Cell tradition and identification
Cell tradition: Rat BMSCs (CM-R131, Procell) have been cultured in α-MEM medium (SH30265.01, HyClone, Thermo Fisher Scientific, USA) supplemented with 15% fetal bovine serum (FBS; 10091148, Thermo Fisher Scientific, USA) and 100 U/mL penicillin–streptomycin answer (10378016, Thermo Fisher Scientific, USA), and maintained at 37 °C and 5% CO2 in a tradition incubator. When the confluence price of BMSCs reaches 80%, passaging ought to be performed. The third technology BMSCs have been used for differentiation detection and extraction of EVs [33]. BMSCs identification: After washing with PBS (E607008-0500, Sangon Biotech, China), a single-cell suspension was ready at a focus of 1 × 106/mL. Grouped cells have been incubated with fluorescent-tagged antibodies: CD44-FTITC (ab30405, Abcam), CD90-PE (ab24904, Abcam), CD45-FITC (ab210220, Abcam), CD34-PE (ab23830, Abcam) and IgG (ab172730, Abcam) at 4 °C for 30 min. Subsequently, unbound antibodies have been washed off with PBS, and the expression of corresponding labeled antibodies within the pattern was analyzed utilizing a move cytometer. In line with the directions of the reagent equipment (PD-003/4/5, Procell) for inducing differentiation (osteogenic, adipogenic, and chondrogenic) of BMSCs, the osteogenic, adipogenic, and chondrogenic differentiation potential of BMSCs was noticed by staining with Alizarin Purple S (ARS), Oil Purple O and Alcian Blue respectively [34].
Cell transfection
In line with the sequences of miR-19b-3p and WWP1 revealed by NCBI, Shanghai Sangon Biotech was commissioned to assemble the next plasmids: oe-NC, oe-WWP1, NC mimic, miR-19b-3p mimic, NC inhibitor, and miR-19b-3p inhibitor. The gene vectors for overexpression and silence have been constructed utilizing the next plasmids: pGPU6/Neo (Genomeditech, Shanghai, China) for silence and pCMV6-AC-GFP (FunGenome, Hunan, China) for overexpression. Digest BMSCs cells with trypsin first, then inoculate 4 × 105 cells per effectively right into a 6-well plate and domesticate them to kind a monolayer. Subsequent, take away the tradition medium and transfect in line with the Lipofectamine 2000 handbook (11668-019, Invitrogen, New York, California, USA). After transfection, the cells have been cultured at 37 °C underneath 5% CO2 for six–8 h, then modified the entire tradition medium and incubated for one more 48 h to extract RNA and protein for subsequent experiments [35].
Isolation, purification, and identification of EVs derived from BMSCs
Technique for isolation of EVs derived from BMSCs: BMSCs transfected with inhibitor NC and miR-19b-3p inhibitor have been cultured till 80–90% confluence. Take away the supernatant, wash twice with PBS, change with FBS tradition medium containing 10% EVs depletion, and proceed to tradition for 48 h in a 37 °C CO2 incubator. Then, the collected supernatant could be centrifuged stepwise. Firstly, centrifuge at 500g for 15 min at 4 °C to take away the mobile particles. Then, centrifuge at 2000g for 15 min at 4 °C to take away apoptotic our bodies or mobile particles. Lastly, centrifuge at 10,000g for 20 min at 4 °C to take away massive vesicles. Subsequent, filter by way of a 0.22 μm filter and take away EVs by ultracentrifugation at 110,000g for 70 min at 4 °C. After resuspending the remaining materials, use 100 μL of sterile PBS for downstream experiments. Beckman ultracentrifuge (Optima L-90K, bio-thing) was used for the high-speed centrifugation steps, whereas Beckman Allegra X-15R benchtop centrifuge (Beckmancoulter) [11] was used for the remaining low-speed centrifugation steps.
NanoSight nanoparticle monitoring evaluation (NTA): 20 μg of EVs have been dissolved in 1 mL of PBS, vortexed for 1 min, and measured utilizing a NanoSight nanoparticle monitoring analyzer (Malvern Devices Ltd, Malvern Panalytical) and the corresponding software program Zetaview 8.04.02. Calibrate utilizing 110 nm polystyrene particles. The temperature is maintained at 27.65 °C to instantly observe and measure the scale distribution of EVs [11].
