Cell preparation and tradition
BMSCs have been remoted from 8-week-old male Sprague–Dawley (SD) rats by flushing the bone marrow from femurs and tibias with phosphate-buffered saline (PBS; HyClone, USA). BMSCs have been cultured in α‐minimal important medium (Gibco, USA) supplemented with 10% (v/v) foetal bovine serum (FBS; HyClone), 1% (v/v) penicillin/streptomycin (P/S; HyClone). Human embryonic kidney 293T cells (HEK 293T) have been bought from ATCC. HEK 293T have been cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) supplemented with 10% (v/v) FBS and 1% (v/v) P/S. All cells have been cultured at 37 °C in a humidified environment containing 5% CO2/95% air.
Plasmid building
Two synthetic plasmids (Bmp2 and NoBody) have been designed on this experiment based mostly on the properties of NoBody protein to inhibit the mRNA translation course of. The sequences of MS2/Linker/intrinsic ribosomal entry web site (IRES)/rat BMP2(rBMP2)/Flag/NoBody/MCP are proven in Further file 1: Desk S1. The fragments have been cloned into the NheI and BamHI websites of pcDNA3.1(−) vector to generate the plasmids pCDNA3.1(−)-MS2-Linker-MS2-Linker-IRES-rBMP2 and pCDNA3.1(−)-Flag-NoBody-Linker-MCP.
Transfection
Two synthetic plasmids collectively (with a molar ratio of 1:1) or two synthetic plasmids individually with the empty plasmid pCDNA3.1(−) (with the molar ratio of 1:1) have been dissolved in FBS-free DMEM, combined with Lipofectamine 2000, and incubated at room temperature for 20 min. The plasmids have been transfected into 293T cells at 70 − 80% confluence, and the medium was exchanged with recent 10% FBS-containing medium 6 h later.
Exosome isolation and characterisation
Exosomes from management or Bmp2 mRNA-enriched cells have been remoted utilizing ultracentrifugation. Briefly, cells have been cultured with exosome‐free FBS medium. Cell supernatants have been centrifuged at 3000 ×g for 30 min to get rid of mobile particles. Subsequent, the supernatant was centrifuged at 100,000 ×g for two h to acquire the exosomes. After isolation, all of the exosomes have been resuspended in PBS and saved at − 80 ℃. The scale distribution was analyzed by NanoSight (Malvern Devices Ltd., Malvern, UK). The morphology of remoted exosomes was analyzed by transmission electron microscopy (G2 Spirit Biotwin, TECNAI, USA). Briefly, the exosomes have been utilized on a carbon copper grid and air dried for two min. Subsequent, they have been rinsed with deionized water for 1 min and stained with 2% uranyl acetate for 30 s. The pictures have been analyzed by a transmission electron microscope. To analyze the traits of intracellular internalization of exosomes, cells and DiI-labelled exosomes have been co-cultured for six h, washed 3 times in PBS, and the rat bone marrow mesenchymal stem cells (rBMSCs) have been fastened in 4% paraformaldehyde for 15 min and washed once more. Cell nuclei have been stained with Hoechst for 10 min. The mobile distribution of the exosomes was imaged utilizing a confocal laser scanning microscope (Nikon A1R, Tokyo, Japan).
qRT-PCR
The overall RNA from exosomes was extracted utilizing TRIzol reagent (Invitrogen, USA), in line with the producer’s protocol. Reverse transcription was carried out utilizing the PrimeScript First-Strand cDNA Synthesis Package (Takara, China) for evaluation of mRNA expression. Subsequently, qPCR reactions (20 µL) have been carried out utilizing FastStart Important DNA Inexperienced Grasp. GAPDH was used as an inside management to normalise sign for every goal gene. Relative expression was calculated by the two–ΔΔCt methodology. The sequences of PCR primers for BMP2, OPN, COL1a, ALP, and GAPDH used on this research are listed in Further file 1: Desk S2.
Western blot assay
Whole protein from the donor cells or remoted exosomes was extracted with RIPA Lysis Buffer (Beyotime, China) at 4 ℃ for 30 min. Protein focus was decided utilizing the BCA Protein Assay Package (Pierce, USA) and proteins have been separated utilizing SDS-PAGE with a 6% stacking gel and 12% resolving gel. The proteins have been then transferred to nitrocellulose membranes. After being blocked with 3% bovine serum albumin, the membranes have been incubated with major antibodies in opposition to BMP2 (pa5-69,363, ThermoFisher), FLAG (ab205606, Abcam), GM130 (11,308–1-AP, ProteinTech), TSG101 (ab83, Abcam), CD81 (ab286173, Abcam), Hsp70 (4872T, Cell-Signaling), OPN (sc-73631, Santa Cruz Biotechnology), COL-1 (ab260043, Abcam) and anti-GAPDH (D110016-0100, BBI Life Sciences) at 4 ℃ for 12 h. After being washed 3 times in TBST, the membranes have been incubated with secondary antibodies (anti-mouse [7076, CST] or anti-rabbit [7074, CST]) in Tris-buffered saline at room temperature for 1 h. The pictures have been developed by chemiluminescence (GE Healthcare, Chalfont St. Giles, UK) in a darkish room.
