Cell culture and identification

Cell culture: Rat BMSCs (CM-R131, Procell) were 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 solution (10378016, Thermo Fisher Scientific, USA), and maintained at 37 °C and 5% CO2 in a culture incubator. When the confluence rate of BMSCs reaches 80%, passaging should be conducted. The 3rd generation BMSCs were 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 prepared at a concentration of 1 × 106/mL. Grouped cells were 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 were washed off with PBS, and the expression of corresponding labeled antibodies in the sample was analyzed using a flow cytometer. According to the instructions of the reagent kit (PD-003/4/5, Procell) for inducing differentiation (osteogenic, adipogenic, and chondrogenic) of BMSCs, the osteogenic, adipogenic, and chondrogenic differentiation ability of BMSCs was observed by staining with Alizarin Red S (ARS), Oil Red O and Alcian Blue respectively [34].

Cell transfection

According to the sequences of miR-19b-3p and WWP1 published by NCBI, Shanghai Sangon Biotech was commissioned to construct the following 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 were constructed using the following 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 well into a 6-well plate and cultivate them to form a monolayer. Next, remove the culture medium and transfect according to the Lipofectamine 2000 manual (11668-019, Invitrogen, New York, California, USA). After transfection, the cells were cultured at 37 °C under 5% CO2 for 6–8 h, then changed the complete culture medium and incubated for another 48 h to extract RNA and protein for subsequent experiments [35].

Isolation, purification, and identification of EVs derived from BMSCs

Method for isolation of EVs derived from BMSCs: BMSCs transfected with inhibitor NC and miR-19b-3p inhibitor were cultured until 80–90% confluence. Remove the supernatant, wash twice with PBS, replace with FBS culture medium containing 10% EVs depletion, and continue to culture for 48 h in a 37 °C CO2 incubator. Then, the collected supernatant would be centrifuged stepwise. Firstly, centrifuge at 500g for 15 min at 4 °C to remove the cellular debris. Then, centrifuge at 2000g for 15 min at 4 °C to remove apoptotic bodies or cellular debris. Finally, centrifuge at 10,000g for 20 min at 4 °C to remove large vesicles. Next, filter through a 0.22 μm filter and remove EVs by ultracentrifugation at 110,000g for 70 min at 4 °C. After resuspending the remaining material, use 100 μL of sterile PBS for downstream experiments. Beckman ultracentrifuge (Optima L-90K, bio-thing) was used for the high-speed centrifugation steps, while Beckman Allegra X-15R benchtop centrifuge (Beckmancoulter) [11] was used for the remaining low-speed centrifugation steps.

NanoSight nanoparticle tracking analysis (NTA): 20 μg of EVs were dissolved in 1 mL of PBS, vortexed for 1 min, and measured using a NanoSight nanoparticle tracking analyzer (Malvern Instruments Ltd, Malvern Panalytical) and the corresponding software Zetaview 8.04.02. Calibrate using 110 nm polystyrene particles. The temperature is maintained at 27.65 °C to directly observe and measure the size distribution of EVs [11].

Transmission electron microscopy (TEM): 20 μL of a freshly prepared sample of ultracentrifuged EVs were loaded onto carbon-coated copper electron microscopy grids, allowed to stand for 2 min, and then negatively stained with ammonium molybdate (Sigma-Aldrich, USA, 12501-23-4) for 5 min. The grids were washed thrice with PBS to remove excess ammonium molybdate, then air-dried on filter paper until partially dry. The image was acquired with a Hitachi H7650 transmission electron microscope (DOLEE observation) at an accelerating voltage of 80 kV [36].

Western blot technique was used to identify the surface markers of EVs: EVs suspension was concentrated, and its protein content was measured using a BCA assay kit (Thermo Fisher Scientific, USA, 23227). The SDS-PAGE gel was prepared, and the proteins were 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), as well as the endoplasmic reticulum marker protein Calnexin (Abcam, ab133615, 1:1000) were detected after transfer [36].

EV labeling method: DiR (Thermo Fisher Scientific, D12731) was added to the EV solution at a concentration of 1:400 and incubated for 30 min. The solution was then ultracentrifuged at 100,000g for 90 min to remove excess dye and obtain DiR-labeled EVs [36].