Transmission electron microscopy (TEM): 20 μL of a freshly ready pattern of ultracentrifuged EVs have been loaded onto carbon-coated copper electron microscopy grids, allowed to face for two min, after which negatively stained with ammonium molybdate (Sigma-Aldrich, USA, 12501-23-4) for five min. The grids have been washed thrice with PBS to take away extra ammonium molybdate, then air-dried on filter paper till partially dry. The picture was acquired with a Hitachi H7650 transmission electron microscope (DOLEE statement) at an accelerating voltage of 80 kV [36].
Western blot method was used to establish the floor markers of EVs: EVs suspension was concentrated, and its protein content material was measured utilizing a BCA assay equipment (Thermo Fisher Scientific, USA, 23227). The SDS-PAGE gel was ready, and the proteins have been denatured and electrophoresed. Then, the EVs-specific marker proteins HSP70 (ab2787, Abcam, USA, 1:1000), CD9 (SBI, USA, EXOAB-CD9A-1, 1:1000), CD81 (SBI, USA, EXOABCD81A-1, 1:1000), in addition to the endoplasmic reticulum marker protein Calnexin (Abcam, ab133615, 1:1000) have been detected after switch [36].
EV labeling methodology: DiR (Thermo Fisher Scientific, D12731) was added to the EV answer at a focus of 1:400 and incubated for 30 min. The answer was then ultracentrifuged at 100,000g for 90 min to take away extra dye and procure DiR-labeled EVs [36].
Gel-OCS/MBGN hydrogel synthesis reagents and supplies
A biomimetic composite hydrogel was ready by uniformly mixing OCS/MBGN combination and gelatin (kind B, with a gel power of about 100 g Bloom, G108398-500 g, Cas(9000-70-8), Aladdin reagent, together with white gelatin, animal gelatin, sinew glue, silver gelatin, Gelatin(Aladdin-e.com)) at a quantity ratio of 1:1 underneath a temperature of 37 °C. OCS is ready from sodium chondroitin sulfate A (CAS 39455-18-0, Aladdin Reagent, CSA, Chondroitin 4-sulfate sodium salt (Aladdin-e.com)) and sodium periodate (AR, ≥ 99.5%, S104090-500 g, CAS 7790-28-5, Aladdin Reagent, sodium periodate, meta-periodate, and sodium meta periodate (Aladdin-e.com)). MBGN was ready by mixing tetraethyl orthosilicate (TEOS, AR, 98%, Sigma-Aldrich), cetyltrimethylammonium bromide (CTAB, AR, ≥ 99%, 30037416, Shanghai State-owned Belongings Chemical Reagent Co., Ltd.), calcium nitrate (CN, AR 99%, Tianjin Fuchen Chemical Reagent Manufacturing facility), and ammonia (AR, 25–28%, A112077-500 ml, Cas(1336-21-6), Aladdin Reagent, ammonium hydroxide answer) collectively [10].
Synthesis of oxidized chondroitin sulfate (OCS)
Dissolve 1.25 g of CS in 20 mL of distilled water and stir at 4 levels Celsius. After the entire dissolution of CS, add 1.93 g of sodium periodate to the answer and react for six h in darkish situations. The molar ratio of CS to sodium periodate is 1:1 for oxidation. Lastly, switch the above answer right into a dialysis bag with a molecular weight cutoff of 3500 and dialyze in 2 L of distilled water at room temperature for twenty-four h. Change the water each 6 h. Afterward, pour the dialysis answer right into a 50 mL centrifuge tube, freeze it at − 20 °C for twenty-four h, then use an FD-10 freeze dryer (made in China) for freeze drying for 7 days to acquire OCS powder [10].
The synthesis of MBGN
Dissolve 2.80 g CTAB in 132 mL deionized water underneath stirring at 35 °C. After utterly dissolving CTAB, add 40 mL of EA and proceed stirring for 30 min. Then add 28 mL of ammonia answer (1 mol/L) and stir for 15 min. Then add 14.40 mL TEOS and stir for 30 min; lastly, add 6.52 g CN. Additional, stir the above combination for 4 h. Because of the formation of colloids, the combination progressively turns right into a milky white shade. Acquire colloid particles by centrifugation at 8000 rad/s, wash them thrice with water, after which wash them with ethanol. Dry the collected sediment at 60 °C for twenty-four h, then grind it right into a nice powder utilizing a mortar. Lastly, warmth the powder to 700 °C and preserve it for 3 h to take away natural matter and nitrates to acquire MBGNs [10].