Supplies
Gelatin methacryloyl (GM-90, GM-60) and lithium pherryl-2,4,5-trimethylbenzoylphosphinate (LAP) have been bought from Engineering for Life (Suzhou, China). FITC-labelled CP05 and FITC-labelled (allyl-L-glycine)-CP05 have been bought from Bankpeptide (Hefei, China).
Sluggish-release impact of a hydrogel encapsulating exosomes
GelMA (EFL, EFL-GM-60, Suzhou, China) and GelMA (EFL, EFL-GM-90, Suzhou, China) have been dissolved in 0.5% (w/v) PBS. The answer was handled with 0.5 wt% at seen gentle (405 nm), and the combination was incubated at 60 °C for about 30 min to fully dissolve the solids. The answer was filter-sterilised utilizing a 0.22 µm filter. Modified hydrogel options have been ready by dissolving CP05 or (allyl-L-glycine)-CP05 [0.1% (w/v)], and exosome [20% (w/v)] in GelMA answer [10% (w/v)] at 37 °C. (Fig. 3A).
Every hydrogel pattern was incubated in PBS at 37 °C below fixed shaking at 100 rotations/min (r/min) to imitate dynamic move of blood and lymph within the physique as described earlier than [29]. Specimens (GM-60, GM-90, GM-60 + Exo, GM-90 + Exo, GM-60/CP05 + Exo, GM-90/CP05 + Exo, GM-60-CP05 + Exo, and GM-90-CP05 + Exo, n = 3/group) have been weighed at baseline (W0), which represented 0 h. Samples have been renewed with recent answer each 10 h. On the particular time factors (10, 20, 30, 40, 50, and 60 h), the specimens have been faraway from the answer, washed twice with sterile deionized water, drained via filter paper, and reweighed (Wt). The remaining mass ratio (%) of every hydrogel pattern was calculated as follows: remaining mass ratio = (Wt/W0) × 100%. Within the GM-60(90)/CP05 + Exo group, the exosomes weren’t covalently linked to the hydrogel; nonetheless, within the GM-60(90)-CP05 + Exo group, the exosomes have been covalently linked to the hydrogel. Amongst them, 500 µL of GM-90-CP05 + Exo hydrogel was injected subcutaneously below the center dorsal areas of the rat pores and skin to research the degradation charges of the experimental group in vivo. On the particular time factors (0, 2, 4, 6, 8, and 10 d), the specimens have been faraway from the rat, drained via filter paper, and reweighed (Wt). The load residual (%) of every hydrogel pattern was calculated as follows: weight residual = (Wt/W0) × 100%. The experimental animal process was carried out below strict supervision and permitted by the Animal Care and Use Committee of Fourth Navy Medical College.
DiI-labelled exosomes (500 µg/mL) have been loaded into management and experimental teams of hydrogel and incubated in 1 mL PBS at 37 °C at 100 r/min to watch exosomes. Cumulative exosome launch was monitored by eradicating and changing the buffer each 10 h and exosome-associated fluorescence was analyzed by confocal laser scanning microscope (Nikon A1R, Tokyo, Japan). The exosome focus was detected in PBS prior to every change utilizing the ELISA equipment (Elabscience, China).
Every pattern was positioned at − 80 °C in a single day after which taken out and positioned on a silicon wafer and freeze dried (GOLD-SIM, US) at − 80 °C. Samples have been examined utilizing a scanning electron microscope (S-4800, Hitachi, Japan) at an accelerating voltage of 5 kV. The samples have been loaded on prime of the conductive tape and sputter-coated with gold for 60 s with a magnetron sputtering equipment (E-1045, Hitachi).
Analysis of the biocompatibility between cells and hydrogel in vitro
Hydrogel (1 mL) was injected into the confocal dish and irradiated with gentle at 405 nm for 10 s. rBMSCs have been seeded on the cured hydrogel and cultured for 1 and three days. The cells have been rinsed 3 times with PBS answer and incubated with Calcein/PI Cell Viability/Cytotoxicity Assy Package (Beyotime) for 30 min at 37 ℃. The cells have been then rinsed 3 times with PBS answer and noticed utilizing a confocal laser scanning microscope (Nikon A1R, Tokyo, Japan) and analysed by Picture J model 1.48 (USA).