Gel-OCS/MBGN hydrogel synthesis reagents and materials

A biomimetic composite hydrogel was prepared by uniformly mixing OCS/MBGN mixture and gelatin (type B, with a gel strength of about 100 g Bloom, G108398-500 g, Cas(9000-70-8), Aladdin reagent, including white gelatin, animal gelatin, sinew glue, silver gelatin, Gelatin(Aladdin-e.com)) at a volume ratio of 1:1 under a temperature of 37 °C. OCS is prepared 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 prepared by mixing tetraethyl orthosilicate (TEOS, AR, 98%, Sigma-Aldrich), cetyltrimethylammonium bromide (CTAB, AR, ≥ 99%, 30037416, Shanghai State-owned Assets Chemical Reagent Co., Ltd.), calcium nitrate (CN, AR 99%, Tianjin Fuchen Chemical Reagent Factory), and ammonia (AR, 25–28%, A112077-500 ml, Cas(1336-21-6), Aladdin Reagent, ammonium hydroxide solution) together [10].

Synthesis of oxidized chondroitin sulfate (OCS)

Dissolve 1.25 g of CS in 20 mL of distilled water and stir at 4 degrees Celsius. After the complete dissolution of CS, add 1.93 g of sodium periodate to the solution and react for 6 h in dark conditions. The molar ratio of CS to sodium periodate is 1:1 for oxidation. Finally, transfer the above solution into a dialysis bag with a molecular weight cutoff of 3500 and dialyze in 2 L of distilled water at room temperature for 24 h. Change the water every 6 h. Afterward, pour the dialysis solution into a 50 mL centrifuge tube, freeze it at − 20 °C for 24 h, then use an FD-10 freeze dryer (made in China) for freeze drying for 7 days to obtain OCS powder [10].

The synthesis of MBGN

Dissolve 2.80 g CTAB in 132 mL deionized water under stirring at 35 °C. After completely dissolving CTAB, add 40 mL of EA and continue stirring for 30 min. Then add 28 mL of ammonia solution (1 mol/L) and stir for 15 min. Then add 14.40 mL TEOS and stir for 30 min; finally, add 6.52 g CN. Further, stir the above mixture for 4 h. Due to the formation of colloids, the mixture gradually turns into a milky white color. Collect colloid particles by centrifugation at 8000 rad/s, wash them three times with water, and then wash them with ethanol. Dry the collected sediment at 60 °C for 24 h, then grind it into a fine powder using a mortar. Finally, heat the powder to 700 °C and maintain it for 3 h to remove organic matter and nitrates to obtain MBGNs [10].

Preparation of composite Gel-OCS/MBGNs hydrogels

Dissolve OCS powder in 0.05 M borate buffer solution to prepare a 10% (w/v) OCS solution. Subsequently, ultrasound dispersion was conducted at room temperature for 10 min, and various concentrations of MBGNs were added to the OCS solution to prepare MBGNs-contained OCS solutions. Heat gelatin to 60 °C to dissolve and prepare a 30% (w/v) gelatin solution. Next, mix the OCS/MBGN mixture with gelatin solution in a 1:1 volume ratio, and then pour into the mold to form a hybrid gel-OCS/MBGN hydrogel at 37 °C. The final concentration of MBGNs in the resulting hydrogels was 0% and 15% (w/w) [10].

OCS characterization

Dissolve CS and OCS separately in deuterium oxide (D2O, 99.9% purity, Adamasβ) for nuclear magnetic resonance (NMR) analysis (AVANCE III HD 400, Bruker) and use tetramethylsilane (TMS) as an internal standard at 25 °C. The chemical structure and functional groups of MBGNs and OCS could be detected with higher resolution (4 cm−1) using Fourier transform infrared spectroscopy (FTIR) in transmission mode, with 16 scans taken in the wavelength range of 400 to 4000 cm−1, using a Nicolet 6700 instrument from Thermo Fisher, USA [10].

Field emission scanning electron microscope (FE-SEM)

We used the FE-SEM (Ultra 55) from the German company Carl Zeiss AG to investigate the morphology of MBGNs and Gel-OCS-MBGN composites. Before characterization, we dispersed the MBGNs in ethanol and then dropped them onto a silicon wafer. Before observing SEM, we freeze-dried the hydrogel sample for three days. We used gold (SC7620) from Quorum Technologies in the UK for a 60-s sputter coating. During SEM observation, we examined samples of MBGNs and Gel OCS/MBGN composite hydrogels by energy-dispersive spectroscopy (EDS) [10].