Preparation of composite Gel-OCS/MBGNs hydrogels
Dissolve OCS powder in 0.05 M borate buffer answer to organize a ten% (w/v) OCS answer. Subsequently, ultrasound dispersion was performed at room temperature for 10 min, and numerous concentrations of MBGNs have been added to the OCS answer to organize MBGNs-contained OCS options. Warmth gelatin to 60 °C to dissolve and put together a 30% (w/v) gelatin answer. Subsequent, combine the OCS/MBGN combination with gelatin answer in a 1:1 quantity ratio, after which pour into the mould to kind a hybrid gel-OCS/MBGN hydrogel at 37 °C. The ultimate focus of MBGNs within the ensuing hydrogels was 0% and 15% (w/w) [10].
OCS characterization
Dissolve CS and OCS individually in deuterium oxide (D2O, 99.9% purity, Adamasβ) for nuclear magnetic resonance (NMR) evaluation (AVANCE III HD 400, Bruker) and use tetramethylsilane (TMS) as an inside customary at 25 °C. The chemical construction and practical teams of MBGNs and OCS might be detected with larger decision (4 cm−1) utilizing Fourier rework infrared spectroscopy (FTIR) in transmission mode, with 16 scans taken within the wavelength vary of 400 to 4000 cm−1, utilizing a Nicolet 6700 instrument from Thermo Fisher, USA [10].
Subject emission scanning electron microscope (FE-SEM)
We used the FE-SEM (Extremely 55) from the German firm Carl Zeiss AG to analyze the morphology of MBGNs and Gel-OCS-MBGN composites. Earlier than characterization, we dispersed the MBGNs in ethanol after which dropped them onto a silicon wafer. Earlier than observing SEM, we freeze-dried the hydrogel pattern for 3 days. We used gold (SC7620) from Quorum Applied sciences within the UK for a 60-s sputter coating. Throughout SEM statement, we examined samples of MBGNs and Gel OCS/MBGN composite hydrogels by energy-dispersive spectroscopy (EDS) [10].
X-ray diffraction (XRD) and Fourier rework infrared spectroscopy (FTIR)
Underneath a generator voltage of 40 kV and a tube present of 40 mA, the mineralized layer of MBGNs and hydrogels was analyzed utilizing XRD (Ultima III, Japan). The scanning pace is 2°/min, and the 2θ vary is between 10° and 80°. Utilizing FTIR in transmission mode, the composite hydrogel’s chemical construction and practical teams have been detected with a decision of 4 cm−1 and 16 scans (wavelength vary 500–4000 cm−1). Earlier than analyzing the gel-OCS/MBGN hydrogel, it should first be freeze-dried [10].
Rheology and dynamic mechanical, and thermal evaluation
Firstly, the gelation time of the hydrogel is decided by recording the storage modulus and loss modulus (elastic modulus) by way of an oscillatory time scan experiment carried out by setting the oscillation frequency to 1 Hz and making use of a 5% shear pressure. Use Anton Paar’s rotational rheometer (Physica MCR301) to judge the rheological properties of combined hydrogels. Place the cross-linked hydrogel (Φ25 mm × 2 mm) on the pattern stage and carry out the check utilizing a 25 mm diameter plate system at 37 °C by acquiring the storage modulus (G’) and the loss modulus (G”) within the vary of 0.1 and 100 rad/s from the frequency-modulus curve, with a pressure amplitude of 5.0%. As well as, the compressive power of the saturated hybrid hydrogel was studied utilizing Dynamic Mechanical Evaluation (DMA, TA Devices, Q800) at a stress price of three N/min and 25 °C. Lastly, the compressive modulus was calculated within the DMA check, utilizing the stress–pressure curve’s linear area to judge the cross-linked hydrogel’s compressive modulus (Φ10 mm × 5 mm) [10].