Hydrogel (100 µL) was injected into 96-well tradition plates and irradiated with gentle at 405 nm for 10 s. BMSCs have been seeded on the cured hydrogel and cultured for 1, 3, 5, and seven days. The proliferation of rBMSCs seeded on hydrogel was measured with the CCK8 Assay Package (Beyotime) and a microplate reader (Tecan, Spark 20 M, Shanghai, China) at 450 nm. Every group was examined in triplicate.
Mobile uptake and intracellular internalization of exosomes
To additional discover the exosome uptake motion by cells on the hydrogel, rBMSCs have been seeded onto hydrogel conjugated with DiI-labelled exosomes and incubated for 48 h. After washing 3 times in PBS, the rBMSCs have been fastened in 4% paraformaldehyde for 15 min after which washed once more. Cell nuclei have been stained with Hoechst for 10 min. The mobile distribution of the exosomes was imaged utilizing a confocal laser scanning microscope (Nikon A1R).
ALP stain, ALP exercise, Alizarin purple stain, and qualification
For investigating the extent of osteogenic differentiation within the 2D and 3D setting, BMSCs with and with out hydrogel have been cultured in osteogenic medium with out exosomes for a set time interval. To measure ALP expression, cells have been fastened with 10% formalin, stained utilizing BCIP/NBT ALP Color Growth Package (Beyotime), and imaged with an inverted fluorescence microscope (DMI6000 B, Leica, Germany). To quantitate ALP exercise, cells have been examined with an ALP Assay Package (Beyotime). Absorbance was measured at 405 nm. To analyze mineral deposition, cells have been fastened with formalin and stained with Alizarin Pink S Staining Package for Osteogenesis (Beyotime) for about 20 min, washed 3 times with dH2O and stained with ARS for 20 min at room temperature. Cells have been imaged with a Leica MDI6000 B fluorescence microscope. After a number of washes with dH2O, the stain was desorbed with 200 µL 10% cetylpyridinium chloride (Sigma, Germany) for 1 h. The dye was collected, and the absorbance was learn at 590 nm utilizing a spectrophotometer.
Institution of rat calvarial defect mannequin
The experimental animal process was carried out below strict supervision and permitted by the Animal Care and Use Committee of Fourth Navy Medical College. A complete of 72 eight-week-old male SD rats have been used on this research. The rats have been randomly divided into NC, GM90, GM90 + ExoNone, GM90-CP05 + ExoNone, GM90 + ExoBMP2, and GM90-CP05 + ExoBMP2 (n = 6/group). Rats have been anesthetized with pentobarbital sodium (50 mg/kg). Bilateral vital full-thickness cranial defects (d: 5.0 mm) have been drilled utilizing a trephine drill. Hydrogel (100 µL) was then injected into every faulty area, layered, and closed with 5–0 absorbable sutures. Consuming water containing trimethoprim-sulfamethoxazole was supplied for 7 days to stop infections. The rats have been euthanized at weeks 4 and eight post-surgery, and the samples have been collected and glued for evaluation.
Micro-CT evaluation
To guage bone regeneration within the faulty space, the harvested calvarias have been scanned by high-resolution Micro-CT (Siemens, Inveon MM Micro CT, USA). Samples have been reconstructed and analyzed utilizing supporting software program (Inveon Analysis Office 2.2). The varied bone parameters, together with BS/TV (%), Tb. Sp (mm), and Tb. T (mm), have been analyzed.
Histology, immunohistochemistry, and immunofluorescence analyses
The samples have been decalcified in 10% disodium ethylenediaminetetraacetic acid (Solarbio, China) at 4 °C for 30 d. Afterwards, decalcified samples have been longitudinally embedded in paraffin wax and sliced into 5 μm sections. Then, the tissue sections have been stained with haematoxylin and eosin (Solarbio) and TRAP staining equipment (Solarbio) based mostly on the producer’s directions. The stained sections have been photographed below the microscope (DM6000 B, Leica) and analyzed with Picture ProPlus 6.0 Software program (Media Cybernetics, Silver Spring, USA). OCN (PB1009, Boster) and COL-1a (PB0981, Boster) staining was carried out with an anti-rabbit HRP/DAB Detection Package (18653 s, CST), following the producer’s protocol. The pictures have been photographed below the microscope. Semiquantitative evaluation was measured by Picture Proplus 6.0 software program. For histology analysis, 3 skilled orthopedical pathologists have been invited.
Statistical evaluation
Knowledge are expressed because the imply ± SEM and have been analyzed by one-way ANOVA for a three-group comparability and log rank take a look at for survival curves. Variations have been thought-about vital at P < 0.05. All statistical analyses have been carried out with GraphPad Prism 8.0.