X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR)

Under a generator voltage of 40 kV and a tube current of 40 mA, the mineralized layer of MBGNs and hydrogels was analyzed using XRD (Ultima III, Japan). The scanning speed is 2°/min, and the 2θ range is between 10° and 80°. Using FTIR in transmission mode, the composite hydrogel's chemical structure and functional groups were detected with a resolution of 4 cm−1 and 16 scans (wavelength range 500–4000 cm−1). Before analyzing the gel-OCS/MBGN hydrogel, it must first be freeze-dried [10].

Rheology and dynamic mechanical, and thermal analysis

Firstly, the gelation time of the hydrogel is determined by recording the storage modulus and loss modulus (elastic modulus) through an oscillatory time scan experiment performed by setting the oscillation frequency to 1 Hz and applying a 5% shear strain. Use Anton Paar's rotational rheometer (Physica MCR301) to evaluate the rheological properties of mixed hydrogels. Place the cross-linked hydrogel (Φ25 mm × 2 mm) on the sample stage and perform the test using a 25 mm diameter plate device at 37 °C by obtaining the storage modulus (G') and the loss modulus (G'') in the range of 0.1 and 100 rad/s from the frequency-modulus curve, with a strain amplitude of 5.0%. In addition, the compressive strength of the saturated hybrid hydrogel was studied using Dynamic Mechanical Analysis (DMA, TA Instruments, Q800) at a pressure rate of 3 N/min and 25 °C. Finally, the compressive modulus was calculated in the DMA test, using the stress–strain curve's linear region to evaluate 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 every other day. After incubation for 7 days, remove the sample from SBF and rinse it with deionized water to eliminate excess SBF. As described above, the samples were characterized by SEM–EDS, FTIR, and XRD before undergoing freeze-drying treatment. Measure the mass loss of the hydrogel in PBS (pH 7.4) to evaluate it’s in vitro degradation. Add about 1 mL of hydrogel to 3 mL of PBS at 37 °C. Remove PBS and record the quality daily to evaluate the degradation of the hydrogel. The following equation could calculate the percentage of quality loss (%): quality loss (%) = (M1-M2)/M1 * 100%, where M1 and M2 represent the quality of the hydrogel before and after soaking in PBS, respectively. Meanwhile, the pH value of PBS-containing water gel was also measured for changes [10].

Gel-OCS/MBGNs hydrogel loaded with EVs

Mix the prepared Gel-OCS/MBGNs hydrogel with EVs isolated from BMSCs labeled with DiR (D12731, Thermo Fisher Scientific), place the mixture in an ice bath until dissolved, and store the mixture at 4 °C. Place the cells in a culture incubator containing 5% CO2 and incubate in the dark for 12 h. Prepare a 1:1000 DAPI nuclear staining buffer, wash cells with PBS three times, place cells on ice, and incubate in the dark for 30 min [37]. Four images with different fields of view were randomly selected under an optical microscope (Leica®, Germany) for statistical analysis.

The in vitro release 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 different time points (1, 3, 6, 9, 12, 15, 18, and 21 days), 100 μL of supernatant was replaced with 100 μL of fresh PBS. The concentration of EVs in the supernatant was evaluated by detecting the fluorescence intensity of DiR (Ex: 754 nm, Em: 778 nm) in the supernatant. Draw release curve, determined by the ratio of supernatant to total fluorescence intensity [38].

CCK-8 and dead cell apoptosis kit are used to evaluate cell viability and proliferation

The live/dead staining experiment for cell viability was performed using the Live/Dead assay kit (L10119, Invitrogen, USA) following the manufacturer's instructions. BMSCs were seeded at a density of 2 × 104 cells/well into 48-well plates containing different hydrogels. Prepare a staining solution with a concentration of 2 μM Calcein-AM and 4 μM ethidium homodimer-1, and add it to the wells—culture the cells in a CO2 incubator at 37 °C and 5% CO2 for 30 min. Imaging of live cells (green, Ex: 480 nm, Em: 530 nm) and dead cells (red, Ex: 530 nm, Em: 645 nm) was performed using an inverted fluorescence microscope (Olympus IMT-2/).