Extracellular mineralization and degradation
Soak the cross-linked hydrogel (Φ8 mm × 2 mm) in 10 mL SBF in a centrifuge tube at 37 °C. Change the SBF each different day. After incubation for 7 days, take away the pattern from SBF and rinse it with deionized water to remove extra SBF. As described above, the samples have been characterised by SEM–EDS, FTIR, and XRD earlier than present process freeze-drying remedy. Measure the mass lack of the hydrogel in PBS (pH 7.4) to judge it’s in vitro degradation. Add about 1 mL of hydrogel to three mL of PBS at 37 °C. Take away PBS and document the standard every day to judge the degradation of the hydrogel. The next equation might calculate the share of high quality loss (%): high quality loss (%) = (M1-M2)/M1 * 100%, the place M1 and M2 characterize the standard of the hydrogel earlier than and after soaking in PBS, respectively. In the meantime, the pH worth of PBS-containing water gel was additionally measured for adjustments [10].
Gel-OCS/MBGNs hydrogel loaded with EVs
Combine the ready Gel-OCS/MBGNs hydrogel with EVs remoted from BMSCs labeled with DiR (D12731, Thermo Fisher Scientific), place the combination in an ice bathtub till dissolved, and retailer the combination at 4 °C. Place the cells in a tradition incubator containing 5% CO2 and incubate at the hours of darkness for 12 h. Put together a 1:1000 DAPI nuclear staining buffer, wash cells with PBS thrice, place cells on ice, and incubate at the hours of darkness for 30 min [37]. 4 pictures with completely different fields of view have been randomly chosen underneath an optical microscope (Leica®, Germany) for statistical evaluation.
The in vitro launch of EVs in Gel-OCS/MBGNs@EVs
Soak the Gel-OCS/MBGNs@EVs in a 15 mL centrifuge tube containing 4 mL of PBS. At completely different time factors (1, 3, 6, 9, 12, 15, 18, and 21 days), 100 μL of supernatant was changed with 100 μL of recent PBS. The focus of EVs within the supernatant was evaluated by detecting the fluorescence depth of DiR (Ex: 754 nm, Em: 778 nm) within the supernatant. Draw launch curve, decided by the ratio of supernatant to whole fluorescence depth [38].
CCK-8 and useless cell apoptosis equipment are used to judge cell viability and proliferation
The dwell/useless staining experiment for cell viability was carried out utilizing the Dwell/Useless assay equipment (L10119, Invitrogen, USA) following the producer’s directions. BMSCs have been seeded at a density of two × 104 cells/effectively into 48-well plates containing completely different hydrogels. Put together a staining answer with a focus of two μM Calcein-AM and 4 μM ethidium homodimer-1, and add it to the wells—tradition the cells in a CO2 incubator at 37 °C and 5% CO2 for 30 min. Imaging of dwell cells (inexperienced, Ex: 480 nm, Em: 530 nm) and useless cells (pink, Ex: 530 nm, Em: 645 nm) was carried out utilizing an inverted fluorescence microscope (Olympus IMT-2/).
The cell proliferation experiment was performed utilizing CCK-8 reagent (CA1210, Solarbio) in line with the next steps. BMSCs have been seeded at a density of 1 × 104 cells/effectively in 48-well plates containing completely different hydrogels. On the primary, third, fifth, and seventh days of cultivation, the recent tradition medium was added to the 48-well plate with 10% CCK8 reagent and incubated for 1 h underneath darkish situations. Afterward, switch 100 μL of the supernatant to a 96-well plate for absorbance measurement at 450 nm utilizing a microplate spectrophotometer (Bio-Tek, UK) [38].
The cell adhesion experiment was carried out utilizing iFluor™ 488-phalloidin (ab176753, Abcam) and DAPI (ab285390, Abcam) for immunofluorescence staining. BMSCs have been seeded at a density of 1 × 105 cells/effectively into 35 mm tradition dishes with completely different hydrogels. Repair cells in 4% paraformaldehyde and permeabilize with PBS buffer containing 1% Triton X-100. Afterward, stain with phalloidin answer for 1 h at room temperature, adopted by staining with DAPI answer for five min. Lastly, cells have been imaged utilizing a laser scanning confocal microscope (Olympus FV1000, BX61W1).
Induction and identification of osteoblast differentiation
Osteoblast differentiation induction: BMSCs have been 3D cultured in hydrogel supplemented with 10 mM β-glycerophosphate, 100 nM dexamethasone, and 50 mg/mL ascorbic acid-2-phosphate within the tradition medium to acquire osteogenic induction medium. Within the means of BMSC osteogenic differentiation, the tradition medium is changed each three days. Use GelMA digestion equipment (EFL-GM-LS-001, Cellendes) and trypsin (R001100, Thermo Fisher Scientific) to degrade the hydrogel for cell retrieval.
