, CA, USA). A P worth < 0.05 was thought-about statistically vital. Energy evaluation was carried out by G*energy software program (Model 3.1).