The cell proliferation experiment was conducted using CCK-8 reagent (CA1210, Solarbio) according to the following steps. BMSCs were seeded at a density of 1 × 104 cells/well in 48-well plates containing different hydrogels. On the first, third, fifth, and seventh days of cultivation, the fresh culture medium was added to the 48-well plate with 10% CCK8 reagent and incubated for 1 h under dark conditions. Afterward, transfer 100 μL of the supernatant to a 96-well plate for absorbance measurement at 450 nm using a microplate spectrophotometer (Bio-Tek, UK) [38].

The cell adhesion experiment was performed using iFluor™ 488-phalloidin (ab176753, Abcam) and DAPI (ab285390, Abcam) for immunofluorescence staining. BMSCs were seeded at a density of 1 × 105 cells/well into 35 mm culture dishes with different hydrogels. Fix cells in 4% paraformaldehyde and permeabilize with PBS buffer containing 1% Triton X-100. Afterward, stain with phalloidin solution for 1 h at room temperature, followed by staining with DAPI solution for 5 min. Finally, cells were imaged using a laser scanning confocal microscope (Olympus FV1000, BX61W1).

Induction and identification of osteoblast differentiation

Osteoblast differentiation induction: BMSCs were 3D cultured in hydrogel supplemented with 10 mM β-glycerophosphate, 100 nM dexamethasone, and 50 mg/mL ascorbic acid-2-phosphate in the culture medium to obtain osteogenic induction medium. In the process of BMSC osteogenic differentiation, the culture medium is replaced every three days. Use GelMA digestion kit (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 performed using the alkaline phosphatase staining kit (40749ES60, Yeasen, China) following the manufacturer's instructions. After induced for 7 days, fix the BMSCs in 4% paraformaldehyde, washed with PBS, and stain for 30 min. Observe stained cells using a microscope (IX73, OLYMPUS, Japan). To evaluate the ALP activity of BMSCs, the ALP activity assay kit (MAK411, Sigma-Aldrich) was used to characterize the cell lysate. The alkaline phosphatase activity was measured by incubating with a dinitrophenylphosphate solution and then measuring the absorbance at 520 nm with a microplate spectrophotometer (Bio-Tek, Thermo Fisher Scientific).

Alizarin Red S (ARS) staining: staining of the calcium deposited in BMSCs induced for 21 days with ARS. Fix BMSCs with 4% formaldehyde, stain them with ARS solution (pH 1.354, PHYGENE) at room temperature for 30 min, and wash them with PBS. Observe stained cells using an inverted microscope (IX73, OLYMPUS). Calcium deposits stained with 10% cetylpyridinium chloride (CPC, C0732, Sigma-Aldrich) were dissolved, and the mineralization process was measured using a microplate spectrophotometer at 562 nm by determining absorbance [38].

Immunofluorescence co-staining

Fix cells with 4% formaldehyde at room temperature for 15 min, followed by two washes with PBS. Next, cells were treated with 0.5% Triton X-100 (P0096, Beyotime) for 10 min to increase cellular permeability. Then incubate overnight at 4 °C with primary anti-OPN rabbit antibody (ab11503, 1:200, Abcam) and react with it. After incubation, wash the slices three times with PBS solution, then incubate with secondary antibody (ab150129/ab150077, 1:200, Abcam) conjugated with Alexa Fluor 488 for 1 h. Then, wash the sections three times with PBS and stain the cells with 10 μg/mL DAPI (D3571, Thermo Fisher, USA) at room temperature [39]. Finally, save the slices at 4 °C and observe the cells using a fluorescence microscope (IMT-2, Olympus).