Alkaline phosphatase (ALP) staining: ALP staining was carried out utilizing the alkaline phosphatase staining equipment (40749ES60, Yeasen, China) following the producer’s directions. After induced for 7 days, repair the BMSCs in 4% paraformaldehyde, washed with PBS, and stain for 30 min. Observe stained cells utilizing a microscope (IX73, OLYMPUS, Japan). To guage the ALP exercise of BMSCs, the ALP exercise assay equipment (MAK411, Sigma-Aldrich) was used to characterize the cell lysate. The alkaline phosphatase exercise was measured by incubating with a dinitrophenylphosphate answer after which measuring the absorbance at 520 nm with a microplate spectrophotometer (Bio-Tek, Thermo Fisher Scientific).
Alizarin Purple S (ARS) staining: staining of the calcium deposited in BMSCs induced for 21 days with ARS. Repair BMSCs with 4% formaldehyde, stain them with ARS answer (pH 1.354, PHYGENE) at room temperature for 30 min, and wash them with PBS. Observe stained cells utilizing an inverted microscope (IX73, OLYMPUS). Calcium deposits stained with 10% cetylpyridinium chloride (CPC, C0732, Sigma-Aldrich) have been dissolved, and the mineralization course of was measured utilizing a microplate spectrophotometer at 562 nm by figuring out absorbance [38].
Immunofluorescence co-staining
Repair cells with 4% formaldehyde at room temperature for 15 min, adopted by two washes with PBS. Subsequent, cells have been handled with 0.5% Triton X-100 (P0096, Beyotime) for 10 min to extend mobile permeability. Then incubate in a single day at 4 °C with main anti-OPN rabbit antibody (ab11503, 1:200, Abcam) and react with it. After incubation, wash the slices thrice with PBS answer, then incubate with secondary antibody (ab150129/ab150077, 1:200, Abcam) conjugated with Alexa Fluor 488 for 1 h. Then, wash the sections thrice with PBS and stain the cells with 10 μg/mL DAPI (D3571, Thermo Fisher, USA) at room temperature [39]. Lastly, save the slices at 4 °C and observe the cells utilizing a fluorescence microscope (IMT-2, Olympus).
Transcriptome sequencing
This research was permitted by Institutional Animal Ethics Committee of The Third Hospital of Hebei Medical College. Complete RNA was extracted from three samples of rat BMSCs and three osteoblast cells (OB) utilizing TRI reagent (Sigma, USA) following the producer’s directions. The purity and focus of DNA have been then measured utilizing a Nanodrop 2000 spectrophotometer (DeNovix, USA) and Qubit 2.0 fluorometer with the Quant-IT dsDNA HS assay equipment (Thermo Fisher Scientific). Messenger RNA is extracted from whole RNA and purified utilizing oligo-dT magnetic beads. 1 μg DNA was sheared into 250-kb fragments by ultrasonic remedy, adopted by finish restore, dA addition, and ligation to listed Illumina sequencing adapters. Partial enrichment was carried out utilizing DNA seize probes (NimbleGen, US), and deep sequencing was performed on the Illumina NexSeq CN500 platform. The library was checked utilizing Qubit 2.0 and real-time polymerase chain response (PCR), and dimension distribution have been analyzed by a biophysical analyzer. The uncooked information (in fastq format) is first processed by an inside perl script, which removes reads containing adapters, poly-N sequences, and low-quality reads, making certain clear information. All downstream evaluation relies on high-quality information. The reference genome was listed utilizing Hisat2 v2.0.5, and the paired-end clear reads have been aligned to the reference genome. Calculate the learn counts mapped to every gene utilizing featureCounts v1.5.0-p3, after which calculate the fragments per kilobase of transcript per million mapped fragments (FPKM) of every gene based mostly on its size and mapped learn counts [40, 41].
Differential expression evaluation
We performed differential expression evaluation of sequencing information utilizing the “limma” package deal in R language. We extracted differentially expressed genes underneath the situation that |log2FC| > 2 and advert.P.Val < 0.05. We used R language to attract heatmaps and volcano plots of differentially expressed genes [42,43,44].
GO and KEGG practical enrichment evaluation
Carry out practical enrichment evaluation on differentially expressed genes utilizing the ClusterProfiler R package deal. This software program package deal might be used for gene ontology (GO) practical enrichment evaluation, masking three ranges of organic course of (BP), mobile part (CC), and molecular perform (MF) [42]. As well as, we used the Enrichr web site for KEGG evaluation of genes, with a significance threshold of P < 0.05.
Lasso regression is used to display for characteristic genes
The “glmnet” package deal in R language is used to carry out regression evaluation on differentially expressed genes in sequencing information to establish the core characteristic genes for osteogenic differentiation of BMSCs [45].
Twin-luciferase reporter assay
Through the use of TargetScan evaluation, potential binding websites between miR-19b-3p and WWP1 have been predicted. Insert the three’-UTR sequence of WWP1 (wild kind, WT) into the pGL3-Fundamental vector (Promega) to assemble the recombinant vector WWP1-WT. Additional, the miR-19b-3p binding web site mutation sequence of WWP1 was cloned into the a number of cloning web site area of the pGL3-Fundamental vector (Promega) to generate the recombinant vector WWP1-MUT. The Twin-Luciferase Reporter Assay System equipment (Promega, USA) carried out transfection experiments for the wild-type and mutant luciferase reporter plasmids. Cells have been transfected with mimic NC and miR-19b-3p mimic individually and harvested 48 h post-transfection. Cell lysates have been collected and luciferase exercise was measured with Luminometer TD-20/20 Detector (Mannequin: E5311, Promega, USA). Relative fluorescence values have been obtained by dividing RLU values measured by Renilla luciferase utilizing firefly luciferase as an inside management [46]. The experiment was repeated 3 occasions in whole.
RT-qPCR
Complete mobile RNA was extracted utilizing Trizol (16096020, Invitrogen, USA). The absorbance of the answer at 260 and 280 nm was measured utilizing spectrophotometry to judge the purity and focus of the obtained RNA. The A260/A280 ratio of the pattern ought to be ≥ 1.8. The reverse transcription equipment (11483188001, Roche, Switzerland) was used to reverse transcribe mRNA, and cDNA was ready. For miRNA, a PolyA tailing equipment (B532451, Sangon Biotech Co., Ltd., China) was used to acquire cDNA with PolyA-tailed miRNA. The reverse transcription response was carried out at 42 °C for 15 min, adopted by a 5-s inactivation response of the reverse transcriptase at 85 °C. The reverse transcribed cDNA was diluted to 50 ng/μL and subsequently used for fluorescence quantitative PCR evaluation. PCR was carried out utilizing LightCycler 480 SYBR Inexperienced I Grasp. The response situations have been as follows: preliminary denaturation at 95 °C for 10 min, amplification at 95 °C for 15 s, 60 °C for 20 s, 72 °C for 20 s, for 40 cycles. Utilizing GAPDH as the inner reference for mRNA, U6 as the inner reference for miRNA, and miR16 as the inner reference for miRNA in EVs [47]. Calculate 2-ΔΔCt to characterize the goal gene expression fold change between experimental and management teams. The primer sequences are proven in Further file 2: Desk S2. Every experiment was repeated thrice.
Western blot
Complete protein was extracted from tissues or cells utilizing high-efficiency RIPA lysis buffer (C0481, Sigma-Aldrich Aldrich, USA) containing 1% protease inhibitor and 1% phosphatase inhibitor (ST019-5 mg, Beyotime, Shanghai, China). After being lysed at 4 °C for 15 min, the lysate was centrifuged at 15,000 r/min for 15 min. The supernatant was collected, and the protein focus of every pattern was measured utilizing a BCA assay equipment (23,227, TH&Ermo, USA). Quantify the pattern by including 5 occasions of loading buffer (P0015, Bi Yun Tian, China) at completely different concentrations, then separate the protein by polyacrylamide gel electrophoresis and switch it to a PVDF membrane (IPVH00010, Millipore, Billerica, MA, USA). Block with 5% BSA at room temperature for 1 h. Incubate with the primary antibody at 4 °C in a single day. On the second day, wash the membrane thrice utilizing TBST for five min every time. Then, incubate with a diluted answer of goat anti-rabbit IgG (1:2000, ab205718, Abcam, UK) or goat anti-mouse IgG (1:2000, ab6789, Abcam, UK) labeled with HRP at room temperature for 1.5 h. After washing the TBST membrane thrice for 5 minutes every, add the growing answer (NCI4106, Pierce, Rockford, IL, USA) and perform the event course of. Protein quantification evaluation was carried out utilizing ImageJ software program. The grayscale values of every protein have been in comparison with the grayscale ratio of the inner management GAPDH, and every experiment was repeated thrice [48].