Transcriptome sequencing

This study was approved by Institutional Animal Ethics Committee of The Third Hospital of Hebei Medical University. Total RNA was extracted from three samples of rat BMSCs and three osteoblast cells (OB) using TRI reagent (Sigma, USA) following the manufacturer's instructions. The purity and concentration of DNA were then measured using a Nanodrop 2000 spectrophotometer (DeNovix, USA) and Qubit 2.0 fluorometer with the Quant-IT dsDNA HS assay kit (Thermo Fisher Scientific). Messenger RNA is extracted from total RNA and purified using oligo-dT magnetic beads. 1 μg DNA was sheared into 250-kb fragments by ultrasonic treatment, followed by end repair, dA addition, and ligation to indexed Illumina sequencing adapters. Partial enrichment was performed using DNA capture probes (NimbleGen, US), and deep sequencing was conducted on the Illumina NexSeq CN500 platform. The library was checked using Qubit 2.0 and real-time polymerase chain reaction (PCR), and size distribution were analyzed by a biophysical analyzer. The raw data (in fastq format) is first processed by an internal perl script, which removes reads containing adapters, poly-N sequences, and low-quality reads, ensuring clean data. All downstream analysis is based on high-quality data. The reference genome was indexed using Hisat2 v2.0.5, and the paired-end clean reads were aligned to the reference genome. Calculate the read counts mapped to each gene using featureCounts v1.5.0-p3, and then calculate the fragments per kilobase of transcript per million mapped fragments (FPKM) of each gene based on its length and mapped read counts [40, 41].

Differential expression analysis

We conducted differential expression analysis of sequencing data using the "limma" package in R language. We extracted differentially expressed genes under the condition that |log2FC| > 2 and ad.P.Val < 0.05. We used R language to draw heatmaps and volcano plots of differentially expressed genes [42,43,44].

GO and KEGG functional enrichment analysis

Perform functional enrichment analysis on differentially expressed genes using the ClusterProfiler R package. This software package could be used for gene ontology (GO) functional enrichment analysis, covering three levels of biological process (BP), cellular component (CC), and molecular function (MF) [42]. In addition, we used the Enrichr website for KEGG analysis of genes, with a significance threshold of P < 0.05.

Lasso regression is used to screen for feature genes

The "glmnet" package in R language is used to perform regression analysis on differentially expressed genes in sequencing data to identify the core feature genes for osteogenic differentiation of BMSCs [45].

Dual-luciferase reporter assay

By using TargetScan analysis, potential binding sites between miR-19b-3p and WWP1 were predicted. Insert the 3'-UTR sequence of WWP1 (wild type, WT) into the pGL3-Basic vector (Promega) to construct the recombinant vector WWP1-WT. Further, the miR-19b-3p binding site mutation sequence of WWP1 was cloned into the multiple cloning site region of the pGL3-Basic vector (Promega) to generate the recombinant vector WWP1-MUT. The Dual-Luciferase Reporter Assay System kit (Promega, USA) performed transfection experiments for the wild-type and mutant luciferase reporter plasmids. Cells were transfected with mimic NC and miR-19b-3p mimic separately and harvested 48 h post-transfection. Cell lysates were collected and luciferase activity was measured with Luminometer TD-20/20 Detector (Model: E5311, Promega, USA). Relative fluorescence values were obtained by dividing RLU values measured by Renilla luciferase using firefly luciferase as an internal control [46]. The experiment was repeated 3 times in total.

RT-qPCR

Total cellular RNA was extracted using Trizol (16096020, Invitrogen, USA). The absorbance of the solution at 260 and 280 nm was measured using spectrophotometry to evaluate the purity and concentration of the obtained RNA. The A260/A280 ratio of the sample should be ≥ 1.8. The reverse transcription kit (11483188001, Roche, Switzerland) was used to reverse transcribe mRNA, and cDNA was prepared. For miRNA, a PolyA tailing kit (B532451, Sangon Biotech Co., Ltd., China) was used to obtain cDNA with PolyA-tailed miRNA. The reverse transcription reaction was performed at 42 °C for 15 min, followed by a 5-s inactivation reaction of the reverse transcriptase at 85 °C. The reverse transcribed cDNA was diluted to 50 ng/μL and subsequently used for fluorescence quantitative PCR analysis. PCR was performed using LightCycler 480 SYBR Green I Master. The reaction conditions were as follows: initial 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. Using GAPDH as the internal reference for mRNA, U6 as the internal reference for miRNA, and miR16 as the internal reference for miRNA in EVs [47]. Calculate 2-ΔΔCt to represent the target gene expression fold change between experimental and control groups. The primer sequences are shown in Additional file 2: Table S2. Each experiment was repeated three times.