Developing rat fashions with femoral bone defects
We bought 24 SD male rats (7 weeks previous, weighing 250–300 g) with pressure code 101 from Beijing Important River Laboratory Animal Expertise Co., Ltd. These rats have been raised in an SPF-grade animal laboratory with a humidity of 60% to 65%, a temperature of twenty-two–25 °C, and free entry to meals and water underneath a 12-h mild–darkish cycle. After one week of adaptive feeding, we noticed the well being situations of the rats earlier than beginning the experiment. This experiment has been permitted by the Animal Ethics Committee of The Third Hospital of Hebei Medical College and conforms to the ideas for the administration and use of experimental animals within the native space.
When performing the femoral defect surgical procedure, we carried out normal anesthesia on every rat. We made a 1.5 cm longitudinal incision within the heart of the palpable bone protrusion on the outer facet of the femoral condyle of every leg whereas taking adequate sterile precautions. Then we rigorously dissected the subcutaneous tissue, fascia, muscle tissue, and periosteum, exposing the underlying bone. We induce bone defects utilizing an oral micromotor to kind a 4 mm deep gap within the subchondral bone under with a 4 mm annular bone drill. Subsequent, we’ll implant corresponding hydrogels into the defect space, adopted by publicity to 405 nm mild for 90 s (25 mW/cm2) to kind a secondary community. Lastly, we rigorously layered and sutured the comfortable tissues and pores and skin. After 8 weeks, we euthanized rats to acquire femurs. We mounted it utilizing over 4% formaldehyde answer (24 h) and preserved it in 75% ethanol for subsequent evaluation [38, 49, 50].
We randomly divided the rats into 4 teams, with 6 rats in every group: (1) PBS group; (2) Gel-OCS/MBGNs@EVs group (injection of Gel-OCS/MBGN@EVs hydrogel); (3) Gel-OCS/MBGNs@EVs-inhibitor NC group (transfecting inhibitor NC obtained EVs into BMSCs, and Gel-OCS/MBGNs@EVs-inhibitor NC hydrogel was ready for injection); (4) Gel-OCS/MBGNs@EVs-miR-19b-3p inhibitor group (EVs have been obtained by transfecting miR-19b-3p inhibitor into BMSCs, and Gel-OCS/MBGNs@EVs-miR-19b-3p inhibitor hydrogel was ready for injection). Inject 100 μL water gel per group, and the ultimate focus of EVs is 0.2 μg/μL.
Micro-CT and X-ray
Bruker’s Micro-CT used a supply present of 280 µA, a supply voltage of 90 kV, and an publicity time of 550 ms. Every defect area was scanned and analyzed in sagittal and axial planes with the identical calibration parameters and reconstructed utilizing NRecon software program. To quantify the newly mineralized tissue, bone mineral content material (BMC) and new bone quantity (BV/TV%) have been calculated. After the specimen was taken, X-ray pictures have been additionally taken [38, 50, 51].
H&E staining and Masson staining
After decalcification in ethylenediaminetetraacetic acid (EDTA) for six weeks, the femur tissue was dehydrated and embedded in paraffin by way of a graded ethanol sequence. After processing, reduce it into 6 μm thick slices for staining. In line with the directions, use Su Mu Jing & Yi Hong (H&E, G1076-500ML, Servicebio) and Masson’s trichrome staining equipment (G1340, Solarbio, Beijing, China) to stain the slices [38, 39, 52].
Statistical evaluation
Knowledge statistical evaluation required for this research was performed utilizing the SPSS 21.0 software program produced by IBM. The imply ± customary deviation represents the measurement information. Firstly, normality and homoscedasticity exams are performed. Suppose the information match the conventional distribution and has equal variances. In that case, the t-test is used to check two teams of knowledge, and a one-way evaluation of variance is used to check a number of information teams. Tukey’s methodology is used for post-hoc exams. When evaluating completely different information teams at completely different time factors, repeated measures evaluation of variance is adopted, and Tukey’s methodology is used for the post-hoc check. When the P worth is lower than 0.05, it signifies that the distinction is statistically important.