Western blot

Total protein was extracted from tissues or cells using 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 concentration of each sample was measured using a BCA assay kit (23,227, TH&Ermo, USA). Quantify the sample by adding 5 times of loading buffer (P0015, Bi Yun Tian, China) at different concentrations, then separate the protein by polyacrylamide gel electrophoresis and transfer it to a PVDF membrane (IPVH00010, Millipore, Billerica, MA, USA). Block with 5% BSA at room temperature for 1 h. Incubate with the first antibody at 4 °C overnight. On the second day, wash the membrane thrice using TBST for 5 min each time. Then, incubate with a diluted solution 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 three times for five minutes each, add the developing solution (NCI4106, Pierce, Rockford, IL, USA) and carry out the development process. Protein quantification analysis was performed using ImageJ software. The grayscale values of each protein were compared to the grayscale ratio of the internal control GAPDH, and each experiment was repeated three times [48].

Constructing rat models with femoral bone defects

We purchased 24 SD male rats (7 weeks old, weighing 250–300 g) with strain code 101 from Beijing Vital River Laboratory Animal Technology Co., Ltd. These rats were raised in an SPF-grade animal laboratory with a humidity of 60% to 65%, a temperature of 22–25 °C, and free access to food and water under a 12-h light–dark cycle. After one week of adaptive feeding, we observed the health conditions of the rats before starting the experiment. This experiment has been approved by the Animal Ethics Committee of The Third Hospital of Hebei Medical University and conforms to the principles for the management and use of experimental animals in the local area.

When performing the femoral defect surgery, we performed general anesthesia on each rat. We made a 1.5 cm longitudinal incision in the center of the palpable bone protrusion on the outer side of the femoral condyle of each leg while taking sufficient sterile precautions. Then we carefully dissected the subcutaneous tissue, fascia, muscles, and periosteum, exposing the underlying bone. We induce bone defects using an oral micromotor to form a 4 mm deep hole in the subchondral bone below with a 4 mm annular bone drill. Next, we will implant corresponding hydrogels into the defect area, followed by exposure to 405 nm light for 90 s (25 mW/cm2) to form a secondary network. Finally, we carefully layered and sutured the soft tissues and skin. After 8 weeks, we euthanized rats to obtain femurs. We fixed it using over 4% formaldehyde solution (24 h) and preserved it in 75% ethanol for subsequent analysis [38, 49, 50].

We randomly divided the rats into 4 groups, with 6 rats in each 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 prepared for injection); (4) Gel-OCS/MBGNs@EVs-miR-19b-3p inhibitor group (EVs were obtained by transfecting miR-19b-3p inhibitor into BMSCs, and Gel-OCS/MBGNs@EVs-miR-19b-3p inhibitor hydrogel was prepared for injection). Inject 100 μL water gel per group, and the final concentration of EVs is 0.2 μg/μL.

Micro-CT and X-ray

Bruker's Micro-CT used a source current of 280 µA, a source voltage of 90 kV, and an exposure time of 550 ms. Each defect region was scanned and analyzed in sagittal and axial planes with the same calibration parameters and reconstructed using NRecon software. To quantify the newly mineralized tissue, bone mineral content (BMC) and new bone volume (BV/TV%) were calculated. After the specimen was taken, X-ray images were also taken [38, 50, 51].

H&E staining and Masson staining

After decalcification in ethylenediaminetetraacetic acid (EDTA) for 6 weeks, the femur tissue was dehydrated and embedded in paraffin through a graded ethanol series. After processing, cut it into 6 μm thick slices for staining. According to the instructions, use Su Mu Jing & Yi Hong (H&E, G1076-500ML, Servicebio) and Masson's trichrome staining kit (G1340, Solarbio, Beijing, China) to stain the slices [38, 39, 52].

Statistical analysis

Data statistical analysis required for this study was conducted using the SPSS 21.0 software produced by IBM. The mean ± standard deviation represents the measurement data. Firstly, normality and homoscedasticity tests are conducted. Suppose the data fit the normal distribution and has equal variances. In that case, the t-test is used to compare two groups of data, and a one-way analysis of variance is used to compare multiple data groups. Tukey's method is used for post-hoc tests. When comparing different data groups at different time points, repeated measures analysis of variance is adopted, and Tukey's method is used for the post-hoc test. When the P value is less than 0.05, it indicates that the difference is statistically significant.